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WO2018140827A1 - Reporter microorganisms and uses thereof - Google Patents

Reporter microorganisms and uses thereof Download PDF

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Publication number
WO2018140827A1
WO2018140827A1 PCT/US2018/015602 US2018015602W WO2018140827A1 WO 2018140827 A1 WO2018140827 A1 WO 2018140827A1 US 2018015602 W US2018015602 W US 2018015602W WO 2018140827 A1 WO2018140827 A1 WO 2018140827A1
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WO
WIPO (PCT)
Prior art keywords
reporter
acinetobacter
atcc
microorganism
vol
Prior art date
Application number
PCT/US2018/015602
Other languages
French (fr)
Inventor
Dante RICCI
Ami Patel
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Achaogen, Inc.
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Publication date
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Publication of WO2018140827A1 publication Critical patent/WO2018140827A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • C07K14/212—Moraxellaceae, e.g. Acinetobacter, Moraxella, Oligella, Psychrobacter
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
    • C07K16/1267—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-positive bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/21—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Pseudomonadaceae (F)
    • G01N2333/212—Moraxellaceae, e.g. Acinetobacter, Moraxella, Oligella or Psychrobacter

Definitions

  • the present disclosure provides in some aspects reporter polynucleotides comprising a sequence comprising a regulatory region of an outer membrane (OM) stress-responsive gene of an Acinetobacter bacterium operably linked to a sequence encoding a reporter molecule.
  • the disclosure further relates to reporter vectors, genetically engineered reporter microorganisms, and uses thereof.
  • Multidrug-resistant bacteria have emerged worldwide and are increasing in prevalence, creating a substantial public health concern.
  • the Centers for Disease Control and Prevention attributes at least 23,000 deaths in the U.S. each year to antibiotic-resistant infections, with some infection types associated with mortality rates as high as 50%.
  • infections such as Acinetobacter spp. and Pseudomonas aeruginosa
  • rates of multi-drug resistance in the U.S. have been reported as 63% and 13%, respectively.
  • the continued prevalence of these multidrug-resistant isolates has left clinicians with few treatment options for the patients with life-threatening infections. Addressing this urgent need for new antibiotics to treat multidrug-resistant Gram-negative infections is critical. There is a need in the art for methods of identifying antibiotics specific for pathogenic bacteria. Provided are methods and articles of manufacture that meet such need.
  • reporter polynucleotides comprising a sequence comprising a regulatory region of an outer membrane (OM) stress-responsive gene of an Acinetobacter bacterium operably linked to a sequence encoding a reporter molecule, wherein the OM stress- responsive gene is modulated in response to a stress to the outer membrane of the Acinetobacter bacterium.
  • OM outer membrane
  • the Acinetobacter bacterium is a Acinetobacter apis, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii,
  • Acinetobacter brisouii Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter organizerri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus, Acinetobacter harbinensis, Acinetobacter indicus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter kookii, Acinetobacter Iwoffii, Acinetobacter nectaris, Acinetobacter nosocomialis, Acinetobacter parvus, Acinetobacter pakistanensis, Acinetobacter pittii, Acinetobacter puyangensis, Acinetobacter qingfengensis, Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii
  • Acinetobacter soli Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis, or Acinetobacter venetianus.
  • the Acinetobacter bacterium is Acinetobacter baumannii.
  • the Acinetobacter bacterium is ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA- 1605, ATCC BAA-1709, ATCC BAA- 1710, ATCC BAA- 1789, ATCC BAA-1790, ATCC BAA-1791, ATCC BAA-1792, ATCC BAA-1793, ATCC BAA-1794, ATCC BAA- 1795, ATCC BAA- 1796, ATCC BAA-1797, ATCC BAA-1798, ATCC BAA-1799, ATCC BAA-
  • Acinetobacter bacterium is ATCC 17978, Ab307-0294, AABA041, or AABA046.
  • the stress to the outer membrane of the Acinetobacter bacterium is or is caused by depletion of BamA or treatment with polymyxin B nonapeptide (PMBN). In certain embodiments, the stress to the outer membrane of the Acinetobacter bacterium is or is caused by depletion of BamA and treatment with PMBN.
  • PMBN polymyxin B nonapeptide
  • stress to the outer membrane of Acinetobacter bacterium involves depletion of BamA and depletion of BamA is performed by removing or decreasing an amount of an inducer that controls expression of BamA from a culture or composition containing Acinetobacter bacterium.
  • the inducer is removed or decreased for more than or more than about 5, 10, 15, 20, 30, 45, 60, 75, 90, or 120 minutes.
  • the inducer is removed or decreased for more than or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours.
  • the inducer is arabinose.
  • stress to the outer membrane of Acinetobacter bacterium involves treatment with PMBN and the Acinetobacter bacterium is treated with PMBN for less than or less than about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, or 120 minutes. In certain embodiments, the Acinetobacter bacterium is treated with PMBN for greater than or greater than about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, or 120 minutes.
  • the OM stress-responsive gene is upregulated or
  • the OM stress-responsive gene is upregulated in response to the stress. In certain embodiments, the OM stress-responsive gene is upregulated in response to the stress at least or about at least 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, 10-fold or more in response to the stress.
  • the OM stress-responsive gene is A1SJ3012, A1SJ3023, A1S_0027, A1S_0028, A1S_0029, A1S_0030, A1S_0031, A1S_0032, A1S_0033, A1S_0037, A1SJ3040, A1SJ3041, A1S_0044, A1S_0066, A1S_0092, A1S_0093, A1S_0109, A1S_0110, A1S_0112, A1S_0113, A1S_0114, A1S_0115, A1S_0116, A1S_0117, A1S_0118, A1S_0126, A1S_0158, A1S_0170, A1S_0175, A1S_0178, A1S_0189, A1S_0224, A1S_0245, A1S_0256, A
  • A1S_ _0680, A1S_ _0683 A1S_ _0714, A1S_ _0717 A1S_ _0718, A1S_ _0719 A1S_ _0736, A1S_ _0738,
  • the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or A1S_3127.
  • the OM stress-responsive gene is
  • the OM stress-responsive gene is downregulated in response to the stress. In certain embodiments, the OM stress-responsive gene is downregulated in response to the stress at least or about at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more in response to the stress.
  • the OM stress-responsive gene is A1S_0009, A1S_0010,
  • A1S_ _1072 A1S_ _1079 A1S_ _1080, A1S_ _1088 A1S_ .1089, A1S_ .1091 A1S_ . 1092, A1S_ .1093,
  • the OM stress-responsive gene is A1S_0103, A1S_0645, A1S_1266, A1S_1268, A1S_1335, A1S_1336, A1S_1337, A1S_1338, A1S_1339, A1S_1340, A1S_1341, A1S_1342, A1S_1343, A1S_1344, A1S_1345, A1S_1791, A1S_1792, A1S_1794, A1S_1796, A1S_1835, A1S_1836, A1S_1837, A1S_1838, A1S_1839, A1S_2449, A1S_2450, A1S_2452, A1S_3540, A1S_3541, A1S_3542, A1S_3543, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_380
  • the OM stress- responsive gene is A1S_1336, A1S_1836, A1S_1838, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, or A1S_3809.
  • the regulatory region comprises a contiguous sequence of nucleotides within 500 base pairs upstream or 5' of the open reading frame (ORF) of the OM stress-responsive gene.
  • the contiguous sequence of nucleotides comprises at least or at least about 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400 or more base pairs.
  • the regulatory region comprises a promoter.
  • the regulatory region comprises a sequence to further promote translation of the encoded reporter molecule.
  • the sequence further promoting translation is or comprises a bacterial ribosome binding site.
  • the ribosome binding site is a Shine-Dalgarno sequence.
  • the Shine-Dalgarno sequence is native to the regulatory region of the OM stress-responsive gene.
  • the Shine-Dalgarno sequence is synthetic and/or heterologous to the regulatory region of the OM stress-responsive gene.
  • the Shine- Dalgarno sequence comprises the sequence set forth in SEQ K) NO: 14 or a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 14. In certain embodiments, the Shine-Dalgarno sequence comprises a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 14.
  • the regulatory region comprises the sequence set forth in any of SEQ ID NOS: 1-13 or a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NOs: 1-13. In certain embodiments, the regulatory region comprises a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NOs: 1- 13.
  • the reporter molecule is a fluorescent protein, a luminescent protein, a chromoprotein, or an enzyme.
  • the fluorescent protein is Sirius, SBFP2, Azurite, mAzurite, EBFP2, moxBFP, mKalamal, mTagBFP2, Aquamarine, ECFP, Cerulean, mCerulean, mCerulean3, moxCerulean3, SCFP3A, mTurquoise2, CyPet, AmCyanl, MiCy (Midoriishi-Cyan), iLOV, AcGFPl, sfGFP, moxGFP, mEmerald, EGFP, mEGFP, AzamiGreen, cfSGFP2, ZsGreen, SGFP2, Clover, mClover2, mClover3, EYFP, Topaz, mTopaz, mVenus, mox Venus, SYFP
  • the fluorescent protein is sfGFP, mClover3, or mRuby2. In certain embodiments, the fluorescent protein is sfGFP.
  • the sequence encoding the reporter molecule comprises the sequence set forth in SEQ ID NO: 15, 33, or 40 or a sequence with at least or at least about 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 15, 33, or 40. In certain embodiments, the sequence encoding the reporter molecule comprises a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 15.
  • the reporter polynucleotide comprises the sequence set forth in any of SEQ ID NOs: 16-28 or 34-36 or a sequence with at least or at least about 85%, 90%, 95%, 99%, or more sequence identity to any of SEQ ID NOs: 16-28 or 34-36. In certain embodiments, the reporter polynucleotide comprises a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NOs: 16-28.
  • the luminescent protein is North American firefly luciferase, Genji-botaru luciferase, Italian firefly luciferase, Heike luciferase, East European firefly luciferase, Pennsylvania firefly luciferase, Click beetle luciferase, Railroad worm luciferase, Renilla luciferase, Rluc8, Green Renilla luciferase, Gaussia luciferase, Gaussia-Dura luciferase, Cypridina luciferase, Vargula luciferase, Metridia luciferase, OLuc, bacterial luciferase
  • the enzyme is
  • chloramphenicol acetyltransferase CAT
  • ⁇ -galactosidase alkaline phosphatase
  • ⁇ - glucuronidase alkaline phosphatase
  • ⁇ -lactamase neomycin phosphotransferase
  • neomycin phosphotransferase or a modified version thereof.
  • reporter vectors comprising any of the reporter polynucleotide described herein.
  • the reporter polynucleotide is a first reporter polynucleotide and the reporter vector further comprises a second reporter polynucleotide that is any of the reporter polynucleotides described herein.
  • the first reporter polynucleotide and the second reporter polynucleotide are different.
  • the regulatory region of the first reporter polynucleotide and the regulatory region of the second reporter polynucleotide are from different OM stress-responsive genes.
  • the reporter molecule encoded by the first reporter polynucleotide is different from the reporter molecule encoded by the second reporter polynucleotide. In certain embodiments, the reporter molecule encoded by the first reporter polynucleotide and the reporter molecule encoded by the second reporter polynucleotide do not exhibit an overlapping emission and absorption spectra and/or are distinguishably detectable.
  • the reporter vector is capable of being expressed in a host microorganism. In certain embodiments, the reporter vector is capable of being expressed in a Gram-negative bacterium. In certain embodiments, the host microorganism is Acinetobacter, Bdellovibrio, Burkholderia, Chlamydia, Enter obacter, Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella, Neisseria, Pantoea, Pseudomonas, Salmonella, Shigella, Stenotrophomonas, Vibrio, or Yersinia.
  • the host microorganism is Acinetobacter. In certain embodiments, the host microorganism is Acinetobacter apis, Acinetobacter baumannii,
  • Acinetobacter baylyi Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii,
  • the host microorganism is Acinetobacter baumannii.
  • the host microorganism is ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA- 1605, ATCC BAA-1709, ATCC BAA-1710, ATCC BAA-1789, ATCC BAA-1790, ATCC BAA- 1791, ATCC BAA- 1792, ATCC BAA-1793, ATCC BAA-1794, ATCC BAA-1795, ATCC BAA- 1796, ATCC BAA-1797, ATCC BAA-1798, ATCC BAA- 1799, ATCC BAA-1800, ATCC
  • the reporter polynucleotide is comprised in a backbone vector, and the backbone vector is pACH106, pWH1266, or pET-RA.
  • the reporter polynucleotide is comprised in a backbone vector, and the backbone vector is pACH106, pWH1266, or pET-RA.
  • a nucleotide sequence comprising the reporter polynucleotide, optionally the first reporter polynucleotide and the second reporter
  • polynucleotide is inserted into or replaces a portion of the nucleotide sequence of the backbone vector.
  • a nucleotide sequence comprising the reporter polynucleotide is inserted into or replaces a portion of the nucleotide sequence of the backbone vector.
  • the backbone vector comprises the sequence of nucleotides set forth in SEQ ID NO: 29 and a nucleotide sequence comprising the reporter polynucleotide replaces nucleotides 5,715-7,395 of the backbone vector.
  • the reporter vector comprises the sequence set forth in any of SEQ ID NOs: 30-32 or a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NOs: 30-32. In certain embodiments, the reporter vector comprises a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NOs: 30-32.
  • reporter microorganisms comprising any of the reporter polynucleotides or any of the reporter vectors described herein.
  • reporter microorganisms comprising one or more reporter polynucleotide of any described herein or one or more reporter vector of any described herein.
  • the one or more reporter polynucleotides comprise a first reporter polynucleotide and a second reporter polynucleotide that are different.
  • the regulatory region of the first reporter polynucleotide and the regulatory region of the second reporter polynucleotide are from different OM stress-responsive genes.
  • the reporter molecule encoded by the first reporter polynucleotide is different from the reporter molecule encoded by the second reporter polynucleotide. In certain embodiments, the reporter molecule encoded by the first reporter polynucleotide and the reporter molecule encoded by the second reporter polynucleotide do not exhibit an overlapping emission and absorption spectra and/or are distinguishably detectable. In certain embodiments, the first reporter polynucleotide and second reporter polynucleotide are comprised in the same reporter vector. In certain embodiments, the first reporter polynucleotide and second reporter polynucleotide are comprised in different reporter vectors.
  • the reporter microorganism is a Gram-negative bacterium. In certain embodiments, the reporter microorganism is Acinetobacter, Bdellovibrio,
  • Burkholderia Chlamydia, Enterobacter, Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella, Neisseria, Pantoea, Pseudomonas, Salmonella, Shigella, Stenotrophomonas, Vibrio, or Yersinia.
  • the reporter microorganism is Acinetobacter.
  • the reporter microorganism is Acinetobacter apis, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii,
  • the reporter microorganism is ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA- 1605, ATCC BAA-1709, ATCC BAA-1710, ATCC BAA-1789, ATCC BAA- 1790, ATCC BAA- 1791, ATCC BAA-1792, ATCC BAA-1793, ATCC BAA-1794, ATCC BAA- 1795, ATCC BAA-1796, ATCC BAA-1797, ATCC BAA- 1798, ATCC BAA-1799, ATCC BAA-1800, ATCC BAA- 1878, ATCC BAA-2093, ATCC BAA-747, S
  • each of at least two reporter microorganisms in the plurality comprise a different reporter polynucleotide in which comprises the same regulatory region of an OM-responsive gene but that is operatively linked to a different reporter molecule.
  • the different reporter molecules do not exhibit overlapping emission and absorption spectra and/or are distinguishably detectable.
  • the plurality of reporter microorganisms comprises 2, 3, 4, 5, or more different reporter microorganisms.
  • the at least two reporter microorganisms are derived from the same host strain.
  • each of the at least two reporter microorganisms is derived from a different host strain, optionally wherein each of the at least two reporter microorganisms is derived from a different isolate or subtype of the strain.
  • compositions comprising a reporter microorganism described herein or a plurality of any of the reporter microorganisms described herein.
  • the composition comprises one or more components capable of activating the complement pathway.
  • the composition comprises serum.
  • the serum is human serum, rabbit serum, bovine serum, or mouse serum.
  • the concentration of serum is at least or at least about 2.5% (vol/vol), 5.0% (vol/vol), 7.5% (vol/vol), 10.0% (vol/vol), 15.0% (vol/vol), 20% (vol/vol), or 25% (vol/vol).
  • the concentration of serum is from or from about 2.5% (vol/vol) to 25% (vol/vol), 2.5% (vol/vol) to 15% (vol/vol), 2.5% (vol/vol) to 10% (vol/vol), 2.5% (vol/vol) to 5.0% (vol/vol), 5.0% (vol/vol) to 25% (vol/vol), 5.0% (vol/vol) to 15% (vol/vol), 5.0% (vol/vol) to 10% (vol/vol), 10.0% (vol/vol) to 25% (vol/vol), 10.0% (vol/vol) to 15% (vol/vol), or 15% (vol/vol) to 25% (vol/vol).
  • the composition comprises an agent, optionally a candidate antibacterial agent.
  • the agent is a small molecule compound, a peptide or a protein.
  • the agent is an antibody or antigen-binding fragment thereof.
  • microdroplets comprising a microorganism described herein, a plurality of microorganisms described herein, or a composition described herein.
  • microdroplets comprising a reporter microorganism described herein.
  • the microdroplet comprises an agent, such as a candidate antibacterial agent, including agents known to cause or suspected of causing OM stress to the reporter
  • microorganism and/or that may impact the integrity or biogenesis of the outer membrane of the reporter microorganism.
  • the microdroplet further comprises a cell that produces or secretes an agent.
  • the cell is an antibody-producing cell.
  • the cell is a B cell.
  • the cell is a plasma cell or a plasmablast.
  • the cell is a mammalian cell.
  • the cell is a microorganism.
  • the cell is a fungal or bacterial cell.
  • the agent is a candidate antibacterial agent.
  • the agent is a small molecule compound, a peptide, or a protein.
  • the agent is an antibody or antigen-binding fragment thereof.
  • the agent is a small molecule antibiotic or peptide antibiotic.
  • the microdroplet comprises agarose, carrageenan, alginate, alginate-polylysine, collagen, cellulose, methylcellulose, gelatin, chitosan, extracellular matrix, dextran, starch, inulin, heparin, hyaluronan, fibrin, polyvinyl alcohol, poly(N-vinyl-2- pyrrolidone), polyethylene glycol, poly(hydroxyethyl methacrylate), acrylate polymers and sodium polyacrylate, polydimethyl siloxane, cis- polyisoprene, PuramatrixTM, poly- divenylbenzene, polyurethane, polyacrylamide or combinations thereof.
  • agarose carrageenan
  • alginate alginate-polylysine
  • collagen cellulose
  • methylcellulose gelatin
  • gelatin chitosan
  • extracellular matrix extracellular matrix
  • dextran starch
  • inulin heparin
  • hyaluronan fibrin
  • the microdroplet comprises agarose.
  • the microdroplet comprises growth media.
  • the microdroplet comprises serum.
  • the serum is human serum, rabbit serum, bovine serum, or mouse serum.
  • the concentration of serum is at least or at least about 2.5% (vol/vol), 5.0% (vol/vol), 7.5% (vol/vol), 10.0% (vol/vol), 15.0% (vol/vol), 20% (vol/vol), or 25% (vol/vol).
  • the concentration of serum is from or from about 2.5% (vol/vol) to 25% (vol/vol), 2.5% (vol/vol) to 15% (vol/vol), 2.5% (vol/vol) to 10% (vol/vol), 2.5% (vol/vol) to 5.0% (vol/vol), 5.0% (vol/vol) to 25% (vol/vol), 5.0% (vol/vol) to 15% (vol/vol), 5.0% (vol/vol) to 10% (vol/vol), 10.0% (vol/vol) to 25% (vol/vol), 10.0% (vol/vol) to 15% (vol/vol), or 15% (vol/vol) to 25% (vol/vol).
  • compositions comprising a microdroplet described herein or a plurality of microdroplets described herein.
  • kits comprising: a reporter polynucleotide described herein, a reporter vector described herein, a reporter microorganism described herein, a plurality of reporter microorganisms described herein, a composition described herein, or a microdroplet described herein; and instructions for use.
  • the method further comprises (c) determining if there a change in the level of the detectable signal from the reporter molecule(s) compared to in the absence of exposing the reporter microorganism to the condition, wherein a change in the level of the detectable signal indicates the condition causes OM stress to the microorganism.
  • exposing the reporter microorganism to a condition or agent is carried out in suspension, in an array, or in a microdroplet.
  • the condition is treatment with an agent.
  • kits for screening an agent comprising: (a) contacting a reporter microorganism described herein, a plurality of reporter microorganisms described herein, or a composition described herein with an agent; and b) detecting the presence, absence, or level of a detectable signal from the reporter molecule(s).
  • the method further comprises (c) identifying the agent that causes a change in the level of the detectable signal from the reporter molecule compared to in the absence of contacting the reporter microorganism to the agent.
  • kits for screening an agent comprising: (a) contacting an agent with a first reporter microorganism described herein; (b) contacting the agent with at least one additional reporter microorganism described herein, wherein the at least one additional reporter microorganism is not the same as the first reporter microorganism; and (c) detecting the presence, absence, or level of a detectable signal from the reporter molecule from the first and/or at least one additional reporter microorganism.
  • the contacting in (a) and (b) is carried out separately. In certain embodiments, the contacting in (a) and (b) is carried out together.
  • the first microorganism, the at least one additional reporter microorganism, and the agent are encapsulated together in a microdroplet.
  • the first and the at least one additional reporter microorganism comprise a different reporter polynucleotide in which comprises the same regulatory region of an OM-responsive gene but that is operatively linked to a different reporter molecule.
  • the first and the at least one additional microorganism comprise a different reporter polynucleotide in which comprises a different regulatory region of an OM-responsive gene and is operatively linked to a different reporter molecule.
  • the different reporter molecules do not exhibit overlapping emission and absorption spectra and/or are distinguishably detectable.
  • the first and the at least one additional microorganism are derived from the same host strain. In certain embodiments, the first and the at least one additional microorganism are derived from a different host strain, optionally wherein each of the first and the at least one additional microorganism is derived from a different isolate or subtype of the strain. In certain embodiments, the contacting is carried out in suspension, in an array, or in a microdroplet. In certain embodiments, the contacting is carried out for at least or about at least 5 minutes, 10 minutes, 30 minutes, 60 minutes, 2 hours or 3 hours.
  • step (a) is carried out with a plurality of agents, wherein the reporter microorganism is contacted with each of the plurality of agents.
  • kits for screening an agent comprising: (a) encapsulating in a microdroplet: (i) a reporter microorganism described herein, a plurality of reporter
  • microorganisms described herein, or a composition described herein and (ii) a cell, wherein the cell produces an agent; and (b) detecting, in the microdroplet, the presence, absence, or level of a detectable signal from the reporter molecule(s).
  • identifying an agent that modulates an activity or property of a reporter microorganism comprising: (a) encapsulating in a microdroplet: (i) a reporter microorganism comprising a reporter polynucleotide described herein or a reporter vector described herein; and (ii) a cell, wherein the cell produces an agent; and (b) identifying a reporter microorganism in which there is a change in a detectable signal from the reporter molecule; thereby identifying an agent that modulates the activity or property of the target microorganism.
  • the method further comprises (c) isolating the cell from the microdroplet in which there is a change in the level of the detectable signal from the reporter molecule compared to in the absence of exposing the reporter microorganism to the agent.
  • the isolating is carried out using a micromanipulator or an automated sorter.
  • the method further comprises identifying the agent produced by the cell.
  • the identifying comprises determining the sequence of the agent, optionally using single cell PCR and nucleic acid sequencing.
  • step (a) is repeated with a plurality of agents.
  • the microorganism if there is a change in the presence, absence, or level of the detectable signal, the microorganism is identified as potentially not being resistant to the agent; and if there is not a change in the presence, absence, or level of the detectable signal, the microorganism is identified as potentially being resistant to the agent.
  • determining the drug resistance of a reporter microorganism comprising: (a) contacting a reporter microorganism comprising a reporter polynucleotide described herein or a reporter vector described herein with a drug; and (b) identifying a reporter microorganism in which there is a change in a detectable signal from the reporter molecule compared to in the absence of contacting the reporter microorganism with the drug, wherein if there is a change in the detectable signal, the reporter microorganism is not resistant to the drug and if there is not a change in the detectable signal, the reporter
  • microorganism is identified as potentially being resistant to the drug.
  • the OM-stress responsive gene that is operably linked to the reporter molecule in the reporter vector comprised in the reporter microorganism is
  • the OM-stress responsive gene that is operably linked to the reporter molecule in the reporter vector comprised in the reporter microorganism is downregulated in response to an outer membrane stress and the level of the detectable signal decreases.
  • the OM stress- responsive gene is A1S_0009, A1S_0010, A1S_0025, A1S_0027, A1S_0038, A1S_0067,
  • A1S_ _nn A1S_ _1719 A1S_ _1724, A1S_ _1729 A1S_ _1730, A1S_ _1731 A1S_ _1732, A1S_ _1734,
  • A1S_ _3912, A1S_ _3914, or A1S_3915 are A1S_ _3912, A1S_ _3914, or A1S_3915.
  • the OM stress- responsive gene is A1S_0103, A1S_0645, A1S_1266, A1S_1268, A1S_1335, A1S_1336, A1S_1337, A1S_1338, A1S_1339, A1S_1340, A1S_1341, A1S_1342, A1S_1343, A1S_1344, A1S_1345, A1S_1791, A1S_1792, A1S_1794, A1S_1796, A1S_1835, A1S_1836, A1S_1837, A1S_1838, A1S_1839, A1S_2449, A1S_2450, A1S_2452, A1S_3540, A1S_3541, A1S_3542, A1S_3543, A1S_35
  • the OM stress-responsive gene is A1S_1336, A1S_1836, A1S_1838, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, or A1S_3809.
  • the OM-stress responsive gene that is operably linked to the reporter molecule in the reporter vector comprised in the reporter microorganism is upregulated in response to an outer membrane stress and the change in the detectable signal is an increase in the detectable signal.
  • the OM-stress responsive gene that is operably linked to the reporter molecule in the reporter vector comprised in the reporter microorganism is upregulated in response to an outer membrane stress and the level of the detectable signal increases.
  • the OM stress- responsive gene is A1S_0012, A1S_0023, A1S_0027, A1S_0028, A1S_0029, A1S_0030,
  • the OM stress- responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_0189, A1S_0516, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, A1S_3127, A1S_3492, or AIS_3791.
  • the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or A1S_3127.
  • the OM stress-responsive gene is A1S_0032, A1S_2885, A1S_2889, A1S_3127, A1S_3492, A1S_3791.
  • the OM stress-responsive gene is A1S_0032, A1S_2885, or A1S_2889.
  • the OM stress-responsive gene is A1S_0113 or A1S_1751. In certain embodiments, the OM stress-responsive gene is A1S_0189, A1S_0516, A1S_1224, A1S_2093, or A1S_2271.
  • a method of assessing OM stress comprising: (a) contacting a reporter microorganism described herein or a composition described herein with an agent; and (b) identifying a reporter microorganism producing a detectable signal from the reporter molecule, thereby assessing OM stress.
  • step (a) is carried out with a plurality of agents, wherein the reporter microorganism is contacted with each of the plurality of agents.
  • kits for identifying an agent that modulates an activity or property of a microorganism comprising: (a) encapsulating in a microdroplet: (i) a reporter microorganism comprising a reporter polynucleotide described herein or a reporter vector described herein; and (ii) a cell, wherein the cell produces an agent; and (b) identifying a reporter microorganism producing a detectable signal from the reporter molecule.
  • a reporter microorganism comprising a reporter polynucleotide described herein or a reporter vector described herein with a drug; and (b) identifying a reporter microorganism producing a detectable signal from the reporter molecule, wherein if the reporter microorganism produces a detectable signal, the reporter microorganism is not resistant to the drug and if the reporter microorganism does not produce a detectable signal, the reporter microorganism is identified as potentially being resistant to the drug.
  • the OM-stress responsive gene that is operably linked to the reporter molecule in the reporter vector comprised in the reporter microorganism is downregulated in response to an outer membrane stress.
  • the OM stress- responsive gene is A1S_0009, A1S_0010, A1S_0025, A1S_0027, A1S_0038, A1S_0067,
  • A1S_ _3912, A1S_ _3914, or A1S_3915 are A1S_ _3912, A1S_ _3914, or A1S_3915.
  • the OM stress- responsive gene is A1S_0103, A1S_0645, A1S_1266, A1S_1268, A1S_1335, A1S_1336, A1S_1337, A1S_1338, A1S_1339, A1S_1340, A1S_1341, A1S_1342, A1S_1343, A1S_1344, A1S_1345, A1S_1791, A1S_1792, A1S_1794, A1S_1796, A1S_1835, A1S_1836, A1S_1837, A1S_1838, A1S_1839, A1S_2449, A1S_2450, A1S_2452, A1S_3540, A1S_3541, A1S_3542, A1S_3543, A1S_35
  • the OM stress-responsive gene is A1S_1336, A1S_1836, A1S_1838, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, or A1S_3809.
  • the OM-stress responsive gene that is operably linked to the reporter molecule in the reporter vector comprised in the reporter microorganism is upregulated in response to an outer membrane stress.
  • the OM stress- responsive gene is A1S_0012, A1S_0023, A1S_0027, A1S_0028, A1S_0029, A1S_0030,
  • the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or A1S_3127.
  • the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or A1S_3127.
  • the OM stress-responsive gene is
  • the contacting is carried out in suspension, in an array, or in a microdroplet.
  • the reporter is carried out in suspension, in an array, or in a microdroplet.
  • microorganism of (a) is encapsulated in a microdroplet with a cell.
  • the agent is a candidate antibacterial agent, such as an agent known to cause or suspected of causing OM stress to the reporter microorganism and/or that may impact the integrity or biogenesis of the outer membrane of the reporter microorganism.
  • the agent is a small molecule compound, a peptide or a protein.
  • the agent is an antibiotic.
  • the agent is an antibody or antigen-binding fragment thereof.
  • the agent is an antibody and the cell is an antibody-producing cell.
  • the cell is a microorganism. In certain embodiments, the cell is a fungal or bacterial cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a B cell. In certain embodiments, the cell is a plasma cell or a plasmablast.
  • the cell is obtained from a donor that has been exposed to a target microorganism or an epitope-comprising fragment of a target microorganism or a variant thereof.
  • the donor has been immunized or infected with a target microorganism or an epitope-comprising fragment of a target microorganism or a variant thereof.
  • the donor is an immunized animal or an infected animal.
  • the donor is a mammal or a bird.
  • the donor is a human, a mouse or a chicken.
  • the donor is a human donor who was infected by the target microorganism.
  • the donor is a genetically modified non-human animal that produces partially human or fully human antibodies.
  • the microdroplet is generated by a microfluidics-based method.
  • the microdroplet comprises agarose, carrageenan, alginate, alginate-polylysine, collagen, cellulose, methylcellulose, gelatin, chitosan, extracellular matrix, dextran, starch, inulin, heparin, hyaluronan, fibrin, polyvinyl alcohol, poly(N-vinyl-2- pyrrolidone), polyethylene glycol, poly(hydroxyethyl methacrylate), acrylate polymers and sodium polyacrylate, polydimethyl siloxane, cis- polyisoprene, PuramatrixTM, poly- divenylbenzene, polyurethane, polyacrylamide, or combinations thereof.
  • the microdroplet comprises agarose.
  • the method further comprises: (c) isolating the cell from the microdroplet in which there is a change in the level of the detectable signal from the reporter molecule compared to in the absence of exposing the reporter microorganism to the agent.
  • the isolating is carried out using a micromanipulator or an automated sorter.
  • the method further comprises identifying the agent produced by the cell.
  • the identifying comprises determining the sequence of the agent, optionally using single cell PCR and nucleic acid sequencing.
  • the method further comprises: (c) isolating the microdroplet comprising the cell producing the identified agent. In certain embodiments, the method further comprises: (d) isolating polynucleotides encoding the agent, wherein the agent is a polypeptide. In certain embodiments, the method further comprises: (e) determining the sequence of the nucleic acids encoding the polypeptide. In certain embodiments, the method further comprises prior to (a) introducing the reporter polynucleotide or reporter vector described herein into a host microorganism.
  • the host microorganism is a bacterium. In certain embodiments, the host microorganism is a Gram negative bacterium. In certain embodiments, the host microorganism is a proteobacterium. In certain embodiments, the host microorganism is a species of Acinetobacter, Bdellovibrio, Burkholderia, Chlamydia, Enterobacter,
  • Escherichia Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella, Neisseria, Pantoea, Pseudomonas, Salmonella, Shigella, Stenotrophomonas, Vibrio, or Yersinia.
  • the bacterium Acinetobacter apis, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii,
  • Acinetobacter radioresistans Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii, Acinetobacter soli, Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis,
  • the agent is a drug.
  • the drug is a small molecule compound, a peptide or a protein.
  • the drug is an antibiotic.
  • the drug is an antibody or antigen-binding fragment thereof.
  • the drug is an antibody and the cell is an antibody-producing cell.
  • agents identified by any of the methods described herein are agents identified by any of the methods described herein.
  • Fig. 1 shows growth curves of a modified A. baumannii strain that is depleted for BamA unless exogenous arabinose is added.
  • the growth curves compare growth in the presence or absence of added arabinose for up to 9 hours.
  • Circles indicate the time points at which the cells were back-diluted to maintain cells in an exponential growth phase.
  • Rectangles indicate the time points at which samples were taken for analysis by RNA-Seq for comparing differences in gene expression caused by outer membrane stress induced by BamA depletion as described in subsequent Examples and Figs.
  • Fig. 2A-2C show differential gene expression in A. baumannii with or without BamA depletion as determined by RNA-Seq with counts normalized as fragments per kilobase per million reads (FPKM).
  • Fig. 2A depicts results for replicate transcript datasets from A.
  • Fig. 2B depicts results of replicate transcript datasets from A. baumannii cells with BamA depletion cultured in the absence of added arabinose for 6 hours.
  • Fig. 2C shows differences in gene expression when comparing BamA-depleted (+Ara) and non-BamA-depleted (-Ara) cells.
  • Fig. 3A-3C depicts log fold differences in gene expression in A. baumannii over time in BamA depleted cell cultures (incubated without arabinose; -Ara) compared to non-BamA- depleted cell cultures (incubated in the presence of arabinose; +Ara).
  • Fig. 3 A compares the abundance of gene transcripts after 2 hours +Ara or -Ara.
  • Fig. 3B compares the abundance of transcripts after 4 hours -i-Ara or - Ara.
  • Fig. 3C compares the abundance of transcripts after 6 hours -i-Ara or -Ara.
  • Fig. 4 shows the log fold change in gene expression of exemplary transcripts after 2, 4, and 6 hours of BamA depletion in A. baumannii compared to expression of the transcript in A. baumannii expressing BamA.
  • Fig. 5A-5B show A. baumannii gene transcripts whose expression was increased by at least 10-fold after 6 hrs of BamA depletion.
  • a heatmap reflecting the relative change in expression for each gene transcript at 2, 4, or 6 hours of BamA depletion is shown. Predicted functions for encoded protein products are given on the right.
  • FIG. 6A-6D show A. baumannii gene transcripts whose expression decreased by at least 10-fold after 6 hrs of BamA depletion.
  • a heatmap reflecting the relative change in expression for each gene transcript at 2, 4 or 6 hours of BamA depletion is shown. Predicted functions for encoded protein products are given on the right.
  • FIG. 7A-7D show differences in gene expression when comparing non-PMBN-treated cells to cells treated with PMBN for various concentrations and times.
  • FIG. 7A shows gene expression in non-PMBN-treated cells compared to cells treated with 25 ⁇ g/mL for 10 minutes.
  • FIG. 7B shows gene expression in non-PMBN-treated cells compared to cells treated with 250 ⁇ g/mL for 10 minutes.
  • FIG. 7C shows gene expression in non-PMBN-treated cells compared to cells treated with 25 ⁇ g/mL for 30 minutes.
  • FIG. 7D shows gene expression in non-PMBN- treated cells compared to cells treated with or 250 ⁇ g/mL for 30 minutes.
  • Transcripts (genes) whose expression changed by at least 10-fold after treatment with PMBN at the indicated concentration and time are shown in black.
  • Fig. 8 shows the log fold change after treatment of A. baumannii cells with 25 ⁇ g/mL PMBN (gray) or 250 ⁇ g/mL PMBN (black) in exemplary gene transcripts.
  • Fig. 9 shows the backbone vector pACH106 which encodes replication factors and selective markers necessary for autonomous replication in Acinetobacter baumannii and maintenance by antibiotic selection.
  • Fig. 10A shows a histogram of fluorescence intensities induced in P A is_2885-sfGFP reporter cells after treatment with PMBN.
  • Fig. 10B shows fluorescent micrographs of PAIS_28S9- sfGFP reporter cells after treatment in the presence or absence of PMBN.
  • FIG. 11A-11B shows fluorescent FACs histograms of P A is_28S5-sfGFP reporter cells after treatment with PMBN.
  • FIG. 11 A cells were grown in microtiter dishes without shaking or aeration.
  • Fig. 1 IB cells were grown in culture flasks with aeration.
  • Fig. 12A-12B depict fluorescent intensities of A. baumannii P A isj»32-sfGFP and P A is_2889-sfGFP reporter cells that are depleted for BamA, an OM biogenesis factor, unless grown in the presence of arabinose.
  • Fig. 12A depicts mean fluorescent intensities of the reporter cells in the presence or absence of arabinose.
  • Fig. 12B shows a representative flow cytometry fluorescence histogram of PAis_2889-sfGFP reporter cells after growth with or without arabinose for 3 hrs.
  • Fig. 13A-13B show flow cytometry fluorescence histograms of a multi-drug resistant strain of A. baumannii expressing the OM stress transcriptional reporter constructs after culture in the presence of PMBN or vancomycin (vane).
  • Fig. 13A depicts flow cytometry fluorescent histograms of PAis_2885-sfGFP reporter cells.
  • Fig. 13B depicts flow cytometry fluorescent histograms of PAis_2889-sfGFP reporter cells.
  • Fig. 14A-14B show flow cytometry fluorescence histoGrams of A. baumannii reporter cells containing either PAisjxm-sfGFP (Fig. 14A) or PAis_2885-sfGFP (Fig. 14B) after culture in the presence of ACHN-975, an antibiotic that inhibits synthesis of lipopolysaccharide (LPS) by inhibiting the activity of the LpxC enzyme.
  • LPS lipopolysaccharide
  • Fig. 15A shows flow cytometry fluorescence histograms of A. baumannii reporter cells containing PAis_oo32-sfGFP after culture in the presence of ACHN-975, an antibiotic that inhibits synthesis of lipopolysaccharide (LPS) by inhibiting the activity of the LpxC enzyme; colistin, a broad-spectrum antibiotic that binds and perturbs the LPS leaflet of the OM; and EDTA, a chelating agent that disrupts LPS layer by stripping away divalent cations that stabilize the OM by neutralizing the negative charges in LPS.
  • LPS lipopolysaccharide
  • Fig. 15B shows flow cytometry fluorescence histograms of A. baumannii reporter cells containing PAis_2885-sfGFP after culture in the presence of PMBN, EDTA, and EDTA with SDS, a detergent that acts synergistically with EDTA to permeabilize the destabilized OM.
  • Fig. 16 depicts one embodiment of a screening assay for identifying anti-bacterial molecules that induce outer membrane stress.
  • animals are immunized with Acinetobacter cells to generate antibody- secreting B cells, isolated B cells and reporter A. baumannii bacterial cells (containing an outer membrane stress transcriptional reporter construct in which a regulatory region of a gene induced by outer membrane stress is fused to a detectable moiety, such as GFP, as described) are mixed and co-encapsulated to form microparticles using microfluidics, and the microparticles are screened for activated reporter cells expressing a detectable signal (e.g. fluorescence).
  • Fig. 17A-17B show fluorescent micrograph images of A. baumannii reporter cells containing PAis_28S9-sfGFP encapsulated in agarose microparticles after treatment of the reporter cells either with colistin (Fig. 17B) or without colistin (Fig. 17A).
  • Fig. 18A-18C show micrographs of co-encapsulated A. baumannii reporter cells and antibody- secreting murine B cells (plasmablasts) after a one-hour incubation.
  • Fig. 18A is a brightfield image of the co-encapsulated agarose microparticles.
  • Fig. 18B is a fluorescent (FITC) image of the co-encapsulated agarose microparticles.
  • Fig. 18C is an overlay of the brightfield and fluorescent images.
  • Fig. 19A-19D show micrographs of a mixed population of A. baumannii cells expressing inducible mRuby2 or mClover3.
  • Fig. 19A is a brightfield image of the mixed population of A. baumannii cells.
  • Fig. 19B is a fluorescent (RFP) image of the mixed population of A. baumannii cells. Cells expressing mRuby2 can be seen.
  • Fig. 19C is a fluorescent (GFP) image of the mixed population of A. baumannii cells. Cells expressing mClover3 can be seen.
  • Fig. 19D is an overlay of the brightfield and fluorescent images.
  • Fig. 20A depicts mean fluorescent intensities of PAis_oo32-sfGFP, PAis_2889-sfGFP, or PAIS 2885-sfGFP reporter cells in the presence of various concentrations of PMBN.
  • Fig. 20B depicts mean fluorescent intensities of cells expressing P tac -sfGFP (an IPTG-inducible reporter) in the presence of various concentrations of IPTG.
  • Fig. 21A-F shows flow cytometry fluorescence histograms of A. baumannii reporter cells containing PAis_2889-sfGFP after culture without serum (Fig. 21A-C) or with serum (Fig. 21D-F) and in media only (Fig. 21 A, D) or in the presence of an IgG isotype control (Fig. 2 IB, E) or a monoclonal antibody that binds the OM (Fig. 21C, F).
  • the dashed circle highlights a population of fluorescent cells.
  • reporter polynucleotides containing a sequence comprising a regulatory region of an outer membrane (OM) stress-responsive gene of an Acinetobacter bacterium operably linked to a sequence encoding a reporter molecule.
  • reporter vectors genetically engineered reporter microorganisms, and uses thereof.
  • the provided reporter polynucleotides, reporter vectors and engineered reporter microorganisms are useful for detecting outer membrane (OM) stress, including in connection with screening a plurality of candidate molecules or agents to identify molecules or agents that induce and/or modulate outer membrane stress and/or for determining or assessing if a microorganism is drug resistant.
  • reporter polynucleotides, reporter vectors and reporter microorganisms can be used in methods for the rapid identification or characterization of agents, including drug or other therapeutic candidates, to address a multitude of infectious disease threats.
  • the term "effective amount” refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired result, e.g., an enhanced immune response to an antigen, a decrease in tumor growth or metastasis, or a reduction in tumor size.
  • An effective amount can be provided in one or more administrations.
  • Reference to "about” a value or parameter herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. In particular embodiments, reference to about refers to a range within 10% higher or lower than the value or parameter, while in other embodiments, it refers to a range within 5% or 20% higher or lower than the value or parameter. Reference to "about” a value or parameter herein includes (and describes) aspects that are directed to that value or parameter per se.
  • modulating means changing, and includes positive modulating, such as “increasing,” “enhancing,” “inducing” or “stimulating,” as well as negative modulating such as “decreasing,” “inhibiting” or “reducing,” typically in a statistically significant or a physiologically significant amount as compared to a control.
  • An “increased,” “stimulated” or “enhanced” amount is typically a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the amount produced by no treatment as described herein or by a control treatment, including all integers in between.
  • a “decreased,” “inhibited” or “reduced” amount is typically a “statistically significant” amount, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease in the amount produced by no treatment as described herein or by a control treatment, including all integers in between.
  • p-value which is the frequency or probability with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less.
  • antibodies and “immunoglobulin” include antibodies or
  • immunoglobulins of any isotype fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single- chain antibodies, and fusion proteins comprising an antigen- binding portion of an antibody and a non-antibody protein.
  • Antibodies may exist in a variety of other forms including, for example, Fv, Fab, and (Fab')2, as well as bi-functional (i.e. bi- specific) hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in single chains (e.g., Huston et al., Proc. Natl.
  • vector refers to a nucleic acid construct, typically a circular DNA vector, that contains discrete elements that are used to introduce heterologous nucleic acid into cells for either expression of the nucleic acid or replication thereof.
  • the vectors typically remain episomal, but can be designed to effect stable integration of a gene or portion thereof into a chromosome of the genome.
  • vectors contain an origin of replication that allows many copies of the plasmid to be produced in a bacterial or eukaryotic cell without integration of the plasmid into the host cell DNA. Selection and use of such vectors are well known to those of skill in the art.
  • polynucleotide and “nucleic acid molecule” are used interchangeably to refer to a single-stranded and/or double- stranded polynucleotides, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as analogs or derivatives of either RNA or DNA.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the length of a polynucleotide molecule is given herein in terms of nucleotides
  • nucleic acid is also included in the term “nucleic acid” are analogs of nucleic acids such as peptide nucleic acid (PNA), phosphorothioate DNA, and other such analogs and derivatives. Nucleic acids can encode gene products, such as, for example, polypeptides, regulatory RNAs, microRNAs, siRNAs and functional RNAs. Hence, nucleic acid molecule is meant to include all types and sizes of DNA molecules including cDNA, plasmids or vectors and DNA including modified nucleotides and nucleotide analogs.
  • PNA peptide nucleic acid
  • phosphorothioate DNA phosphorothioate DNA
  • Nucleic acids can encode gene products, such as, for example, polypeptides, regulatory RNAs, microRNAs, siRNAs and functional RNAs.
  • nucleic acid molecule is meant to include all types and sizes of DNA molecules including cDNA, plasmids or vectors and DNA including modified nucleot
  • polypeptide and “protein” are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length.
  • Polypeptides may include amino acid residues including natural and/or non-natural amino acid residues.
  • the terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like.
  • the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
  • the term “gene” refers to any and all discrete coding regions of a host genome, or regions that code for a functional RNA only (e.g. , tRNA, rRNA, regulatory RNAs such as ribozymes etc.) as well as associated non-coding regions and optionally regulatory regions.
  • the term “gene” includes within its scope the open reading frame encoding specific polypeptides, introns, and adjacent 5' and 3' non- coding nucleotide sequences involved in the regulation of expression.
  • the gene can further contain control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals.
  • the gene sequences can be cDNA or genomic DNA or a fragment thereof. The gene can be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host.
  • an outer membrane (OM) stress-responsive gene is a gene whose expression or activity is modulated (e.g. increased or decreased) in a microorganism (e.g. an Acinetobacter spp. ) in response to a stress condition to the outer membrane of the
  • the stress condition modulates the biogenesis and/or integrity of the OM, including that disrupts the OM; destabilizes the OM, for example, by inhibiting the synthesis of lipopolysaccharide (LPS) or the peptidoglycan cell wall; or permeabilizes the OM.
  • LPS lipopolysaccharide
  • the expression or activity of the gene is increased or decreased by greater than or greater than about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold or more.
  • regulatory sequence or “regulatory region” as used in reference to a specific gene, refers to the coding or non-coding nucleic acid expression control sequences within that gene that are necessary or sufficient to provide for the regulated expression of the coding region of a gene.
  • the term encompasses promoter sequences, regulatory protein binding sites, upstream activator sequences and the like.
  • Specific nucleotides within a regulatory region may serve multiple functions.
  • a specific nucleotide may be part of a promoter and participate in the binding of a transcriptional activator protein.
  • ORF open reading frame
  • operably linked is meant a functional linkage between a nucleic acid expression control sequence (such as a promoter) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • Percent “identical” or “identity” in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences that are the same or have a specified percentage of nucleic acid residues or amino acid residues, respectively, that are the same, when compared and aligned for maximum similarity, as determined using a sequence comparison algorithm or by visual inspection.
  • Percent sequence identity or “% identity” or “% sequence identity or “% amino acid sequence identity” of a subject amino acid sequence to a reference amino acid sequence means that the subject amino acid sequence is identical (i.e., on an amino acid-by-amino acid basis) by a specified percentage to the reference amino acid sequence over a comparison length when the sequences are optimally aligned.
  • 80% amino acid sequence identity or 80% identity with respect to two amino acid sequences means that 80% of the amino acid residues in two optimally aligned amino acid sequences are identical.
  • reporter molecule refers to a molecule that is directly or indirectly detectable or whose presence is otherwise capable of being measured.
  • reporter molecules include proteins that can emit a detectable signal such as a fluorescence signal, and enzymes that can catalyze a detectable reaction or catalyze formation of a detectable product.
  • Reporter molecules also can include detectable nucleic acids.
  • a reporter molecule is a polypeptide which can be detected when it is expressed in the cell.
  • expression of the detectable reporter may lead to the production of a signal, for example a fluorescent, bio luminescent or colorimetric signal, which can be detected using routine techniques.
  • the signal may be produced directly from the reporter, after expression, or indirectly through a secondary molecule, such as a labelled antibody.
  • reporter cell and “reporter microorganism” are used interchangeably to refer to an engineered microorganism into which an exogenous or heterologous polynucleotide, such as a cDNA or gene, encoding a reporter molecule has been introduced. Therefore, reporter cells are distinguishable from naturally occurring microorganisms which do not contain a recombinantly introduced exogenous polynucleotide. Reporter cells are thus cells having a gene or genes introduced through human intervention and that express an exogenous reporter molecule.
  • heterologous with reference to a polynucleotide or gene refers to a nucleotide sequence that is not native to the organism or a gene contained therein or not normally produced in vivo by an organism, such as bacteria, from which it is expressed.
  • kits are packaged combinations that optionally includes other elements, such as additional reagents and instructions for use of the combination or elements thereof. Kits optionally include instructions for use.
  • a reporter polynucleotide comprising a sequence comprising a regulatory region of an OM stress-responsive gene of an Acinetobacter species (spp). operably linked to a sequence encoding a reporter molecule, wherein the OM stress-responsive gene is modulated in response to a stress to the outer membrane (hereinafter referred to as "OM-stress) of he Acinetobacter spp.
  • the sequence comprising the regulatory region is directly or indirectly linked to the sequence encoding the reporter molecule. In certain embodiments, the sequence comprising the regulatory region is directly linked to the 5' end of the open reading frame (ORF) of the reporter molecule. In certain embodiments, the sequence comprising the regulatory region is indirectly linked to the 5' end of the ORF of the reporter molecule.
  • ORF open reading frame
  • the provided reporter polynucleotides and reporter vectors can be produced or generated by standard DNA techniques. Genes or nucleotide sequences of interest can be obtained by amplification of nucleic acids, such as by polymerase chain reaction methods; generated de novo by synthetic construction (e.g. overlapping PCR and/or oligonucleotide hybridization); or desired sequences can be removed from already existing sources such as commercially available vectors. Synthesized or harvested sequences can be further modified by site directed mutagenesis, PCR, or other methods known to those of skill in the art.
  • Such modifications include, but are not limited to optimize codon usage for a host microorganism, modification of a coding sequence to reduce or enhance a desired activity, and addition of restriction sequences for cloning purposes.
  • codon optimization can be employed to optimize expression in a host microorganism, such as by modification of certain codons to reflect particular codon usage of the reporter polynucleotide containing the reporter molecule to that which is found more frequently in particular species in which it is being expressed.
  • Nucleic acids molecules can be synthesized by methods known to one of skill in the art using synthetic gene synthesis.
  • individual oligonucleotides corresponding to fragments of a polynucleotide sequence vector are synthesized by standard automated methods and mixed together in an annealing or hybridization reaction.
  • synthetic genes are assembled from a large number of short partially overlapping DNA oligonucleotides or fragments, generally each independently about or about at least 20, 30, 40, 50, 60, 70, 80, 90 100, 200, 300, 400, 500 or more nucleotides in length.
  • DNA fragments with overlapping ends can be generated by restriction digest, by PCR or by gene synthesis methods.
  • oligonucleotides can be commercially obtained, such as from Integrated DNA Technologies (Coralville, IA).
  • overlapping segments are allowed to anneal and then are assembled into longer double- stranded DNA, for example, by ligation and/or polymerase extension reactions, either alone or in combination.
  • Single nucleotide "nicks" in the duplex DNA are sealed using ligation, for example with bacteriophage T4 DNA ligase.
  • Such strategies are variously referred to as "assembly PCR,” “splicing by overlap extension,” “polymerase chain assembly” and others.
  • Gibson Assembly methods can be employed.
  • reporter polynucleotide A description of the regulatory region and reporter molecule of the provided reporter polynucleotides is provided below. It is within the level of one of skill in the art to generate and design a reporter polynucleotide, and reporter vectors containing such reporter polynucleotides, as described herein. Exemplary reporter polynucleotides and reporter vectors are provided.
  • the provided reporter polynucleotides contain a regulatory region of an Acinetobacter spp. gene that is responsive to and/or whose expression is modulated in response to stress to the outer membrane (hereinafter called "OM stress-responsive gene").
  • the stress to the outer membrane is caused by altering, mutating, depleting, or removing a component from the OM.
  • the stress to the OM can be caused by genetically altering the Acinetobacter spp. to reduce expression of an OM lipid, polysaccharide, or protein.
  • the OM stress responsive gene is responsive to stress caused by altering, mutating, depleting, or removing BamA, LptD, FhuA, PldA, OmpT, PagP, OstA, OmpA, OmpF, Omp200, Ompl21, Omp71, Ompl l7, OprF, PhoE, OmpC, OmpF, NmpC; PorA, PorB, OprA, OprM, OprN, OprJ, OprB, NspA, PagL, OmpW, OpcA, NalP, NupA, OmpG, FadL, PhoE, LamB, FhaC, SucY, FepA, FecA, BtuB, TolC, Porin P, Porin D, SmeC, SmeF, MepC, SrpC, TtgC, TtgF, AdeC, AdeK, or MexA.
  • the stress to the OM of the Acinetobacter bacterium is or is caused by depletion of BamA.
  • the genetic alteration e.g. deletion
  • an outer membrane gene can be deleted from a microorganism and, instead, can be exogenously expressed under the control of an inducer.
  • the outer membrane gene is expressed only in the presence of the inducer but is not expressed, such as is not expressed on the cell surface, in the absence of the inducer.
  • the inducer is arabinose and the genetic alteration is induced by the addition of an inducer. In some embodiments, the inducer is arabinose.
  • the concentration of the arabinose is about 0.0004 -0.2%, such as about 0.0006%-0.02%, 0.0008- 0.02%, or about 0.001%-0.02%.
  • Acinetobacter spp. is AABA046, which carries a deletion of the bamA gene by replacement with a selective marker conferring resistance to kanamycin and contains an exogenous polynucleotide encoding bamA fused to the araBAD promoter from E. coli which allows regulation of transcription by addition of the inducing sugar arabinose.
  • AABA041 which carries a deletion of the bamA gene by replacement with a selective marker conferring resistance to carbenicillin and contains an exogenous polynucleotide encoding bamA fused to the araBAD promoter from E. coli which allows regulation of transcription by addition of the inducing sugar arabinose.
  • the stress to the outer membrane can be caused by treatment with a molecule or combination of molecules that causes temporary or permanent damage to the OM.
  • the stress to the OM is or is caused by treatment with polymyxin B nonapeptide (PMBN), vancomycin, ACHN-975, colistin,
  • the stress to the OM is or is caused by treatment with PMBN.
  • the concentration of the molecule is a sub-lethal concentration. In certain embodiments the concentration of the molecule is below the MIC of the molecule.
  • the concentration of the molecule is about 0.1 ⁇ g/mL - 1000 ⁇ g/mL, such as about 1 ⁇ g/mL - 750 ⁇ g/mL, 10 ⁇ g/mL - 500 ⁇ g/mL, 25 ⁇ g/mL - 500 ⁇ g/mL, or 25 ⁇ g/mL - 250 ⁇ g/mL. In certain embodiments, the concentration of the molecule is about 0.1 mM- 500 mM, such as about 0.1 mM - 500 mM, 1 mM - 50 mM, or 1 mM - 10 mM.
  • the concentration of the molecule is about 0.1% - 5%, such as about 0.5% - 2.5% or 0.75% - 2%.
  • the OM stress-responsive gene is modulated in response to more than 1, 2, 3, 4, 5, or 6 stresses. In certain embodiments, the OM stress-responsive gene is modulated in response to depletion of BamA and treatment with PMBN.
  • the OM stress-responsive gene is identified using a method that includes a) inducing OM stress in Acinetobacter spp. by exposing or subjecting an
  • Acinetobacter spp. to one or more stress conditions, such as any described above and b) identifying one or more genes that is modulated in response to the OM stress (e.g. using RNA- Seq or other method).
  • A. bacterium genes such as Acinetobacter spp. genes in the presence of one or more stress conditions, such as described above.
  • expression levels can be assessed or determined using any method known to a skilled artisan, such as by using quantitative PCR, microarrays, RNA-Seq, northern blotting, or SAGE.
  • genes whose sequences, or portions or fragments of sequences, have been identified as having been modulated can be identified using a reference sequence of the A. bacterium genome.
  • Exemplary A. bacterium genome sequences are known and readily available online on the world wide web at tigr.org, kegg.jp, or ncbi.nlm.nih.gov/genbank.
  • baumannii species or isolates include GENBANKTM Accession Nos. CP00521.1 or
  • the Acinetobacter spp. is an Acinetobacter apis, Acinetobacter baumannii, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii, Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter citri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus,
  • Acinetobacter harbinensis Acinetobacter indicus, Acinetobacter junii, Acinetobacter kookii, Acinetobacter Iwoffii, Acinetobacter nectaris, Acinetobacter nosocomialis, Acinetobacter parvus, Acinetobacter pakistanensis, Acinetobacter pittii, Acinetobacter puyangensis,
  • Acinetobacter qingfengensis Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii, Acinetobacter soli, Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis, or Acinetobacter venetianus.
  • the Acinetobacter spp. is Acinetobacter baumannii.
  • the Acinetobacter spp. is ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA- 1605, ATCC BAA-1709, ATCC BAA-1710, ATCC BAA-1789, ATCC BAA-1790, ATCC BAA- 1791, ATCC BAA- 1792, ATCC BAA-1793, ATCC BAA-1794, ATCC BAA-1795, ATCC BAA- 1796, ATCC BAA-1797, ATCC BAA-1798, ATCC BAA- 1799, ATCC B
  • the Acinetobacter spp. is a multi-drug resistant bacterium.
  • the OM stress-responsive gene is one that is upregulated or downregulated in response to the stress to the outer membrane of Acinetobacter spp.
  • the OM stress-responsive gene is downregulated in response to the stress.
  • the OM stress-responsive gene is downregulated by at least or at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20- fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold or more in response to the stress compared to the expression level of the gene in the absence of the stress condition.
  • the OM stress-responsive gene such as an OM-stress responsive gene that is downregulated in response to OM stress, is A1S_0009, A1S_0010, A1S_0025, A1S_0027, A1S_0038, A1S_0067, A1S_0070, A1S_0071, A1S_0073, A1S_0076, A1SJ3077, A1SJ3079, A1S_0087, A1S_0090, A1S_0091, A1S_0095, A1S_0096, A1S_0097, A1S_0098, A1S_0099, A1S_0103, A1S_0104, A1S_0105, A1S_0106, A1S_0107, A1S_0108, A1S_0109, A1S_0121, A1S_0128, A1S_0129, A1S_0141
  • the OM stress-responsive gene such as an OM-stress responsive gene that is downregulated in response to OM stress, is A1S_0103, A1S_0645, A1S_1266, A1S_1268, A1S_1335, A1S_1336, A1S_1337, A1S_1338, A1S_1339, A1S_1340, A1S_1341, A1S_1342, A1S_1343, A1S_1344, A1S_1345, A1S_1791, A1S_1792, A1S_1794, A1S_1796, A1S_1835, A1S_1836, A1S_1837, A1S_1838, A1S_1839, A1S_2449, A1S_2450, A1S_2452, A1S_3540, A1S_3541, A1S_3542, A1S_3543, A1S_3586, A1S_
  • such an OM stress- responsive gene is A1S_1336, A1S_1836, A1S_1838, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, or A1S_3809.
  • the OM stress-responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any of the gene described herein.
  • the OM stress- responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with A1S_0103, A1S_0645, A1S_1266, A1S_1268, A1S_1335, A1S_1336, A1S_1337, A1S_1338, A1S_1339, A1S_1340, A1S_1341, A1S_1342, A1S_1343, A1S_1344, A1S_1345, A1S_1791, A1S_1792, A1S_1794, A1S_1796, A1S_1835, A1S_1836, A1S_1837, A1S_1838, A1S_1839, A1S_2449, A1S_2450, A1S_2452, A1S_3540, A1S_3541, A
  • the OM stress-responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with A1S_1336, A1S_1836, A1S_1838, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, or A1S_3809.
  • the OM stress-responsive gene is one that is upregulated in response to the stress to the outer membrane of Acinetobacter spp. In some embodiments, the OM stress-responsive gene is upregulated in response to the stress by at least or at least about 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50- fold, 60-fold, 70-fold, 80, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold or more in response to the stress compared to the expression level of the gene in the absence of the stress condition.
  • the OM stress-responsive gene such as an OM-stress responsive gene that is upregulated in response to OM stress, is A1S_0012, A1S_0023,
  • A1S_ _3 16 A1S_ _3725, A1S_ _3726, A1S_ _3727, A1S_ _3728, A1S_ _3736, A1S_ _3738, A1S_ _3739,
  • the OM stress-responsive gene such as an OM-stress responsive gene that is upregulated in response to OM stress, is A1S_0032, A1S_0033, A1S_0113, A1S_0189, A1S_0516, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, A1S_3127, A1S_3492, or AIS_3791.
  • A1S_0032, A1S_0033 A1S_0113, A1S_0189, A1S_0516, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, A1S_3127, A1S_3492, or AIS_3791.
  • the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or A1S_3127.
  • the OM stress-responsive gene is A1S_0032, A1S_2885, A1S_2889, A1S_3127, A1S_3492, or A1S_3791.
  • the OM stress- responsive gene is A1S_0032, A1S_2885, or A1S_2889.
  • the OM stress-responsive gene is A1S_0113 or A1S_1751. In certain embodiments, the OM stress- responsive gene is A1S_0189, A1S_0516, A1S_1224, A1S_2093, or A1S_2271.
  • the OM stress-responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any of the genes described herein.
  • the OM stress- responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with A1SJ3032, A1S_0033, A1S_0113, A1S_0189, A1SJ3516, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, A1S_3127, A1S_3492, or AIS_3791.
  • the OM stress-responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or A1S_3127.
  • the OM stress-responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with A1S_0032, A1S_2885, A1S_2889, A1S_3127, A1S_3492, AIS_3791. In some embodiments, the OM stress-responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with A1S_0032, A1S_2885, or A1S_2889.
  • the OM stress-responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with A1S_0113 or
  • the OM stress-responsive gene is A1S_0189, A1S_0516, A1S_1224, A1S_2093, or A1S_2271.
  • the provided reporter polynucleotides contain a regulatory region or portion thereof of an OM stress-responsive gene, such as any described above, operatively linked to a reporter molecule.
  • the regulatory region or portion thereof comprises a sequence upstream or 5' of the open reading frame (ORF) of the OM stress- responsive gene, such as any described above.
  • ORF open reading frame
  • the sequence of the regulatory region or portion thereof is sufficient to provide for regulated expression of the coding region of the reporter molecule operatively linked thereto, such as upon induction or in the presence of an OM stress condition.
  • the regulatory region is or comprises a native promoter of the OM-responsive gene.
  • One of skill in the art can identify a regulatory region through standard techniques. For example, one could identify a regulatory region by fusing a putative regulatory region or sequence upstream of an OM stress-responsive gene to a sequence encoding a reporter molecule, introducing the construct using standard techniques into an Acinetobacter spp., inducing the putative regulatory region or upstream sequence by causing OM stress, and determining if the reporter molecule is induced. Putative regulatory regions can often be shortened or lengthened without influencing activity or inducibility. One of skill in the art can systematically test the effect of removing nucleotides from putative regulatory region sequence to determine what putative regulatory elements are critical or required for the OM stress-responsive behavior.
  • the regulatory region comprises a sequence to further promote translation of the encoded reporter molecule.
  • the sequence to further promote translation of the encoded reporter molecule is directly linked to the 5' end of the open reading frame (ORF) of the reporter molecule.
  • the sequence to further promote translation of the encoded reporter molecule is indirectly linked to the 5' end of the ORF of the reporter molecule.
  • the sequence further promoting translation is or comprises a bacterial ribosome binding site.
  • the ribosome binding site is a Shine-Dalgarno (SD) sequence, which is a sequence usually found in natural prokaryotic genes 5' of the ATG translation start codon that acts as a binding site on the mRNA molecule for the ribosome, thereby facilitating translation of the mRNA.
  • the regulatory region is one that contains a SD sequence in which, when operatively linked to the sequence encoding the reporter molecule, such SD sequence is about 2 to about 15 nucleotides 5' of the ATG of the sequence encoding the reporter molecule, such as about 5 to about 7 nucleotides upstream of the ATG.
  • the SD sequence contained in the regulatory region can be any variant of the consensus sequence AGGAGG that retains the characteristic of facilitating translation of the sequence encoding the reporter molecule.
  • the SD is from about 3 nucleotides to about 9 nucleotides in length.
  • the Shine- Dalgarno sequence is native to the regulatory region of the OM stress-responsive gene.
  • the Shine-Dalgarno sequence is synthetic and/or heterologous to the regulatory region of the OM stress-responsive gene.
  • the Shine- Dalgarno sequence comprises a sequence with at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14.
  • the regulatory region such as the promoter, is modified in its sequence compared to the native sequence of an OM stress-responsive gene, such as any described above.
  • the regulatory region is modified or altered in its sequence, for example to alter the configuration of regulatory binding regions, such as for transcription factors and/or can be made to have specific regions deleted.
  • Such mutational and/or deletional analysis can be rationally or empirically performed and the resulting constructs tested by one of skill in the art.
  • the various constructs containing a modified regulatory region can be tested in a construct in which expression of an operatively linked reporter molecule (e.g. GFP) can be used to determine the activity of the modified regulatory region under different conditions, such as one or more different OM stress conditions. In some cases, this approach can be used to identify the smallest region capable of conferring or regulating expression of an operatively linked reporter molecule.
  • an operatively linked reporter molecule e.g. GFP
  • the regulatory region includes a contiguous sequence of nucleotides within or within about 1000, 750, 500, 450, 400, 350, 300, or 250 base pairs upstream or 5' of the open reading frame (ORF) of the OM stress-responsive gene. In certain embodiments, the regulatory region comprises a contiguous sequence of nucleotides within about 500 base pairs upstream or 5' of the ORF of the OM stress-responsive gene. In certain embodiments, the regulatory region comprises a contiguous sequence of nucleotides within about 250 base pairs upstream or 5' of the ORF of the OM stress-responsive gene.
  • the contiguous sequence of nucleotides comprises at least or at least about 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400 or more base pairs including any ranges between these values.
  • the regulatory region comprises a sequence with at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 1-13.
  • the regulatory region is or comprises the sequence of nucleotides set forth in any of SEQ ID NOs: 1-13.
  • the regulatory region does not only contain the promoter sequence of the A1S_2889 OM-responsive stress gene. In some embodiments, the regulatory region does not contain a regulatory region of the A1S_2889 OM-responsive stress gene. In some embodiments, the regulatory region does not include the sequence set forth in SEQ ID NO: 11.
  • the regulatory region of an OM stress-responsive gene is operatively linked to a sequence encoding a reporter molecule.
  • Reporter molecules include molecules that express or that are capable of expressing or producing a detectable signal that is assayable or can be detected.
  • the extent of detection of the assayable product indicates the presence, absence or quantity of the reporter molecule which, in turn, indicates the presence, absence, or degree of an OM-stress on a reporter microorganism bearing the reporter molecule.
  • the reporter molecule is any that is capable of producing a detectable signal when expressed in a reporter microorganism, such as Acinetobacter spp. (e.g. Acinetobacter baumannii).
  • Such signals including a fluorescent, bio luminescent or colorimetric signal, can be detected using routine techniques.
  • the reporter molecule is a fluorescent protein, a luminescent protein, a chromoprotein, or an enzyme.
  • the reporter molecule is a fluorescent protein.
  • the fluorescent protein is a red, green, blue, or yellow fluorescent protein.
  • the fluorescent protein is Sirius, SBFP2, Azurite, mAzurite, EBFP2, moxBFP, mKalamal, mTagBFP2, Aquamarine, ECFP, Cerulean, mCerulean, mCerulean3, moxCerulean3, SCFP3A, mTurquoise2, CyPet, AmCyanl, MiCy (Midoriishi-Cyan), iLOV, AcGFPl, sfGFP, moxGFP, mEmerald, EGFP, mEGFP, AzamiGreen, cfSGFP2, ZsGreen, SGFP2, Clover, mClover2, mClover3, EYFP, Topaz, mTopaz, mVenus, mox Venus,
  • the fluorescent protein is sfGFP, mClover3, mRuby2, Ypet, mCerulean, or mTagBFP2. In some embodiments, the fluorescent protein is sfGFP, mClover3, or mRuby2. In some embodiments, the fluorescent protein is sfGFP.
  • Suitable fluorescent reporters are available commercially (Clontech Labs Inc. USA, Evrogen Moscow, RU; MBL Int MA USA; Addgene Inc. MA USA).
  • the sequence encoding the reporter molecule comprises a sequence with at least or at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 15, 33, or 40.
  • the sequence encoding the reporter molecule comprises a sequence with at least or at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 15.
  • the sequence encoding the reporter molecule comprises a sequence with at least or at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 33.
  • the sequence encoding the reporter molecule comprises a sequence with at least or at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 40.
  • the reporter molecule is a luminescent protein.
  • the luminescent protein is North American firefly luciferase, Genji-botaru luciferase, Italian firefly luciferase, Heike luciferase, East European firefly luciferase,
  • the reporter molecule is a chromoprotein.
  • the chromoprotein comprises a heme, riboflavin, or retinal.
  • the chromoprotein is a hemoglobin, hemocyanin, chlorophyll, cytochrome, carotenoid, flavoprotein, or rhodopsin.
  • the reporter molecule is an enzyme.
  • the enzyme is one whose expression in a cell can be detected with a live cell assay (i.e. an assay which does not require cell fixation or lysis).
  • the enzyme is chloramphenicol acetyltransferase (CAT), ⁇ -galactosidase, alkaline phosphatase, ⁇ - glucuronidase, ⁇ -lactamase, neomycin phosphotransferase, or a modified version thereof.
  • the reporter molecule is a gene necessary for the production of essential metabolites such as tryptophan, leucine, uracil, histidine, or methionine.
  • the provided reporter polynucleotides are comprised within a vector (hereinafter called a "reporter vector").
  • a vector hereinafter called a "reporter vector”
  • Suitable vectors can be used for any of the embodiments described herein.
  • a suitable vector can be used that is capable of replicating in a microorganism to which it is introduced.
  • Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences for driving transcription of the coding nucleotide sequence, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate, for expression in a bacterial cell as described herein.
  • the vector comprises a reporter polynucleotide described herein. In some embodiments, the vector comprises a first reporter polynucleotide described herein and a second reporter polynucleotide described herein. In certain embodiments, the vector comprises (i) a first reporter polynucleotide comprising a first sequence comprising a regulatory region of a first OM stress-responsive gene of an Acinetobacter species (spp).
  • spp Acinetobacter species
  • a second reporter polynucleotide comprising a second sequence comprising a regulatory region of a second OM stress-responsive gene of an Acinetobacter species (spp). operably linked to a second sequence encoding a second reporter molecule, wherein the first and second OM stress-responsive genes are modulated in response to a stress to the outer membrane (OM-stress) of the Acinetobacter spp.
  • the first and second reporter polynucleotides share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second reporter polynucleotides are the same. In certain embodiments, the first and second reporter polynucleotides are different.
  • the regulatory region of the first reporter polynucleotide and the regulatory region of the second reporter polynucleotide are from different OM stress- responsive genes.
  • the first OM stress-responsive gene and the second OM stress-responsive gene are different.
  • operatively linking a reporter molecule to different regulatory regions that are both responsive to OM stress can minimize false positives.
  • the different regulatory regions may be responsive to different types of stress, such that the presence of a detectable signal induced from a microorganism carrying both reporter polynucleotides, or a first microorganism comprising a first reporter polynucleotide and a second microorganism comprising a second reporter polynucleotide, can assess different parameters or features of outer membrane stress.
  • an agent is identified as causing or inducing OM stress only if a change in a detectable signal by or from both reporter molecules is observed.
  • the different regulatory regions are each operatively linked to the same reporter molecule. In some embodiments, the different regulatory regions are each operatively linked to different reporter molecules.
  • the first and second OM stress-responsive genes are the same or are functional variants. In certain embodiments, the first and second OM stress- responsive genes share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second OM stress-responsive genes are the same. In certain embodiments, the first and second OM stress-responsive genes are different.
  • the first and second sequences comprising the regulatory regions are the same or are functional variants. In certain embodiments, the first and second sequences comprising the regulatory regions share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second sequences comprising the regulatory regions are the same. In certain embodiments, the first and second sequences comprising the regulatory regions are different.
  • the reporter molecule encoded by the first reporter polynucleotide, i.e. first reporter molecule, and the reporter molecule encoded by the second reporter polynucleotide, i.e. second reporter molecule are different.
  • the first reporter molecule and the second reporter molecule are different.
  • the first reporter molecule and the second reporter molecule do not exhibit an overlapping emission and absorption spectra. Reporter molecules capable of producing different, such as non-overlapping signals, can be separately or distinguishably detected.
  • the use of several reporters can provide a multiplexed system to simultaneously measure or assess the presence or absence of a microorganism to one or more stress condition or potential stress condition and/or ensure that a response to a condition, e.g. caused by a physical condition or by an agent, is real or not likely to be a false positive.
  • the first and second reporter molecule is independently sfGFP and mRuby2.
  • the first and second sequences encoding the first and second reporter molecules are the same or are functional variants. In certain embodiments, the first and second sequences encoding the reporter molecules share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second sequences encoding the first and second reporter molecules are the same. In certain
  • the first and second sequences encoding the first and second reporter molecules are different. In certain embodiments, the first and second reporter molecules are the same.
  • the first reporter vector includes a first reporter polynucleotide comprising a first sequence comprising a regulatory region of a first OM stress-responsive gene of an Acinetobacter species (spp). operably linked to a first sequence encoding a first reporter molecule.
  • the second reporter vector includes a second reporter polynucleotide comprising a second sequence comprising a regulatory region of a second OM stress-responsive gene of an Acinetobacter species (spp).
  • the first and second reporter vector can be introduced into the same microorganism. In some embodiments, the first and second reporter vector can be introduced into different microorganisms.
  • reporter vectors are designed to contain certain components which optimize gene expression for certain microorganisms.
  • optimization components include, but are not limited to, origins of replication, promoters, and enhancers.
  • the vector contains an origin of replication in the IncQ incompatibility group that is proficient for replication in a broad host range, including in Acinetobacter spp.
  • the plasmid is a low-copy plasmid.
  • the vector is a high-copy plasmid.
  • the reporter vector contains a selective marker.
  • selectable markers include, but are not limited to, antibiotic resistance nucleic acids (e.g., kanamycin, ampicillin, carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin, neomycin, or chloramphenicol) and/or nucleic acids that confer a metabolic advantage, such as a nutritional advantage on the reporter microorganism.
  • the provided reporter vector is capable of being expressed in any microorganism or progeny thereof that can be used to heterologously express genes.
  • the reporter vector is capable of being expressed in any microorganisms described herein.
  • the reporter vector is capable of being expressed in a Gram- negative bacterium. In certain embodiments, the reporter vector is capable of being expressed in a multi-drug resistant bacterium. In certain embodiments, the vector is capable of being expressed in Acinetobacter, Bdellovibrio, Burkholderia, Chlamydia, Enterobacter, Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella, Neisseria, Pantoea, Pseudomonas, Salmonella, Shigella, Stenotrophomonas, Vibrio, or Yersinia. In particular embodiments, the reporter vector is capable of being expressed in Acinetobacter. In certain embodiments, the reporter vector is capable of being expressed in Acinetobacter apis,
  • Acinetobacter baumannii Acinetobacter baylyi, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii,
  • Acinetobacter brisouii Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter organizerri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus, Acinetobacter harbinensis, Acinetobacter indicus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter kookii, Acinetobacter Iwoffii, Acinetobacter nectaris, Acinetobacter nosocomialis, Acinetobacter parvus, Acinetobacter pakistanensis, Acinetobacter pittii, Acinetobacter puyangensis, Acinetobacter qingfengensis, Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii
  • Acinetobacter soli Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis, or Acinetobacter venetianus.
  • the reporter vector is capable of being expressed in
  • the reporter vector is capable of being expressed in ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA-1605, ATCC BAA-1709, ATCC BAA-1710, ATCC BAA- 1789, ATCC BAA-1790, ATCC BAA-1791, ATCC BAA-1792, ATCC BAA- 1793, ATCC BAA-1794, ATCC BAA-1795, ATCC BAA- 1796, ATCC BAA-1797, ATCC BAA-1798, ATCC BAA-1799, ATCC BAA-1800, ATCC BAA-1878, ATCC BAA-2093, ATCC BAA
  • the reporter polynucleotide as described herein is comprised in a backbone vector.
  • a nucleotide sequence comprising the reporter polynucleotide is inserted into or replaces a portion of the nucleotide sequence of the backbone vector.
  • Backbone vectors that can be expressed in specific microorganism are known in the art, for example pWH1266, or pET-RA (GenBank: M36473.1 and HM219006.1) are known to be capable of expression in Acinetobacter.
  • the backbone vector is pACH106 (SEQ ID NO: 29), pWH1266, or pET-RA.
  • the backbone vector has sequence with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:29.
  • the backbone vector comprises the sequence of nucleotides set forth in SEQ ID NO: 29 and a nucleotide sequence comprising the reporter polynucleotide replaces nucleotides 5,715-7,395 of the backbone vector.
  • the provided reporter vector comprises the sequence of nucleotides set forth in SEQ ID NO:29 in which nucleotides 5,715-7,395 thereof are replaced with a nucleotide sequence comprising a reporter polynucleotide comprising any of SEQ ID NOS: 1- 13, or a reporter polynucleotide with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOS: l-13; or a sequence with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of such reporter vectors.
  • the provided reporter vector comprises the sequence of nucleotides set forth in SEQ ID NO:29 in which nucleotides 5,715-7,395 thereof are replaced with a nucleotide sequence comprising a reporter polynucleotide comprising any of SEQ ID NOS: 16-28 or 34-36, or a reporter polynucleotide with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOS: 16-28 or 34-36; or a sequence with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of such reporter vectors.
  • the provided reporter vector comprises the sequence of nucleotides set forth in SEQ ID NO:29 in which nucleotides 5,715-7,395 thereof are replaced with a nucleotide sequence comprising any of SEQ ID NOS: 1-13, or a sequence with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of such reporter vectors.
  • the reporter vector is or comprises a sequence with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 30-32 or 37-39.
  • the reporter vector is or comprises the sequences set forth in any of SEQ ID NOS: 30-32 or 37-39.
  • the reporter vector is or comprises a sequence with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 30-32.
  • the reporter vector is or comprises the sequences set forth in any of SEQ ID NOS: 30-32. In some embodiments, the reporter vector is or comprises a sequence with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 37-39. In some embodiments, the reporter vector is or comprises the sequences set forth in any of SEQ ID NOS: 37-39.
  • reporter microorganisms comprising any one or more reporter polynucleotide described herein or any one or more reporter vector described herein.
  • the reporter microorganism comprises 1, 2, 3, 4 or more reporter polynucleotides as described herein and/or 1, 2, 3, 4 or more reporter vectors as described herein.
  • the reporter microorganism is a microorganism that is modified by introduction of one or more heterologous or exogenous reporter polynucleotides or reporter vectors as described into a host microorganism.
  • the provided reporter microorganisms include a host microorganism comprising a reporter polynucleotide described herein or a reporter vector described herein.
  • the host microorganism comprises a first reporter polynucleotide and a second reporter polynucleotide.
  • the reporter polynucleotide is integrated into the genome of the host microorganism.
  • one or more reporter polynucleotides present in the provided reporter microorganisms includes as least two different reporter polynucleotides, i.e. a first reporter polynucleotide and a second reporter polynucleotide, as described.
  • the first and second reporter polynucleotides are comprised in or are part of the same reporter vector.
  • the first and second reporter polynucleotides are comprised in or are part of different reporter vectors.
  • a reporter microorganism as provided herein can include two or more reporter vectors in which, for example, at least one reporter vector is a first reporter vector comprising the first reporter polynucleotide and at least one reporter vector is a second reporter vector comprising the second reporter polynucleotide.
  • the reporter microorganism comprises more than one reporter polynucleotide described herein. In some embodiments, the reporter microorganism comprises a first reporter polynucleotide described herein and a second reporter polynucleotide described herein. In some embodiments, the reporter microorganism comprises more than one reporter vector described herein. In some embodiments, the reporter microorganism comprises a first reporter vector comprising a first reporter polynucleotide described herein and a second vector comprising a second reporter polynucleotide described herein. In certain embodiments, multiple copies of the same polynucleotide increase the signal from the reporter microorganism.
  • the reporter microorganism comprises a first reporter polynucleotide described herein and a second reporter polynucleotide described herein.
  • the reporter microorganism comprises (i) a first reporter polynucleotide comprising a first sequence comprising a regulatory region of a first OM stress-responsive gene of an Acinetobacter species (spp). operably linked to a first sequence encoding a first reporter molecule, and (ii) a second reporter polynucleotide comprising a second sequence comprising a regulatory region of a second OM stress-responsive gene of an Acinetobacter species (spp). operably linked to a second sequence encoding a second reporter molecule, wherein the first and second OM stress-responsive genes are modulated in response to a stress to the outer membrane (OM-stress) of the Acinetobacter spp.
  • OM-stress outer membrane
  • a reporter vector comprises the first reporter polynucleotide and the second reporter polynucleotide.
  • a first reporter vector comprises a first reporter polynucleotide and a second reporter vector comprises the second reporter polynucleotide.
  • the first and second vectors are the same or are functional variants. In certain embodiments, multiple copies of the same vector increases signal from the reporter molecule. In certain embodiments, the first and second vectors share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second vectors are the same. In certain embodiments, the first and second vectors are different.
  • the first and second reporter polynucleotides are the same or functional variants. In certain embodiments, the first and second reporter polynucleotides share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second reporter polynucleotides are the same. In certain embodiments,
  • the first and second reporter polynucleotides are different. In certain embodiments, the first and second reporter polynucleotides are different.
  • different reporter polynucleotides allows for assaying different genes or strains in the same assay.
  • the first and second OM stress-responsive genes are the same or are functional variants.
  • the first and second OM stress-responsive genes share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity.
  • the first and second OM stress-responsive genes are the same.
  • the first and second OM stress-responsive genes are different.
  • different OM stress-responsive genes allow for the identification of broader spectrum or more potent agents.
  • different OM stress- responsive genes allow for identification of more selective agents.
  • first OM stress-responsive gene was responsive to PMBN but not BamA depletion and the second OM stress-responsive gene was responsive to BamA depletion but not PMBN, different agents may be identified as including the first and/or second OM stress-responsive gene.
  • the reporter microorganism includes at least two different reporter polynucleotides as described in which the regulatory region of a first reporter polynucleotide and the regulatory region of a second reporter polynucleotide are from different OM stress-responsive genes.
  • the first OM stress-responsive gene and the second OM stress-responsive gene are different.
  • such reporter microorganisms are capable of modulating a detectable signal in response to at least two different stress conditions, e.g. a physical condition or by an agent.
  • the ability of a single reporter microorganism to produce or modulate a detectable signal in response to one or more stress condition or potential stress condition can provide a multiplexed system to simultaneously measure or assess the susceptibility or resistance of a microorganism to one or more stress condition or potential stress condition and/or ensure that a response to a condition, e.g. caused by a physical condition or by an agent, is real or is not likely to be a false positive.
  • the different regulatory regions are each operatively linked to the same reporter molecule, such that the reporter microorganism is capable of producing a single detectable signal.
  • the different regulatory regions are each operatively linked to different reporter molecules, such that the reporter microorganism is capable of producing different detectable signals, e.g. induced by the different regulatory regions.
  • the first and second sequences comprising the regulatory regions are the same or functional variants. In certain embodiments, the first and second sequences comprising the regulatory regions share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second sequences comprising the regulatory regions are the same.
  • the reporter microorganism is capable of producing at least two different detectable signals.
  • the reporter microorganism contains at least two reporter polynucleotides as described, in which, for example, a regulatory region of the first OM-responsive stress gene is operatively linked to a first reporter molecule and the regulatory region of the second OM-responsive gene is operatively linked to a second reporter molecule that is different from the first reporter molecule.
  • the first reporter molecule and the second reporter molecule do not exhibit an overlapping emission and absorption spectra. Reporter molecules capable of producing different, such as non-overlapping signals, can be separately or distinguishably detected.
  • the reporter molecules capable of producing different, such as non-overlapping signals, can be separately or distinguishably detected.
  • the reporter molecules capable of producing different, such as non-overlapping signals, can be separately or distinguishably detected.
  • microorganism is capable of producing a detectable signal from a first and second reporter molecule that is independently sfGFP and mRuby2.
  • the first and second sequences encoding the first and second reporter molecules are the same or functional variants. In certain embodiments, the first and second sequences encoding the reporter molecules share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second sequences encoding the first and second reporter molecules are the same. In certain embodiments,
  • the first and second sequences encoding the first and second reporter molecules are different. In certain embodiments, the first and second reporter molecules are the same.
  • each of the plurality of reporter microorganisms includes any one or more reporter polynucleotides as described or any one or more reporter vectors as described.
  • each of the plurality of reporter microorganisms includes a reporter polynucleotide that includes the same regulatory region of an OM-responsive gene.
  • each of the plurality of microorganisms includes the same reporter
  • each of the plurality of microorganisms includes a reporter polynucleotide containing the same regulatory region of the same OM responsive gene operatively linked to the sequence encoding a reporter molecule.
  • each of the plurality of microorganisms includes a different reporter polynucleotide.
  • each of the plurality of reporter microorganisms is capable of producing a different detectable signal, whereby each contains a reporter polynucleotide that includes the same regulatory region of an OM-responsive gene, but that is operatively linked to a sequence encoding different reporter molecules.
  • the different reporter molecules do not exhibit overlapping emission and absorption spectra.
  • Reporter molecules capable of producing different, such as non-overlapping signals can be separately or distinguishably detected.
  • a plurality of microorganisms may be useful for comparing the affect or impact of various outer membrane stress conditions capable of engaging a common regulatory region in different microorganisms.
  • the plurality of microorganisms include microorganisms across different strains, isolates or subtypes, such as any as described.
  • each of the plurality of reporter microorganisms includes a different reporter polynucleotide that includes a regulatory region of a different OM-responsive gene.
  • the microorganisms are of the same strain, isolate or subtype of a microorganism, e.g. bacteria.
  • the plurality of microorganisms represents different strains, isolate or subtype of a microorganism, e.g. bacteria.
  • such a plurality of reporter microorganisms can be screened together in response to the same stress condition, e.g. physical or via an agent.
  • the plurality of microorganisms can be co- encapsulated in a gel microdroplet as described.
  • a change in a detectable signal in or from each of the plurality of microorganisms in response to the same stress condition can indicate that the microorganisms are universally responsive or susceptible to the outer membrane stress condition and/or that there is one or more feature (e.g. conserved targeting of an outer membrane protein) that is similar or the same to each of the plurality of microorganisms rendering each similarly susceptible to the outer membrane stress.
  • microorganisms in response to the same stress condition can indicate that susceptibility to the stress condition is specific to one or more microorganism, such as one or more strain, isolate or subtype.
  • the vectors may be used to transfect and transform a host microorganism. Any procedure able to introduce the genetic material into the host microorganism for modulation (e.g. increase or decrease) of expression of the reporter molecule in the presence of OM-stress can be employed.
  • such methods include calcium phosphate transfection, electroporation, retroviral mediated transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasmid vectors, viral vectors and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host
  • Exemplary host microorganisms, and thus the resulting reporter microorganisms include yeast, bacteria, and archaea. Bacteria cells, including Gram positive or Gram negative bacteria can be used to express any of the reporter polynucleotides described above.
  • the host or reporter microorganism is a multi-drug resistant bacterium.
  • the host or reporter microorganism is a Gram-negative bacterium. Non-limiting examples include strains of Acinetobacter, Bdellovibrio, Burkholderia, Chlamydia,
  • reporter polynucleotides or reporter vectors described above can be expressed in Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae,
  • Pseudomonas aeruginosa Salmonella typhimurium, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Vibrio cholera, or Yersinia pestis.
  • the host microorganism, and hence the resulting reporter microorganism is Acinetobacter.
  • the host or reporter microorganism is Acinetobacter apis, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii, Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter organizerri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus, Acinetobacter harbinensis, Acinetobacter indicus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter kookii
  • the host or reporter microorganism is Acinetobacter baumannii.
  • the host microorganism is or is derived from ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA-1605, ATCC BAA-1709, ATCC BAA- 1710, ATCC BAA-1789, ATCC BAA-1790, ATCC BAA-1791, ATCC BAA-1792, ATCC BAA-1793, ATCC BAA- 1794, ATCC BAA- 1795, ATCC BAA-1796, ATCC BAA-1797, ATCC BAA-1798, ATCC BAA- 1799, ATCC B
  • the host microorganism is ATCC 17978, AABA041, AABA046, or Ab307-0294.
  • the reporter microorganism is derived from ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA- 1605, ATCC BAA-1709, ATCC BAA-1710, ATCC BAA-1789, ATCC BAA- 1790, ATCC BAA-1791, ATCC BAA-1792, ATCC BAA- 1793, ATCC BAA-1794, ATCC BAA-1795, ATCC BAA-1796, ATCC BAA-1797, ATCC BAA-1798, ATCC BAA- 1799
  • the reporter microorganism is derived from ATCC 17978, AABA041, AABA046, or Ab307-0294.
  • the transformed reporter strains may be selected utilizing a selectable marker present on the vector (e.g. a drug resistance gene such as a Kanamycin resistance gene) and cultured by a variety of means known to those of skill in the art (see, e.g. Good et al. Clin. Chest Med. 10: 315-322 (1984), Heifets Ann. Rev. Respir. Dis. , 137: 1217-1222 (1988), and Sommers et al. in Color Atlas and Textbook of Diagnostic Microbiology, Third .Edition, J.B. Lippincott Co., Philadelphia, PA (1988)).
  • a selectable marker present on the vector e.g. a drug resistance gene such as a Kanamycin resistance gene
  • microdroplets comprising a reporter microorganism or a plurality of reporter microorganisms as described herein.
  • the microdroplet comprises (i) an agent, such as a candidate agent, which, in some cases, can be an agent- producing cell, e.g. an antibody-producing cell and (ii) a reporter microorganism or a plurality of reporter microorganisms as provided herein.
  • the microdroplet may comprise multiple copies of reporter microorganisms, such as multiple copies of the reporter microorganism or multiple copies and/or agent or agent-producing cell.
  • the microdroplets may contain a single agent or agent-producing cell (e.g. antibody-producing cell) and multiple reporter
  • the average number of reporter microorganisms per microdroplet can be between about 5 and about 500, such as about 10 and about 250, about 50 and about 200, about 50 and about 150, about 50 and about 100, or about 80 and about 120, such as about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200.
  • the number of reporter microorganisms per microdroplet may be lower on average for microorganisms that are larger in cell size, e.g., a fungus or a parasite.
  • the agent can be any agent as described herein.
  • the agent is a candidate antibacterial agent.
  • the agent is a small molecule, a peptide, or a protein, such as an antibody or antigen-binding fragment thereof.
  • the agent is a small molecule antibiotic or peptide antibiotic.
  • the peptide is a fragment of a larger protein.
  • the peptide comprises about 50 or fewer amino acids, such as about 45, 40, 35, 30, 25, 20, or fewer amino acids.
  • the agent is bound to a solid support, such as a bead.
  • a cell comprises the agent.
  • a cell produces or secretes the agent.
  • the cell is an antibody-producing cell.
  • the cell is a B cell, a plasma cell, or a plasmablast.
  • the cell is a mammalian cell or a microorganism, such as a fungal or bacterial cell.
  • the microdroplet comprises (i) an agent, such as a candidate agent, which, in some cases, can be an agent-producing cell, e.g. an antibody-producing cell; (ii) a first reporter microorganism as provided herein; and (iii) a second reporter microorganism as provided herein.
  • an agent such as a candidate agent
  • an agent-producing cell e.g. an antibody-producing cell
  • a first reporter microorganism as provided herein
  • a second reporter microorganism as provided herein.
  • the microdroplet comprises (i) an agent, such as a candidate agent, which, in some cases, can be an agent-producing cell, e.g. an antibody-producing cell; (ii) a first reporter microorganism comprising a first reporter polynucleotide comprising a first sequence comprising a regulatory region of a first OM stress-responsive gene of an
  • Acinetobacter species operably linked to a first sequence encoding a first reporter molecule; and (iii) a second reporter microorganism comprising a second reporter
  • polynucleotide comprising a second sequence comprising a regulatory region of a second OM stress-responsive gene of an Acinetobacter species (spp). operably linked to a second sequence encoding a second reporter molecule, wherein the first and second OM stress-responsive genes are modulated in response to a stress to the outer membrane (OM-stress) of the Acinetobacter spp.
  • spp Acinetobacter species
  • the first reporter microorganism and the second reporter microorganism are clonal. In certain embodiments, the first reporter microorganism and the second reporter microorganism are not clonal. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from the same host strain. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from different host strains. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from different host species. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from the same host species.
  • the first reporter microorganism and the second reporter microorganism are derived from the same host strain and are not clonal. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from the same host species and are derived from different host strains. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from the same host species and are not clonal.
  • a first vector comprises a first reporter polynucleotide and a second vector comprises the second reporter polynucleotide.
  • the first and second vectors share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity.
  • the first and second vectors are the same. In certain embodiments, the first and second vectors are different.
  • the first and second reporter polynucleotides share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second reporter polynucleotides are the same. In certain embodiments, the first and second reporter polynucleotides are different.
  • the first and second OM stress-responsive genes share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second OM stress-responsive genes are the same. In certain embodiments, the first and second OM stress-responsive genes are different.
  • the first and second sequences comprising the regulatory regions share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second sequences comprising the regulatory regions are the same. In certain embodiments, the first and second sequences comprising the regulatory regions are different.
  • the first and second sequences encoding the reporter molecules share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second sequences encoding the first and second reporter molecules are the same. In certain embodiments, the first and second sequences encoding the first and second reporter molecules are different. In certain embodiments, the first and second reporter molecules are the same. In certain embodiments, the first and second reporter molecules are different.
  • the first reporter microorganism and the second reporter microorganism are derived from different host strains and the first and second reporter polynucleotides are different. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from different host strains; the first and second reporter molecules are different; and the first and second OM stress-responsive genes are the same.
  • the first reporter microorganism and the second reporter microorganism are derived from the same host strain and the first and second reporter polynucleotides are different. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from the same host strain; the first and second reporter molecules are different; and the first and second OM stress-responsive genes are different.
  • the microdroplet comprises a polymer matrix and/or a gel matrix.
  • microdroplet comprises agarose, carrageenan, alginate, alginate -polylysine, collagen, cellulose, methylcellulose, gelatin, chitosan, extracellular matrix, dextran, starch, inulin, heparin, hyaluronan, fibrin, polyvinyl alcohol, poly(N-vinyl-2- pyrrolidone), polyethylene glycol, poly(hydroxyethyl methacrylate), acrylate polymers and sodium
  • the microdroplet comprises a polymer matrix, which may be e.g., agarose, carrageenan, alginate, alginate-polylysine, collagen, a plant-derived gum, cellulose or a derivatives thereof (e.g., methylcellulose), gelatin, chitosan or an extracellular matrix (ECM), as described by Kleinman (U.S. Pat. No. 4,829,000), or combinations thereof.
  • a polymer matrix which may be e.g., agarose, carrageenan, alginate, alginate-polylysine, collagen, a plant-derived gum, cellulose or a derivatives thereof (e.g., methylcellulose), gelatin, chitosan or an extracellular matrix (ECM), as described by Kleinman (U.S. Pat. No. 4,829,000), or combinations thereof.
  • Synthetic hydrogels that may be used in the microdroplet include but are not limited to polyvinyl alcohol, block copolymer of ethylene- vinylalcohol, sodium polystyrene sulfonate, vinyl-methyl-tribenzyl ammonium chloride and polyphosphazene.
  • microdroplet comprises agarose.
  • the agarose is low gelling temperature agarose, such as an ultra-low gelling temperature agarose.
  • the low gelling temperature agarose allows for the agarose to stay liquid at lower temperatures, e.g., temperatures that permit viability of the agent-producing cells (e.g. antibody-producing cell) and the reporter microorganism and thereby allow live cell and reporter microorganism encapsulation.
  • the gelling temperature of the agarose used in encapsulation is such that the temperature of liquid agarose does not adversely affect viability of the agent-producing cells (e.g.
  • the agarose has a gelling temperature of lower than about 35°C, such as about 30°C, about 25°C, about 20°C, about 15°C, about 10°C or about 5°C.
  • the agarose is an ultra- low gelling temperature agarose, such as those with a gelling temperature of lower than about 20°C, about 15°C, about 10°C or about 5°C.
  • the agarose has a gelling temperature of between about 5°C and about 30°C, about 5°C and about 20°C, about 5°C and about 15°C, about 8°C and about 17°C or about 5°C and about 10°C, such as about 8°C and about 17°C.
  • the microdroplet comprises growth media and/or is surrounded by a non-aqueous environment.
  • the non-aqueous environment In some embodiments, the non-aqueous
  • the environment includes an oil.
  • the oil is gas permeable.
  • the microdroplet comprises serum.
  • the serum is human, bovine, rabbit, or mouse serum.
  • the concentration of serum is at least or at least about 2.5% (vol/vol), 5.0% (vol/vol), 7.5% (vol/vol), 10.0% (vol/vol), 15.0% (vol/vol), 20% (vol/vol) or 25% (vol/vol).
  • the concentration of serum is from or from about 2.5% (vol/vol) to 25% (vol/vol), 2.5% (vol/vol) to 15% (vol/vol), 2.5% (vol/vol) to 10% (vol/vol), 2.5% (vol/vol) to 5.0% (vol/vol), 5.0% (vol/vol) to 25% (vol/vol), 5.0% (vol/vol) to 15% (vol/vol), 5.0% (vol/vol) to 10% (vol/vol), 10.0% (vol/vol) to 25% (vol/vol), 10.0% (vol/vol) to 15% (vol/vol) or 15% (vol/vol) to 25% (vol/vol).
  • populations of microdroplets comprising reporter
  • the microdroplets on average, comprise one or fewer agent or agent-producing cell (e.g. antibody-producing cells).
  • agent or agent-producing cell e.g. antibody-producing cells
  • the average ratio of candidate agents or candidate agent-producing cell (e.g. antibody-producing cell) per microdroplet is less than or less than about 1. In some embodiments, the average ratio of candidate agents or agent-producing cell (e.g.
  • antibody-producing cell) per microdroplet is between about 0.05 and about 1.0, about 0.05 and about 0.5, about 0.05 and about 0.25, about 0.05 and about 0.1, about 0.1 and about 1.0, about 0.1 and about 0.5, about 0.1 and about 0.25, about 0.25 and about 1.0, about 0.25 and about 0.5 or 0.5 and about 1.0, each inclusive.
  • t the average ratio of agent or agent-producing cell (e.g. antibody-producing cells) per microdroplet is or is about 0.1.
  • compositions comprising a reporter polynucleotide, a reporter vector, a reporter microorganism or a microdroplet as described herein.
  • the composition further comprises an excipient.
  • Acceptable excipients are known in the art, such as, for example buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins;
  • the composition comprises media.
  • the composition comprises an agent or a combination of agents as described herein.
  • the composition comprises one or more components capable of activating the complement pathway.
  • the composition comprises serum, such as human, bovine, rabbit, or mouse serum.
  • the composition comprises a plurality of the reporter
  • polynucleotides polynucleotides, reporter vectors, reporter microorganisms or microdroplets as described herein.
  • kits include instructions for use in the provided methods.
  • kits include one or more containers containing one component, and the component is a reporter polynucleotide, a reporter vector, a reporter microorganism, or a composition described herein.
  • the kit further comprises instructions for use in accordance with any of the methods described herein.
  • kits are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. Kits may optionally provide additional components such as buffers and interpretative information. In some embodiments, the components of the kits are contained in one or more containers.
  • the kit contains a reporter microorganism.
  • the reporter microorganism may be in a form suitable for culture or for direct use in an assay, such as a screening assay as described.
  • the kits may contain other materials suitable for the practice of the assays.
  • the kits may also contain buffers, media for culture of the reporter strains, and other components for performing the assay or culturing or maintaining the cells.
  • kits provide a reporter vector suitable for the production of reporter microorganisms.
  • the kits contain various reagents to facilitate the production of reporter microorganisms. Exemplary of such reagents include, but are not limited to, the pathogen to be transformed, culture media, buffers, drugs for selection of transformants, and components for producing a reporter microorganism.
  • the kits are in suitable packaging. Suitable packaging include, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like.
  • reporter microorganisms containing the reporter polynucleotides or reporter vectors can be used in any desired method in which a change (e.g. increase or decrease) in a signal from the reporter molecule provides a readout of a property or activity of a microorganism, including any property or activity that is or may be or is suspected of being modulated in a response to a condition or in the presence of one or more agents compared to in the absence of the condition or the one or more agents.
  • provided herein are methods of assessing outer membrane (OM) stress, methods for identifying an agent that modulates an activity or property of a microorganism, methods of screening agents, including candidate antibacterial agents, and methods of determining the drug resistance of a microorganism. Also provided herein are agents identified by any method described herein.
  • OM outer membrane
  • the provided methods include contacting a reporter microorganism (i) to a condition that is known to cause or suspected of causing OM stress to the reporter microorganism and/or (ii) with one or more agents, including candidate agents, that may impact the integrity or biogenesis of the outer membrane of the reporter microorganism.
  • following the contacting the reporter microorganism can be assessed or monitored for a change in a detectable signal from the reporter molecule contained therein compared to in the absence of the condition or in the absence of contacting the reporter microorganism with the one or more agents or candidate agents.
  • the provided methods include: (a) contacting or exposing the reporter microorganism or a microorganism containing the reporter polynucleotide or reporter vector provided herein with a condition that is known to cause or suspected of causing stress to the outer membrane and (b) determining if there is a change in a detectable signal from the reporter molecule compared to in the absence of the conditions.
  • such methods can be used to assess if the outer membrane of the reporter microorganism is stressed and/or if the integrity or biogenesis of the outer membrane of the reporter microorganism is being impacted in the presence of the condition.
  • the condition can be a physical condition.
  • the condition is caused by the addition of an exogenous agent, such as one or more candidate agents, to which the reporter microorganism is exposed.
  • the provided methods include: (a) contacting the reporter microorganism or a microorganism containing the reporter polynucleotide or reporter vector provided herein with an agent and (b) identifying a reporter microorganism in which there is a change in a detectable signal from the reporter molecule compared to in the absence of contacting with the agent.
  • such methods can be used to identify an agent that causes stress to the outer membrane of a microorganism and/or impacts or modulates the integrity or biogenesis of the outer membrane of a microorganism.
  • the agent is a biological molecule or drug, such as a small molecule compound, peptide, or polypeptide or protein.
  • the agent is produced or secreted from a cell, in which case the reporter microorganism is contacted with an agent-producing cell.
  • the method can be carried out a plurality of agents, such as a plurality or library of candidate agents or a plurality of agent-producing cells, such that the reporter microorganism is contacted with each of the plurality of agents.
  • the reporter microorganism is contacted with each of the plurality of agents.
  • the reporter microorganism is contacted with each of the plurality of agents.
  • microorganism is separately contacted with each of the plurality of agents, such as each of the plurality of agent-producing cells.
  • the provided methods include (a) contacting the reporter microorganism or a microorganism containing the reporter polynucleotide or reporter vector provided herein with a drug and (b) identifying a reporter microorganism in which there is a change in a detectable signal from the reporter molecule compared to in the absence of contacting with the drug.
  • such methods can be used to identify if a microorganism is resistant to the drug.
  • the microorganism if there is a change in the detectable signal, the microorganism is not resistant to the drug and if there is not a change in the detectable signal, the microorganism is identified as potentially being resistant to the drug.
  • the drug is a small molecule antibiotic or peptide antibiotic. In some embodiments, the drug is known.
  • the change in signal is a decrease in signal. In some embodiments, the change in signal is an increase in signal. [0243] In some embodiments, expression of the reporter molecule and/or presence of a signal therefrom is not detected or present in the reporter microorganism or is detected or present at only a low background level when cultured under conditions that optimize or maintain the health of the bacteria and/or under conditions that do not impact the integrity or biogenesis of the outer membrane.
  • expression of the reporter molecule and/or presence of a signal therefrom is induced or increased by OM- stress of the Acinetobacter spp., such as in the presence of an OM stress condition and/or in the presence of one or more agent that causes outer membrane stress and/or impacts the integrity or biogenesis of the outer membrane.
  • expression of the reporter molecule and/or presence of a signal therefrom is increased at least or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100 or more fold.
  • the increased expression is compared to a reporter microorganism that was not contacted with the agent.
  • the increased expression is compared to the reporter microorganism prior to contact with the agent.
  • the reporter molecule is not expressed unless the reporter microorganism is contacted with the agent.
  • the regulatory region present in the provided reporter polynucleotides or vectors contained in the reporter microorganism is from or derived from a non-coding regulatory region of an OM stress- responsive gene whose expression is upregulated or increased in the presence of OM stress.
  • the OM stress-responsive gene is A1S_0012, A1S_0023, A1S_0027, A1S_0028, A1S_0029, A1S_0030, A1S_0031, A1S_0032, A1S_0033, A1S_0037, A1S_0040, A1S_0041, A1SJ3044, A1S_0066, A1S_0092, A1S_0093, A1S_0109, A1S_0110, A1S_0112, A1S_0113, A1S_0114, A1S_0115, A1S_0116, A1S_0117, A1S_0118, A1S_0126, A1S_0158, A1S_0170, A1S_0175, A1S_0178, A1S_0189, A1S_0224, A1S_0245, A1S_0256, A1S_02
  • A1S_ _3604 A1S_ _3605 A1S_ _3606, A1S_ _3607 A1S_ _3608, A1S_ _3609 A1S_ _3610, A1S_ _3611,
  • the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_0189, A1S_0516, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, A1S_3127, A1S_3492, or AIS_3791.
  • the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or
  • the OM stress-responsive gene is A1S_0032, A1S_2885, A1S_2889, A1S_3127, A1S_3492, or A1S_3791. In some embodiments, the OM stress- responsive gene is A1S_0032, A1S_2885, or A1S_2889. In certain embodiments, the OM stress-responsive gene is A1S_0113 or A1S_1751. In certain embodiments, the OM stress- responsive gene is A1S_0189, A1S_0516, A1S_1224, A1S_2093, or A1S_2271.
  • expression of the reporter molecule and/or presence of a signal therefrom is constitutive in the reporter microorganism when cultured under conditions that optimize or maintain the health of the bacteria and/or that do not impact the integrity or biogenesis of the outer membrane.
  • expression of the reporter molecule and/or presence of a signal therefrom is decreased by OM-stress of the Acinetobacter spp., such as in the presence of an OM stress condition and/or in the presence of one or more agent that causes outer membrane stress and/or impacts the integrity or biogenesis of the outer membrane.
  • the expression of the reporter molecule and/or presence of a signal therefrom is decreased at least or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100 or more fold.
  • the decreased expression is compared to a reporter microorganism that was not contacted with the agent.
  • the decreased expression is compared to the reporter microorganism prior to contact with the agent.
  • the regulatory region present in the provided reporter polynucleotides or vectors contained in the reporter microorganism is from or derived from a non-coding regulatory region of an OM stress- responsive gene whose expression is down-regulated in the presence of OM stress.
  • the OM stress-responsive gene is A1SJ3009, A1S_0010, A1SJ3025, A1SJ3027, A1S_0038, A1S_0067, A1S_0070, A1S_0071, A1S_0073, A1S_0076, A1S_0077, A1S_0079, A1SJ3087, A1SJ3090, A1S_0091, A1S_0095, A1S_0096, A1S_0097, A1S_0098, A1S_0099, A1S_ _0103, A1S_ _0104 A1S_ _0105, A1S_ _0106 A1S_ _0107, A1S_ _0108 A1S_ _0109, A1S_ _0121,

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Abstract

Provided herein are polynucleotides containing a regulatory region of an outer membrane (OM) stress-responsive gene of an Acinetobacter bacterium operably linked to a sequence encoding a reporter molecule. Also provided are reporter vectors and genetically engineered reporter microorganisms, and methods and uses thereof.

Description

REPORTER MICROORGANISMS AND USES THEREOF
Cross -Reference to Related Applications
[0001] The application claims the benefit of priority to U.S. provisional patent application 62/451,621, filed January 27, 2017, entitled "REPORTER MICROORGANISMS AND USES THEREOF" and U.S. provisional patent application 62/540,535, filed August 2, 2017, entitled "REPORTER MICROORGANISMS AND USES THEREOF," the contents of which are hereby incorporated by reference in their entirety for all purposes.
Incorporation by Reference of Sequence Listing
[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 757832000240SeqList.TXT, created January 26, 2018, which is 100,464 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.
Field
[0003] The present disclosure provides in some aspects reporter polynucleotides comprising a sequence comprising a regulatory region of an outer membrane (OM) stress-responsive gene of an Acinetobacter bacterium operably linked to a sequence encoding a reporter molecule. The disclosure further relates to reporter vectors, genetically engineered reporter microorganisms, and uses thereof.
Background
[0004] Multidrug-resistant bacteria have emerged worldwide and are increasing in prevalence, creating a substantial public health concern. The Centers for Disease Control and Prevention attributes at least 23,000 deaths in the U.S. each year to antibiotic-resistant infections, with some infection types associated with mortality rates as high as 50%. In difficult- to-treat Gram-negative pathogens, such as Acinetobacter spp. and Pseudomonas aeruginosa, rates of multi-drug resistance in the U.S. have been reported as 63% and 13%, respectively. The continued prevalence of these multidrug-resistant isolates has left clinicians with few treatment options for the patients with life-threatening infections. Addressing this urgent need for new antibiotics to treat multidrug-resistant Gram-negative infections is critical. There is a need in the art for methods of identifying antibiotics specific for pathogenic bacteria. Provided are methods and articles of manufacture that meet such need.
Summary
[0005] Provided herein are reporter polynucleotides comprising a sequence comprising a regulatory region of an outer membrane (OM) stress-responsive gene of an Acinetobacter bacterium operably linked to a sequence encoding a reporter molecule, wherein the OM stress- responsive gene is modulated in response to a stress to the outer membrane of the Acinetobacter bacterium.
[0006] In certain embodiments, the Acinetobacter bacterium is a Acinetobacter apis, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii,
Acinetobacter brisouii, Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter gerneri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus, Acinetobacter harbinensis, Acinetobacter indicus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter kookii, Acinetobacter Iwoffii, Acinetobacter nectaris, Acinetobacter nosocomialis, Acinetobacter parvus, Acinetobacter pakistanensis, Acinetobacter pittii, Acinetobacter puyangensis, Acinetobacter qingfengensis, Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii,
Acinetobacter soli, Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis, or Acinetobacter venetianus.
[0007] In certain embodiments, the Acinetobacter bacterium is Acinetobacter baumannii. In certain embodiments, the Acinetobacter bacterium is ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA- 1605, ATCC BAA-1709, ATCC BAA- 1710, ATCC BAA- 1789, ATCC BAA-1790, ATCC BAA-1791, ATCC BAA-1792, ATCC BAA-1793, ATCC BAA-1794, ATCC BAA- 1795, ATCC BAA- 1796, ATCC BAA-1797, ATCC BAA-1798, ATCC BAA-1799, ATCC BAA- 1800, ATCC BAA-1878, ATCC BAA-2093, ATCC BAA-747, SDF, AYE, ACICU, AB0057, AB307-0294, 1656-2, MDR-ZJ06, TCDC-AB0715, MDR-TJ, TYTH-1, D1279779, BJAB07104, BJAB0715, BJAB0868, ZW85-1, AbH120-A2, AB030, AB031, AC29, LAC-4, Ab307-0294, 1656-2, ID 403, AABA041, AABA046, or a modified strain thereof. In certain embodiments, the
Acinetobacter bacterium is ATCC 17978, Ab307-0294, AABA041, or AABA046.
[0008] In certain embodiments, the stress to the outer membrane of the Acinetobacter bacterium is or is caused by depletion of BamA or treatment with polymyxin B nonapeptide (PMBN). In certain embodiments, the stress to the outer membrane of the Acinetobacter bacterium is or is caused by depletion of BamA and treatment with PMBN.
[0009] In certain embodiments, stress to the outer membrane of Acinetobacter bacterium involves depletion of BamA and depletion of BamA is performed by removing or decreasing an amount of an inducer that controls expression of BamA from a culture or composition containing Acinetobacter bacterium. In certain embodiments, the inducer is removed or decreased for more than or more than about 5, 10, 15, 20, 30, 45, 60, 75, 90, or 120 minutes. In certain embodiments, the inducer is removed or decreased for more than or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 hours. In certain embodiments, the inducer is arabinose.
[0010] In certain embodiments, stress to the outer membrane of Acinetobacter bacterium involves treatment with PMBN and the Acinetobacter bacterium is treated with PMBN for less than or less than about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, or 120 minutes. In certain embodiments, the Acinetobacter bacterium is treated with PMBN for greater than or greater than about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, or 120 minutes.
[0011] In certain embodiments, the OM stress-responsive gene is upregulated or
downregulated in response to the stress. In certain embodiments, the OM stress-responsive gene is upregulated in response to the stress. In certain embodiments, the OM stress-responsive gene is upregulated in response to the stress at least or about at least 2-fold, 3-fold, 4-fold, 5-fold, 6- fold, 7-fold, 8-fold, 9-fold, 10-fold or more in response to the stress.
[0012] In certain embodiments, the OM stress-responsive gene is A1SJ3012, A1SJ3023, A1S_0027, A1S_0028, A1S_0029, A1S_0030, A1S_0031, A1S_0032, A1S_0033, A1S_0037, A1SJ3040, A1SJ3041, A1S_0044, A1S_0066, A1S_0092, A1S_0093, A1S_0109, A1S_0110, A1S_0112, A1S_0113, A1S_0114, A1S_0115, A1S_0116, A1S_0117, A1S_0118, A1S_0126, A1S_0158, A1S_0170, A1S_0175, A1S_0178, A1S_0189, A1S_0224, A1S_0245, A1S_0256, A1SJ3276, A1SJ3293, A1S_0301, A1S_0309, A1S_0310, A1S_0332, A1S_0333, A1S_0363, A1S_ _0372, A1S_ _0376 A1S_ _0391, A1S_ _0392 A1S_ _0401, A1S_ _0441 A1S_ _0462, A1S_ _0463,
A1S_ _0464, A1S_ _0465 A1S_ _0466, A1S_ _0494 A1S_ _0508, A1S_ _0509 A1S_ _0510, A1S_ _0511,
A1S_ _0512, A1S_ _0514 A1S_ _0516, A1S_ _0518 A1S_ _0519, A1S_ _0520 A1S_ _0521, A1S_ _0522,
A1S_ _0523, A1S_ _0527 A1S_ _0535, A1S_ _0536 A1S_ _0537, A1S_ _0538 A1S_ _0547, A1S_ _0559,
A1S_ _0561, A1S_ _0562 A1S_ _0563, A1S_ _0564 A1S_ _0566, A1S_ _0567 A1S_ _0568, A1S_ _0570,
A1S_ _0624, A1S_ _0630 A1S_ _0631, A1S_ _0633 A1S_ _0634, A1S_ _0640 A1S_ _0641, A1S_ _0642,
A1S_ _0643, A1S_ _0644 A1S_ _0645, A1S_ _0646 A1S_ _0647, A1S_ _0650 A1S_ _0663, A1S_ _0664,
A1S_ _0665, A1S_ _0666 A1S_ _0667, A1S_ _0669 A1S_ _0670, A1S_ _0671 A1S_ _0673, A1S_ _0677,
A1S_ _0680, A1S_ _0683 A1S_ _0714, A1S_ _0717 A1S_ _0718, A1S_ _0719 A1S_ _0736, A1S_ _0738,
A1S_ _0739, A1S_ _0749 A1S_ _0770, A1S_ _0772 A1S_ _0779, A1S_ _0780 A1S_ _0781, A1S_ _0800,
A1S_ _0804, A1S_ _0830 A1S_ _0831, A1S_ _0832 A1S_ _0834, A1S_ _0835 A1S_ _0861, A1S_ _0884,
A1S_ _0889, A1S_ _0929 A1S_ _0930, A1S_ _0931 A1S_ _0932, A1S_ _0935 A1S_ _0945, A1S_ _0959,
A1S_ _0980, A1S_ _1003 A1S_ _1009, A1S_ _1010 A1S_ _1027, A1S_ _1028 A1S_ _1030, A1S_ _1031,
A1S_ _1049, A1S_ _1081 A1S_ _1106, A1S_ _1107 A1S_ _1120, A1S_ _1121 A1S_ _1123, A1S_ _1132,
A1S_ _H33, A1S_ _1134 A1S_ _H39, A1S_ _1143 A1S_ _1145, A1S_ _1146 A1S_ _1148, A1S_ _1149,
A1S_ _1150, A1S_ _1151 A1S_ _1152, A1S_ _1153 A1S_ _1155, A1S_ _1156 A1S_ _1157, A1S_ _1158,
A1S_ _1159, A1S_ _1160 A1S_ _1161, A1S_ _1162 A1S_ _1163, A1S_ _1164 A1S_ _1165, A1S_ _1167,
A1S_ _i m, A1S_ _1172 A1S_ _1173, A1S_ _1180 A1S_ _1184, A1S_ _1186 A1S_ _1198, A1S_ _1202,
A1S_ _1203, A1S_ _1223 A1S_ _1224, A1S_ _1225 A1S_ _1230, A1S_ _1236 A1S_ _1237, A1S_ _1248,
A1S_ _1255, A1S_ _1274 A1S_ _1286, A1S_ _1359 A1S_ _1360, A1S_ _1361 A1S_ _1362, A1S_ _1363,
A1S_ _1383, A1S_ _1384 A1S_ _1385, A1S_ _1386 A1S_ _1387, A1S_ _1393 A1S_ _1404, A1S_ _1407,
A1S_ _1422, A1S_ _1454 A1S_ _1472, A1S_ _1481 A1S_ _1494, A1S_ _1512 A1S_ _1515, A1S_ _1526,
A1S_ _1535, A1S_ _1539 A1S_ _1566, A1S_ _1567 A1S_ _1569, A1S_ _1583 A1S_ _1584, A1S_ _1585,
A1S_ _1589, A1S_ _1590 A1S_ _1593, A1S_ _1595 A1S_ _1596, A1S_ _1617 A1S_ _1630, A1S_ _1644,
A1S_ _1645, A1S_ _1647 A1S_ _1648, A1S_ _1649 A1S_ _1651, A1S_ _1655 A1S_ _1658, A1S_ _1662,
A1S_ _1666, A1S_ _1667 A1S_ _1669, A1S_ _1677 A1S_ _1680, A1S_ _1681 A1S_ _1687, A1S_ _1735,
A1S_ _1741, A1S_ _1743 A1S_ _1744, A1S_ _1750 A1S_ _1751, A1S_ _1752 A1S_ _1760, A1S_ _1762,
A1S_ _1767, A1S_ _1778 A1S_ _1813, A1S_ _1827 A1S_ _1829, A1S_ _1831 A1S_ _1843, A1S_ _1876,
A1S_ _1909, A1S_ _1928 A1S_ _1929, A1S_ _1934 A1S_ _1952, A1S_ _1955 A1S_ _1956, A1S_ _1957,
A1S_ _1959, A1S_ _1960 A1S_ _1961, A1S_ _1962 A1S_ _1963, A1S_ _1979 A1S_ _1986, A1S_ _1987,
A1S_ _1988, A1S_ _2006 A1S_ _2026, A1S_ _2033 A1S_ _2034, A1S_ _2035 A1S_ _2036, A1S_ _2038, A1S_ _2039, A1S_ _2061 A1S_ _2074, A1S_ _2079 A1S_ _2082, A1S_ _2092 A1S_ _2093, A1S_ _2106,
A1S_ _2139, A1S_ _2140 A1S_ _2141, A1S_ _2142 A1S_ _2146, A1S_ _2157 A1S_ _2158, A1S_ _2160,
A1S_ _2161, A1S_ _2162 A1S_ _2178, A1S_ _2179 A1S_ _2183, A1S_ _2186 A1S_ _2195, A1S_ _2230,
A1S_ _2247, A1S_ _2252 A1S_ _2257, A1S_ _2258 A1S_ _2259, A1S_ _2262 A1S_ _2271, A1S_ _2272,
A1S_ _2273, A1S_ _2283 A1S_ _2285, A1S_ _2298 A1S_ _2311, A1S_ _2315 A1S_ _2325, A1S_ _2326,
A1S_ _2330, A1S_ _2331 A1S_ _2366, A1S_ _2367 A1S_ _2382, A1S_ _2387 A1S_ _2389, A1S_ _2395,
A1S_ _2396, A1S_ _2414 A1S_ _2434, A1S_ _2445 A1S_ _2446, A1S_ _2447 A1S_ _2448, A1S_ _2454,
A1S_ _2455, A1S_ _2456 A1S_ _2458, A1S_ _2459 A1S_ _2463, A1S_ _2480 A1S_ _2489, A1S_ _2503,
A1S_ _2504, A1S_ _2508 A1S_ _2542, A1S_ _2543 A1S_ _2552, A1S_ _2553 A1S_ _2555, A1S_ _2557,
A1S_ _2558, A1S_ _2573 A1S_ _2577, A1S_ _2578 A1S_ _2580, A1S_ _2586 A1S_ _2588, A1S_ _2593,
A1S_ _2611, A1S_ _2612 A1S_ _2613, A1S_ _2624 A1S_ _2650, A1S_ _2651 A1S_ _2654, A1S_ _2656,
A1S_ _2660, A1S_ _2664 A1S_ _2668, A1S_ _2675 A1S_ _2676, A1S_ _2677 A1S_ _2678, A1S_ _2684,
A1S_ _2705, A1S_ _2729 A1S_ _2734, A1S_ _2756 A1S_ _2768, A1S_ _2786 A1S_ _2798, A1S_ _2801,
A1S_ _2807, A1S_ _2826 A1S_ _2827, A1S_ _2828 A1S_ _2839, A1S_ _2863 A1S_ _2882, A1S_ _2883,
A1S_ _2884, A1S_ _2885 A1S_ _2889, A1S_ _2892 A1S_ _2893, A1S_ _2942 A1S_ _2943, A1S_ _2953,
A1S_ _2959, A1S_ _2960 A1S_ _2968, A1S_ _2976 A1S_ _2992, A1S_ _3011 A1S_ _3026, A1S_ _3027,
A1S_ _3034, A1S_ _3035 A1S_ _3047, A1S_ _3048 A1S_ _3099, A1S_ _3100 A1S_ _3101, A1S_ _3104,
A1S_ _3105, A1S_ _3114 A1S_ _3115, A1S_ _3116 A1S_ _3117, A1S_ _3124 A1S_ _3125, A1S_ _3126,
A1S_ _3127, A1S_ _3139 A1S_ _3146, A1S_ _3147 A1S_ _3175, A1S_ _3206 A1S_ _3224, A1S_ _3253,
A1S_ _3259, A1S_ _3280 A1S_ _3281, A1S_ _3295 A1S_ _3317, A1S_ _3326 A1S_ _3339, A1S_ _3360,
A1S_ _3361, A1S_ _3367 A1S_ _3368, A1S_ _3371 A1S_ _3375, A1S_ _3376 A1S_ _3392, A1S_ _3411,
A1S_ _3412, A1S_ _3463 A1S_ _3466, A1S_ _3468 A1S_ _3469, A1S_ _3471 A1S_ _3479, A1S_ _3480,
A1S_ _3486, A1S_ _3492 A1S_ _3493, A1S_ _3494 A1S_ _3499, A1S_ _3510 A1S_ _3512, A1S_ _3518,
A1S_ _3522, A1S_ _3523 A1S_ _3533, A1S_ _3534 A1S_ _3535, A1S_ _3539 A1S_ _3540, A1S_ _3541,
A1S_ _3542, A1S_ _3543 A1S_ _3544, A1S_ _3545 A1S_ _3546, A1S_ _3548 A1S_ _3552, A1S_ _3553,
A1S_ _3558, A1S_ _3559 A1S_ _3562, A1S_ _3563 A1S_ _3567, A1S_ _3570 A1S_ _3577, A1S_ _3580,
A1S_ _3580, A1S_ _3585 A1S_ _3586, A1S_ _3594 A1S_ _3595, A1S_ _3596 A1S_ _3601, A1S_ _3602,
A1S_ _3603, A1S_ _3604 A1S_ _3605, A1S_ _3606 A1S_ _3607, A1S_ _3608 A1S_ _3609, A1S_ _3610,
A1S_ _3611, A1S_ _3612 A1S_ _3613, A1S_ _3614 A1S_ _3617, A1S_ _3618 A1S_ _3621, A1S_ _3630,
A1S_ _3632, A1S_ _3634 A1S_ _3635, A1S_ _3636 A1S_ _3637, A1S_ _3642 A1S_ _3645, A1S_ _3649,
A1S_ _3654, A1S_ _3658 A1S_ _3661, A1S_ _3662 A1S_ _3666, A1S_ _3682 A1S_ _3686, A1S_ _3687, A1S_3688, A1S_3694, A1S_3695, A1S_3697, A1S_3704, A1S_3707, A1S_3708, A1S_3712, A1S_3716, A1S_3725, A1S_3726, A1S_3727, A1S_3728, A1S_3736, A1S_3738, A1S_3739, A1S_3740, A1S_3750, A1S_3752, A1S_3760, A1S_3768, A1S_3769, A1S_3770, A1S_3771, A1S_3772, A1S_3773, A1S_3776, A1S_3777, A1S_3778, A1S_3782, A1S_3783, A1S_3786, A1S_3789, A1S_3790, A1S_3791, A1S_3792, A1S_3797, A1S_3810, A1S_3818, A1S_3820, A1S_3835, A1S_3837, A1S_3840, A1S_3842, A1S_3844, A1S_3862, A1S_3864, A1S_3865, A1S_3866, A1S_3867, A1S_3868, A1S_3873, A1S_3875, A1S_3879, A1S_3889, A1S_3900, A1S_3901, A1S_3902, A1S_3908, or A1S_3911.
[0013] In certain embodiments, the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or A1S_3127. In certain embodiments, the OM stress-responsive gene is
A1S_0032, A1S_2885, or A1S_2889.
[0014] In certain embodiments, the OM stress-responsive gene is downregulated in response to the stress. In certain embodiments, the OM stress-responsive gene is downregulated in response to the stress at least or about at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more in response to the stress.
[0015] In certain embodiments, the OM stress-responsive gene is A1S_0009, A1S_0010,
A1S_ _0025, A1S_ _0027, A1S_ _0038, A1S_ _0067, A1S_ _0070, A1S_ _0071, A1S_ _0073, A1S_ _0076,
A1S_ _0077, A1S_ _0079, A1S_ _0087, A1S_ _0090, A1S_ _0091, A1S_ _0095, A1S_ _0096, A1S_ _0097,
A1S_ _0098, A1S_ _0099, A1S_ _0103, A1S_ _0104, A1S_ _0105, A1S_ _0106, A1S_ _0107, A1S_ _0108,
A1S_ _0109, A1S_ _0121, A1S_ _0128, A1S_ _0129, A1S_ _0141, A1S_ _0148, A1S_ _0150, A1S_ _0151,
A1S_ _0152, A1S_ _0153, A1S_ _0154, A1S_ _0155, A1S_ _0156, A1S_ _0157, A1S_ _0177, A1S_ _0184,
A1S_ _0200, A1S_ _0201, A1S_ _0209, A1S_ _0218, A1S_ _0238, A1S_ _0239, A1S_ _0253, A1S_ _0257,
A1S_ _0258, A1S_ _0269, A1S_ _0270, A1S_ _0279, A1S_ _0286, A1S_ _0292, A1S_ _0302, A1S_ _0303,
A1S_ _0304, A1S_ _0321, A1S_ _0322, A1S_ _0323, A1S_ _0347, A1S_ _0365, A1S_ _0369, A1S_ _0370,
A1S_ _0388, A1S_ _0408, A1S_ _0410, A1S_ _0427, A1S_ _0429, A1S_ _0447, A1S_ _0448, A1S_ _0474,
A1S_ _0480, A1S_ _0481, A1S_ _0482, A1S_ _0486, A1S_ _0490, A1S_ _0491, A1S_ _0498, A1S_ _0526,
A1S_ _0533, A1S_ _0534, A1S_ _0548, A1S_ _0549, A1S_ _0566, A1S_ _0567, A1S_ _0568, A1S_ _0591,
A1S_ _0594, A1S_ _0624, A1S_ _0625, A1S_ _0626, A1S_ _0627, A1S_ _0629, A1S_ _0630, A1S_ _0631,
A1S_ _0632, A1S_ _0633, A1S_ _0634, A1S_ _0635, A1S_ _0639, A1S_ _0640, A1S_ _0641, A1S_ _0642,
A1S_ _0643, A1S_ _0644, A1S_ _0645, A1S_ _0646, A1S_ _0647, A1S_ _0649, A1S_ _0650, A1S_ _0651, A1S_ _0690, A1S_ _0691 A1S_ _0692, A1S_ _0695 A1S_ _0698, A1S_ _0721 A1S_ .0731, A1S_ _0732,
A1S_ _0771, A1S_ _0785 A1S_ _0786, A1S_ _0787 A1S_ _0788, A1S_ _0818 A1S_ _0822, A1S_ _0846,
A1S_ _0849, A1S_ _0850 A1S_ .0851, A1S_ _0852 A1S_ _0853, A1S_ _0854 A1S_ _0855, A1S_ _0869,
A1S_ _0877, A1S_ _0882 A1S_ _0883, A1S_ _0890 A1S_ .0891, A1S_ _0901 A1S_ .0910, A1S_ .0911,
A1S_ _0912, A1S_ _0913 A1S_ _0960, A1S_ _0965 A1S_ _0973, A1S_ _0984 A1S_ _0996, A1S_ _0997,
A1S_ _0999, A1S_ _1000 A1S_ _1004, A1S_ _1008 A1S_ .1021, A1S_ _1026 A1S_ .1044, A1S_ _1063,
A1S_ _1072, A1S_ _1079 A1S_ _1080, A1S_ _1088 A1S_ .1089, A1S_ .1091 A1S_ .1092, A1S_ .1093,
A1S_ _1094, A1S_ _1109 A1S_ .1113, A1S_ .1139 A1S_ .1142, A1S_ _1182 A1S_ .1193, A1S_ .1195,
A1S_ _1199, A1S_ _1227 A1S_ _1257, A1S_ _1258 A1S_ .1261, A1S_ _1264 A1S_ .1265, A1S_ .1266,
A1S_ _1267, A1S_ _1268 A1S_ _1269, A1S_ _1270 A1S_ .1281, A1S_ _1317 A1S_ .1318, A1S_ .1319,
A1S_ _1327, A1S_ _1334 A1S_ _1335, A1S_ _1336 A1S_ .1337, A1S_ _1338 A1S_ .1339, A1S_ .1340,
A1S_ _1341, A1S_ _1342 A1S_ _1343, A1S_ _1344 A1S_ .1345, A1S_ _1346 A1S_ .1347, A1S_ .1348,
A1S_ _1349, A1S_ _1356 A1S_ _1366, A1S_ _1367 A1S_ _1368, A1S_ _1369 A1S_ .1370, A1S_ .1372,
A1S_ _1373, A1S_ _1374 A1S_ _1375, A1S_ _1376 A1S_ .1377, A1S_ _1378 A1S_ .1379, A1S_ _1380,
A1S_ _1396, A1S_ _1397 A1S_ _1428, A1S_ _1442 A1S_ .1443, A1S_ _1450 A1S_ .1466, A1S_ .1467,
A1S_ _1469, A1S_ _1470 A1S_ _1476, A1S_ _1490 A1S_ .1491, A1S_ _1492 A1S_ .1493, A1S_ .1498,
A1S_ _1499, A1S_ _1505 A1S_ .1510, A1S_ _1523 A1S_ .1528, A1S_ _1530 A1S_ .1532, A1S_ .1543,
A1S_ _1579, A1S_ _1583 A1S_ .1601, A1S_ _1608 A1S_ .1609, A1S_ _1610 A1S_ .1611, A1S_ .1612,
A1S_ _1613, A1S_ _1637 A1S_ _1638, A1S_ _1639 A1S_ _1655, A1S_ _1692 A1S_ .1698, A1S_ .1699,
A1S_ _1700, A1S_ _1701 A1S_ _1703, A1S_ _1705 A1S_ .1717, A1S_ _1719 A1S_ .1724, A1S_ .1729,
A1S_ _1730, A1S_ .1731 A1S_ _1732, A1S_ _1734 A1S_ .1735, A1S_ _1736 A1S_ .1737, A1S_ .1738,
A1S_ _1742, A1S_ _1745 A1S_ _1754, A1S_ _1756 A1S_ .1758, A1S_ _1775 A1S_ .1776, A1S_ .1790,
A1S_ _1791, A1S_ _1792 A1S_ _1794, A1S_ _1795 A1S_ .1796, A1S_ _1797 A1S_ _1805, A1S_ _1806,
A1S_ _1811, A1S_ _1830 A1S_ _1834, A1S_ _1835 A1S_ _1836, A1S_ _1837 A1S_ _1838, A1S_ .1839,
A1S_ _1840, A1S_ .1841 A1S_ _1854, A1S_ _1855 A1S_ _1856, A1S_ _1857 A1S_ _1858, A1S_ .1859,
A1S_ _1860, A1S_ .1861 A1S_ _1862, A1S_ _1863 A1S_ .1864, A1S_ _1865 A1S_ _1866, A1S_ .1879,
A1S_ _1880, A1S_ _1887 A1S_ _1908, A1S_ _1924 A1S_ .1925, A1S_ _1926 A1S_ .1935, A1S_ .1940,
A1S_ _1942, A1S_ _1948 A1S_ .1951, A1S_ _1984 A1S_ .1996, A1S_ _2041 A1S_ _2042, A1S_ .2052,
A1S_ _2053, A1S_ _2068 A1S_ _2072, A1S_ _2081 A1S_ _2084, A1S_ _2098 A1S_ .2100, A1S_ .2101,
A1S_ _2102, A1S_ _2148 A1S_ _2149, A1S_ _2150 A1S_ .2163, A1S_ _2166 A1S_ .2167, A1S_ .2190,
A1S_ _2191, A1S_ _2202 A1S_ _2203, A1S_ _2207 A1S_ _2209, A1S_ _2218 A1S_ .2221, A1S_ .2225, A1S_ _2232, A1S_ _2234 A1S_ _2248, A1S_ _2279 A1S_ _2280, A1S_ _2288 A1S_ _2289, A1S_ _2340,
A1S_ _2341, A1S_ _2342 A1S_ _2348, A1S_ _2353 A1S_ _2354, A1S_ _2415 A1S_ _2416, A1S_ _2417,
A1S_ _2418, A1S_ _2419 A1S_ _2424, A1S_ _2425 A1S_ _2431, A1S_ _2435 A1S_ _2443, A1S_ _2449,
A1S_ _2450, A1S_ _2451 A1S_ _2452, A1S_ _2475 A1S_ _2501, A1S_ _2509 A1S_ _2510, A1S_ _2514,
A1S_ _2531, A1S_ _2532 A1S_ _2533, A1S_ _2535 A1S_ _2601, A1S_ _2602 A1S_ _2633, A1S_ _2662,
A1S_ _2670, A1S_ _2671 A1S_ _2672, A1S_ _2688 A1S_ _2692, A1S_ _2694 A1S_ _2695, A1S_ _2696,
A1S_ _2701, A1S_ _2Ί \ \ A1S_ _2722, A1S_ _2724 A1S_ _2738, A1S_ _2740 A1S_ _2741, A1S_ _2748,
A1S_ _2753, A1S_ _2755 A1S_ _2758, A1S_ _2761 A1S_ _2762, A1S_ _2769 A1S_ _2773, A1S_ _2774,
A1S_ _2785, A1S_ _2788 A1S_ _2789, A1S_ _2793 A1S_ _2809, A1S_ _2814 A1S_ _2815, A1S_ _2820,
A1S_ _2823, A1S_ _2847 A1S_ _2848, A1S_ _2849 A1S_ _2852, A1S_ _2860 A1S_ _2904, A1S_ _2905,
A1S_ _2906, A1S_ _29\ \ A1S_ _2913, A1S_ _2919 A1S_ _2924, A1S_ _2928 A1S_ _2939, A1S_ _2946,
A1S_ _2956, A1S_ _3013 A1S_ _3014, A1S_ _3025 A1S_ _3040, A1S_ _3043 A1S_ _3049, A1S_ _3050,
A1S_ _3051, A1S_ _3074 A1S_ _3084, A1S_ _3110 A1S_ _3120, A1S_ _3121 A1S_ _3122, A1S_ _3128,
A1S_ _3129, A1S_ _3130 A1S_ _3131, A1S_ _3132 A1S_ _3133, A1S_ _3134 A1S_ _3135, A1S_ _3144,
A1S_ _3174, A1S_ _3180 A1S_ _3195, A1S_ _3207 A1S_ _3222, A1S_ _3224 A1S_ _3225, A1S_ _3231,
A1S_ _3232, A1S_ _3236 A1S_ _3238, A1S_ _3248 A1S_ _3250, A1S_ _3268 A1S_ _3269, A1S_ _3273,
A1S_ _3278, A1S_ _3290 A1S_ _3297, A1S_ _3298 A1S_ _3300, A1S_ _3301 A1S_ _3309, A1S_ _3338,
A1S_ _3342, A1S_ _3355 A1S_ _3364, A1S_ _3377 A1S_ _3397, A1S_ _3398 A1S_ _3402, A1S_ _3403,
A1S_ _3404, A1S_ _3405 A1S_ _3406, A1S_ _3407 A1S_ _3410, A1S_ _3413 A1S_ _3414, A1S_ _3415,
A1S_ _3416, A1S_ _3418 A1S_ _3431, A1S_ _3450 A1S_ _3451, A1S_ _3458 A1S_ _3460, A1S_ _3481,
A1S_ _3487, A1S_ _3491 A1S_ _3494, A1S_ _3498 A1S_ _3506, A1S_ _3508 A1S_ _3509, A1S_ _3514,
A1S_ _3518, A1S_ _3519 A1S_ _3520, A1S_ _3521 A1S_ _3522, A1S_ _3523 A1S_ _3524, A1S_ _3526,
A1S_ _3528, A1S_ _3530 A1S_ _3531, A1S_ _3532 A1S_ _3533, A1S_ _3534 A1S_ _3535, A1S_ _3537,
A1S_ _3538, A1S_ _3539 A1S_ _3540, A1S_ _3541 A1S_ _3542, A1S_ _3543 A1S_ _3544, A1S_ _3545,
A1S_ _3546, A1S_ _3547 A1S_ _3548, A1S_ _3549 A1S_ _3550, A1S_ _3552 A1S_ _3553, A1S_ _3554,
A1S_ _3568, A1S_ _3569 A1S_ _3578, A1S_ _3582 A1S_ _3586, A1S_ _3587 A1S_ _3591, A1S_ _3597,
A1S_ _3599, A1S_ _3600 A1S_ _3602, A1S_ _3611 A1S_ _3619, A1S_ _3621 A1S_ _3624, A1S_ _3629,
A1S_ _3633, A1S_ _3640 A1S_ _3641, A1S_ _3644 A1S_ _3647, A1S_ _3651 A1S_ _3652, A1S_ _3659,
A1S_ _3663, A1S_ _3667 A1S_ _3673, A1S_ _3679 A1S_ _3701, A1S_ _3707 A1S_ _3709, A1S_ _3713,
A1S_ _3715, A1S_ _3717 A1S_ _3732, A1S_ _3735 A1S_ _3738, A1S_ _3740 A1S_ _3741, A1S_ _3742,
A1S_ _3759, A1S_ _3774 A1S_ _3779, A1S_ _3787 A1S_ _3788, A1S_ _3794 A1S_ _3801, A1S_ _3802, A1S_3806, A1S_3809, A1S_3811, A1S_3813, A1S_3814, A1S_3816, A1S_3817, A1S_3823, A1S_3829, A1S_3831, A1S_3832, A1S_3836, A1S_3840, A1S_3846, A1S_3857, A1S_3862, A1S_3868, A1S_3870, A1S_3880, A1S_3884, A1S_3886, A1S_3887, A1S_3891, A1S_3894, A1S_3898, A1S_3907, A1S_3908, A1S_3909, A1S_3912, A1S_3914, or A1S_3915.
[0016] In certain embodiments, the OM stress-responsive gene is A1S_0103, A1S_0645, A1S_1266, A1S_1268, A1S_1335, A1S_1336, A1S_1337, A1S_1338, A1S_1339, A1S_1340, A1S_1341, A1S_1342, A1S_1343, A1S_1344, A1S_1345, A1S_1791, A1S_1792, A1S_1794, A1S_1796, A1S_1835, A1S_1836, A1S_1837, A1S_1838, A1S_1839, A1S_2449, A1S_2450, A1S_2452, A1S_3540, A1S_3541, A1S_3542, A1S_3543, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, A1S_3809, or A1S_3908. In certain embodiments, the OM stress- responsive gene is A1S_1336, A1S_1836, A1S_1838, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, or A1S_3809.
[0017] In certain embodiments, the regulatory region comprises a contiguous sequence of nucleotides within 500 base pairs upstream or 5' of the open reading frame (ORF) of the OM stress-responsive gene. In certain embodiments, the contiguous sequence of nucleotides comprises at least or at least about 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400 or more base pairs. In certain embodiments, the regulatory region comprises a promoter.
[0018] In certain embodiments, the regulatory region comprises a sequence to further promote translation of the encoded reporter molecule. In certain embodiments, the sequence further promoting translation is or comprises a bacterial ribosome binding site. In certain embodiments, the ribosome binding site is a Shine-Dalgarno sequence. In certain embodiments, the Shine-Dalgarno sequence is native to the regulatory region of the OM stress-responsive gene. In certain embodiments, the Shine-Dalgarno sequence is synthetic and/or heterologous to the regulatory region of the OM stress-responsive gene. In certain embodiments, the Shine- Dalgarno sequence comprises the sequence set forth in SEQ K) NO: 14 or a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 14. In certain embodiments, the Shine-Dalgarno sequence comprises a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 14.
[0019] In certain embodiments, the regulatory region comprises the sequence set forth in any of SEQ ID NOS: 1-13 or a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NOs: 1-13. In certain embodiments, the regulatory region comprises a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NOs: 1- 13.
[0020] In certain embodiments, the reporter molecule is a fluorescent protein, a luminescent protein, a chromoprotein, or an enzyme. In certain embodiments, the fluorescent protein is Sirius, SBFP2, Azurite, mAzurite, EBFP2, moxBFP, mKalamal, mTagBFP2, Aquamarine, ECFP, Cerulean, mCerulean, mCerulean3, moxCerulean3, SCFP3A, mTurquoise2, CyPet, AmCyanl, MiCy (Midoriishi-Cyan), iLOV, AcGFPl, sfGFP, moxGFP, mEmerald, EGFP, mEGFP, AzamiGreen, cfSGFP2, ZsGreen, SGFP2, Clover, mClover2, mClover3, EYFP, Topaz, mTopaz, mVenus, mox Venus, SYFP2, mCitrine, YPet, ZsYellowl, mPapayal, mKusabira- Orange (mKO), mOrange, mOrange2, mK02, TurboRFP, tdTomato, mScarlet-H, mNectarine, mRuby2, eqFP611, DsRed2, mApple, mScarlet, mStrawberry, FusionRed, mRFPl, mCherry, mCherry2, mCrimson3, HcRedl, dKatushka, mKatel .3, mPlum, mRaspberry, TagRFP675, mNeptune, mCardinal, mMaroon, TagRFP657, smURFP, miRFP670, iRFP670, iRFP682, miRFP703, iRFP702, miRFP709, mIFP 683, IFP2.0, iRFP, iSplit, iRFP720, T-sapphire, mT- sapphire, mAmetrine, LSSmOrange, mKeima Red, dKeima Red, LSSmKatel, LSSmKate2, Phamret, PA-sfGFP, mPA-Emerald, PA-GFP, PATagRFP, PAmCherryl, PAmCherry2, PAmCherry3, PAm ate, PAiRFPl, PAiRFP2, Dendra2, mEos3.2, mEos4a, dEos 505, tdEos 505, mKikGR, Kaede 508, PSmOrange, PSmOrange2, rsTagRFP, rsEmerald, rsGFPl, Dronpa3, mGeos-M, amilGFP, amilCP, or a modified version thereof. In certain embodiments, the fluorescent protein is sfGFP, mClover3, or mRuby2. In certain embodiments, the fluorescent protein is sfGFP. In certain embodiments, the sequence encoding the reporter molecule comprises the sequence set forth in SEQ ID NO: 15, 33, or 40 or a sequence with at least or at least about 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 15, 33, or 40. In certain embodiments, the sequence encoding the reporter molecule comprises a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 15.
[0021] In certain embodiments, the reporter polynucleotide comprises the sequence set forth in any of SEQ ID NOs: 16-28 or 34-36 or a sequence with at least or at least about 85%, 90%, 95%, 99%, or more sequence identity to any of SEQ ID NOs: 16-28 or 34-36. In certain embodiments, the reporter polynucleotide comprises a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NOs: 16-28. [0022] In certain embodiments, the luminescent protein is North American firefly luciferase, Genji-botaru luciferase, Italian firefly luciferase, Heike luciferase, East European firefly luciferase, Pennsylvania firefly luciferase, Click beetle luciferase, Railroad worm luciferase, Renilla luciferase, Rluc8, Green Renilla luciferase, Gaussia luciferase, Gaussia-Dura luciferase, Cypridina luciferase, Vargula luciferase, Metridia luciferase, OLuc, bacterial luciferase
(LuxAB), or a modified version thereof. In certain embodiments, the enzyme is
chloramphenicol acetyltransferase (CAT), β-galactosidase, alkaline phosphatase, β- glucuronidase, β -lactamase, neomycin phosphotransferase, or a modified version thereof.
[0023] Provided herein are reporter vectors comprising any of the reporter polynucleotide described herein. In certain embodiments, the reporter polynucleotide is a first reporter polynucleotide and the reporter vector further comprises a second reporter polynucleotide that is any of the reporter polynucleotides described herein. In certain embodiments, the first reporter polynucleotide and the second reporter polynucleotide are different. In certain embodiments, the regulatory region of the first reporter polynucleotide and the regulatory region of the second reporter polynucleotide are from different OM stress-responsive genes. In certain embodiments, the reporter molecule encoded by the first reporter polynucleotide is different from the reporter molecule encoded by the second reporter polynucleotide. In certain embodiments, the reporter molecule encoded by the first reporter polynucleotide and the reporter molecule encoded by the second reporter polynucleotide do not exhibit an overlapping emission and absorption spectra and/or are distinguishably detectable.
[0024] In certain embodiments, the reporter vector is capable of being expressed in a host microorganism. In certain embodiments, the reporter vector is capable of being expressed in a Gram-negative bacterium. In certain embodiments, the host microorganism is Acinetobacter, Bdellovibrio, Burkholderia, Chlamydia, Enter obacter, Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella, Neisseria, Pantoea, Pseudomonas, Salmonella, Shigella, Stenotrophomonas, Vibrio, or Yersinia.
[0025] In certain embodiments, the host microorganism is Acinetobacter. In certain embodiments, the host microorganism is Acinetobacter apis, Acinetobacter baumannii,
Acinetobacter baylyi, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii,
Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter gerneri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus, Acinetobacter harbinensis, Acinetobacter indicus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter kookii, Acinetobacter Iwoffii, Acinetobacter nectaris, Acinetobacter nosocomialis, Acinetobacter parvus, Acinetobacter pakistanensis, Acinetobacter pittii, Acinetobacter puyangensis, Acinetobacter qingfengensis, Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii, Acinetobacter soli, Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis, or Acinetobacter venetianus.
[0026] In certain embodiments, the host microorganism is Acinetobacter baumannii. In certain embodiments, the host microorganism is ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA- 1605, ATCC BAA-1709, ATCC BAA-1710, ATCC BAA-1789, ATCC BAA-1790, ATCC BAA- 1791, ATCC BAA- 1792, ATCC BAA-1793, ATCC BAA-1794, ATCC BAA-1795, ATCC BAA- 1796, ATCC BAA-1797, ATCC BAA-1798, ATCC BAA- 1799, ATCC BAA-1800, ATCC BAA-1878, ATCC BAA-2093, ATCC BAA-747, SDF, AYE, ACICU, AB0057, AB307-0294, 1656-2, MDR-ZJ06, TCDC-AB0715, MDR-TJ, TYTH-1, D1279779, BJAB07104, BJAB0715, BJAB0868, ZW85-1, AbH120-A2, AB030, AB031, AC29, LAC-4, Ab307-0294, 1656-2, ID 403, AABA041, AABA046, or a modified strain thereof. In certain embodiments, the host microorganism is ATCC 17978, Ab307-0294, AABA041, or AABA046.
[0027] In certain embodiments, the reporter polynucleotide, optionally the first reporter polynucleotide and second reporter polynucleotide, is comprised in a backbone vector, and the backbone vector is pACH106, pWH1266, or pET-RA. In certain embodiments, the reporter polynucleotide is comprised in a backbone vector, and the backbone vector is pACH106, pWH1266, or pET-RA. In certain embodiments, a nucleotide sequence comprising the reporter polynucleotide, optionally the first reporter polynucleotide and the second reporter
polynucleotide, is inserted into or replaces a portion of the nucleotide sequence of the backbone vector. In certain embodiments, a nucleotide sequence comprising the reporter polynucleotide is inserted into or replaces a portion of the nucleotide sequence of the backbone vector. In certain embodiments, the backbone vector comprises the sequence of nucleotides set forth in SEQ ID NO: 29 and a nucleotide sequence comprising the reporter polynucleotide replaces nucleotides 5,715-7,395 of the backbone vector.
[0028] In certain embodiments, the reporter vector comprises the sequence set forth in any of SEQ ID NOs: 30-32 or a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NOs: 30-32. In certain embodiments, the reporter vector comprises a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NOs: 30-32.
[0029] Provided herein are reporter microorganisms comprising any of the reporter polynucleotides or any of the reporter vectors described herein. Provided herein are reporter microorganisms comprising one or more reporter polynucleotide of any described herein or one or more reporter vector of any described herein. In certain embodiments, the one or more reporter polynucleotides comprise a first reporter polynucleotide and a second reporter polynucleotide that are different. In certain embodiments, the regulatory region of the first reporter polynucleotide and the regulatory region of the second reporter polynucleotide are from different OM stress-responsive genes. In certain embodiments, the reporter molecule encoded by the first reporter polynucleotide is different from the reporter molecule encoded by the second reporter polynucleotide. In certain embodiments, the reporter molecule encoded by the first reporter polynucleotide and the reporter molecule encoded by the second reporter polynucleotide do not exhibit an overlapping emission and absorption spectra and/or are distinguishably detectable. In certain embodiments, the first reporter polynucleotide and second reporter polynucleotide are comprised in the same reporter vector. In certain embodiments, the first reporter polynucleotide and second reporter polynucleotide are comprised in different reporter vectors.
[0030] In certain embodiments, the reporter microorganism is a Gram-negative bacterium. In certain embodiments, the reporter microorganism is Acinetobacter, Bdellovibrio,
Burkholderia, Chlamydia, Enterobacter, Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella, Neisseria, Pantoea, Pseudomonas, Salmonella, Shigella, Stenotrophomonas, Vibrio, or Yersinia.
[0031] In certain embodiments, the reporter microorganism is Acinetobacter. In certain embodiments, the reporter microorganism is Acinetobacter apis, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii,
Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter gerneri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus, Acinetobacter harbinensis, Acinetobacter indicus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter kookii, Acinetobacter Iwoffii, Acinetobacter nectaris, Acinetobacter nosocomialis, Acinetobacter parvus, Acinetobacter pakistanensis, Acinetobacter pittii, Acinetobacter puyangensis, Acinetobacter qingfengensis, Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii, Acinetobacter soli, Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis, or Acinetobacter venetianus. In certain embodiments, the reporter microorganism is Acinetobacter baumannii.
[0032] In certain embodiments, the reporter microorganism is ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA- 1605, ATCC BAA-1709, ATCC BAA-1710, ATCC BAA-1789, ATCC BAA- 1790, ATCC BAA- 1791, ATCC BAA-1792, ATCC BAA-1793, ATCC BAA-1794, ATCC BAA- 1795, ATCC BAA-1796, ATCC BAA-1797, ATCC BAA- 1798, ATCC BAA-1799, ATCC BAA-1800, ATCC BAA- 1878, ATCC BAA-2093, ATCC BAA-747, SDF, AYE, ACICU, AB0057, AB307- 0294, 1656-2, MDR-ZJ06, TCDC-AB0715, MDR-TJ, TYTH-1, D1279779, BJAB07104, BJAB0715, BJAB0868, ZW85-1, AbH120-A2, AB030, AB031, AC29, LAC-4, Ab307-0294, 1656-2, ID 403, AABA041, AABA046, or a modified strain thereof. In certain embodiments, the reporter microorganism is ATCC 17978, Ab307-0294, AABA041, or AABA046.
[0033] Provided herein are pluralities of reporter microorganisms comprising two or more reporter microorganisms described herein. In certain embodiments, each of at least two reporter microorganisms in the plurality comprise a different reporter polynucleotide in which comprises the same regulatory region of an OM-responsive gene but that is operatively linked to a different reporter molecule. In certain embodiments, the different reporter molecules do not exhibit overlapping emission and absorption spectra and/or are distinguishably detectable. In certain embodiments, the plurality of reporter microorganisms comprises 2, 3, 4, 5, or more different reporter microorganisms. In certain embodiments, the at least two reporter microorganisms are derived from the same host strain. In certain embodiments, each of the at least two reporter microorganisms is derived from a different host strain, optionally wherein each of the at least two reporter microorganisms is derived from a different isolate or subtype of the strain.
[0034] Provided herein are compositions comprising a reporter microorganism described herein or a plurality of any of the reporter microorganisms described herein. In certain embodiments, the composition comprises one or more components capable of activating the complement pathway. In certain embodiments, the composition comprises serum. In certain embodiments, the serum is human serum, rabbit serum, bovine serum, or mouse serum. In certain embodiments, the concentration of serum is at least or at least about 2.5% (vol/vol), 5.0% (vol/vol), 7.5% (vol/vol), 10.0% (vol/vol), 15.0% (vol/vol), 20% (vol/vol), or 25% (vol/vol). In certain embodiments, the concentration of serum is from or from about 2.5% (vol/vol) to 25% (vol/vol), 2.5% (vol/vol) to 15% (vol/vol), 2.5% (vol/vol) to 10% (vol/vol), 2.5% (vol/vol) to 5.0% (vol/vol), 5.0% (vol/vol) to 25% (vol/vol), 5.0% (vol/vol) to 15% (vol/vol), 5.0% (vol/vol) to 10% (vol/vol), 10.0% (vol/vol) to 25% (vol/vol), 10.0% (vol/vol) to 15% (vol/vol), or 15% (vol/vol) to 25% (vol/vol).
[0035] In certain embodiments, the composition comprises an agent, optionally a candidate antibacterial agent. In certain embodiments, the agent is a small molecule compound, a peptide or a protein. In certain embodiments, the agent is an antibody or antigen-binding fragment thereof.
[0036] Provided herein are microdroplets comprising a microorganism described herein, a plurality of microorganisms described herein, or a composition described herein. Provided herein are microdroplets comprising a reporter microorganism described herein. In certain embodiments, the microdroplet comprises an agent, such as a candidate antibacterial agent, including agents known to cause or suspected of causing OM stress to the reporter
microorganism and/or that may impact the integrity or biogenesis of the outer membrane of the reporter microorganism.
[0037] In certain embodiments, the microdroplet further comprises a cell that produces or secretes an agent. In certain embodiments, the cell is an antibody-producing cell. In certain embodiments, the cell is a B cell. In certain embodiments, the cell is a plasma cell or a plasmablast. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a microorganism. In certain embodiments, the cell is a fungal or bacterial cell. [0038] In certain embodiments, the agent is a candidate antibacterial agent. In certain embodiments, the agent is a small molecule compound, a peptide, or a protein. In certain embodiments, the agent is an antibody or antigen-binding fragment thereof. In certain embodiments, the agent is a small molecule antibiotic or peptide antibiotic.
[0039] In certain embodiments, the microdroplet comprises agarose, carrageenan, alginate, alginate-polylysine, collagen, cellulose, methylcellulose, gelatin, chitosan, extracellular matrix, dextran, starch, inulin, heparin, hyaluronan, fibrin, polyvinyl alcohol, poly(N-vinyl-2- pyrrolidone), polyethylene glycol, poly(hydroxyethyl methacrylate), acrylate polymers and sodium polyacrylate, polydimethyl siloxane, cis- polyisoprene, Puramatrix™, poly- divenylbenzene, polyurethane, polyacrylamide or combinations thereof. In certain
embodiments, the microdroplet comprises agarose.
[0040] In certain embodiments, the microdroplet comprises growth media. In certain embodiments, the microdroplet comprises serum. In certain embodiments, the serum is human serum, rabbit serum, bovine serum, or mouse serum. In certain embodiments, the concentration of serum is at least or at least about 2.5% (vol/vol), 5.0% (vol/vol), 7.5% (vol/vol), 10.0% (vol/vol), 15.0% (vol/vol), 20% (vol/vol), or 25% (vol/vol). In certain embodiments, the concentration of serum is from or from about 2.5% (vol/vol) to 25% (vol/vol), 2.5% (vol/vol) to 15% (vol/vol), 2.5% (vol/vol) to 10% (vol/vol), 2.5% (vol/vol) to 5.0% (vol/vol), 5.0% (vol/vol) to 25% (vol/vol), 5.0% (vol/vol) to 15% (vol/vol), 5.0% (vol/vol) to 10% (vol/vol), 10.0% (vol/vol) to 25% (vol/vol), 10.0% (vol/vol) to 15% (vol/vol), or 15% (vol/vol) to 25% (vol/vol).
[0041] Provided herein are compositions comprising a microdroplet described herein or a plurality of microdroplets described herein.
[0042] Provided herein are kits comprising: a reporter polynucleotide described herein, a reporter vector described herein, a reporter microorganism described herein, a plurality of reporter microorganisms described herein, a composition described herein, or a microdroplet described herein; and instructions for use.
[0043] Provided herein are methods of assessing outer membrane (OM) stress of a microorganism, comprising: (a) exposing a reporter microorganism described herein, a plurality of reporter microorganisms described herein, or a composition described herein to a condition that is known to cause or suspected of causing stress to the outer membrane; and (b) detecting the presence, absence, or level of a detectable signal from the reporter molecule(s). [0044] Provided herein are methods of assessing outer membrane (OM) stress of a microorganism, comprising: (a) exposing a reporter microorganism described herein, a plurality of reporter microorganisms described herein, or a composition described herein to a condition that is known to cause or suspected of causing stress to the outer membrane; and (b) detecting the presence, absence, or level of a detectable signal from the reporter molecule(s).
[0045] In certain embodiments, the method further comprises (c) determining if there a change in the level of the detectable signal from the reporter molecule(s) compared to in the absence of exposing the reporter microorganism to the condition, wherein a change in the level of the detectable signal indicates the condition causes OM stress to the microorganism.
[0046] Provided herein are methods of assessing outer membrane (OM) stress of a microorganism, comprising: (a) exposing a reporter microorganism or a composition described herein to a condition that is known to cause or suspected of causing stress to the outer membrane; and (b) determining if there a change in a detectable signal from the reporter molecule compared to in the absence of exposing the reporter microorganism to the condition, thereby assessing if OM stress is present.
[0047] In certain embodiments, exposing the reporter microorganism to a condition or agent is carried out in suspension, in an array, or in a microdroplet. In certain embodiments, the condition is treatment with an agent.
[0048] Provided herein are methods of screening an agent, comprising: (a) contacting a reporter microorganism described herein, a plurality of reporter microorganisms described herein, or a composition described herein with an agent; and b) detecting the presence, absence, or level of a detectable signal from the reporter molecule(s). In certain embodiments, the method further comprises (c) identifying the agent that causes a change in the level of the detectable signal from the reporter molecule compared to in the absence of contacting the reporter microorganism to the agent.
[0049] Provided herein are methods of screening an agent, comprising: (a) contacting an agent with a first reporter microorganism described herein; (b) contacting the agent with at least one additional reporter microorganism described herein, wherein the at least one additional reporter microorganism is not the same as the first reporter microorganism; and (c) detecting the presence, absence, or level of a detectable signal from the reporter molecule from the first and/or at least one additional reporter microorganism. [0050] In certain embodiments, the contacting in (a) and (b) is carried out separately. In certain embodiments, the contacting in (a) and (b) is carried out together. In certain
embodiments, the first microorganism, the at least one additional reporter microorganism, and the agent are encapsulated together in a microdroplet. In certain embodiments, the first and the at least one additional reporter microorganism comprise a different reporter polynucleotide in which comprises the same regulatory region of an OM-responsive gene but that is operatively linked to a different reporter molecule. In certain embodiments, the first and the at least one additional microorganism comprise a different reporter polynucleotide in which comprises a different regulatory region of an OM-responsive gene and is operatively linked to a different reporter molecule. In certain embodiments, the different reporter molecules do not exhibit overlapping emission and absorption spectra and/or are distinguishably detectable. In certain embodiments, the first and the at least one additional microorganism are derived from the same host strain. In certain embodiments, the first and the at least one additional microorganism are derived from a different host strain, optionally wherein each of the first and the at least one additional microorganism is derived from a different isolate or subtype of the strain. In certain embodiments, the contacting is carried out in suspension, in an array, or in a microdroplet. In certain embodiments, the contacting is carried out for at least or about at least 5 minutes, 10 minutes, 30 minutes, 60 minutes, 2 hours or 3 hours.
[0051] Provided herein are methods of screening an agent, comprising: (a) contacting a reporter microorganism described herein or a composition described herein with an agent; and (b) identifying a reporter microorganism in which there is a change in a detectable signal from the reporter molecule compared to in the absence of contacting the reporter microorganism with the agent. In certain embodiments, step (a) is carried out with a plurality of agents, wherein the reporter microorganism is contacted with each of the plurality of agents.
[0052] Provided herein are methods of screening an agent comprising: (a) encapsulating in a microdroplet: (i) a reporter microorganism described herein, a plurality of reporter
microorganisms described herein, or a composition described herein; and (ii) a cell, wherein the cell produces an agent; and (b) detecting, in the microdroplet, the presence, absence, or level of a detectable signal from the reporter molecule(s).
[0053] Provided herein are methods for identifying an agent that modulates an activity or property of a reporter microorganism comprising: (a) encapsulating in a microdroplet: (i) a reporter microorganism comprising a reporter polynucleotide described herein or a reporter vector described herein; and (ii) a cell, wherein the cell produces an agent; and (b) identifying a reporter microorganism in which there is a change in a detectable signal from the reporter molecule; thereby identifying an agent that modulates the activity or property of the target microorganism.
[0054] In certain embodiments, the method further comprises (c) isolating the cell from the microdroplet in which there is a change in the level of the detectable signal from the reporter molecule compared to in the absence of exposing the reporter microorganism to the agent. In certain embodiments, the isolating is carried out using a micromanipulator or an automated sorter. In certain embodiments, the method further comprises identifying the agent produced by the cell. In certain embodiments, the identifying comprises determining the sequence of the agent, optionally using single cell PCR and nucleic acid sequencing. In certain embodiments, step (a) is repeated with a plurality of agents. In certain embodiments, if there is a change in the presence, absence, or level of the detectable signal, the microorganism is identified as potentially not being resistant to the agent; and if there is not a change in the presence, absence, or level of the detectable signal, the microorganism is identified as potentially being resistant to the agent.
[0055] Provided herein are methods of determining the drug resistance of a reporter microorganism, the method comprising: (a) contacting a reporter microorganism comprising a reporter polynucleotide described herein or a reporter vector described herein with a drug; and (b) identifying a reporter microorganism in which there is a change in a detectable signal from the reporter molecule compared to in the absence of contacting the reporter microorganism with the drug, wherein if there is a change in the detectable signal, the reporter microorganism is not resistant to the drug and if there is not a change in the detectable signal, the reporter
microorganism is identified as potentially being resistant to the drug.
[0056] In certain embodiments, the OM-stress responsive gene that is operably linked to the reporter molecule in the reporter vector comprised in the reporter microorganism is
downregulated in response to an outer membrane stress and the change in the detectable signal is a decrease in the detectable signal. In certain embodiments, the OM-stress responsive gene that is operably linked to the reporter molecule in the reporter vector comprised in the reporter microorganism is downregulated in response to an outer membrane stress and the level of the detectable signal decreases.
[0057] In certain embodiments of the methods, such as in methods in which the OM-stress responsive gene is downregulated in response to an outer membrane stress, the OM stress- responsive gene is A1S_0009, A1S_0010, A1S_0025, A1S_0027, A1S_0038, A1S_0067,
A1S_ _0070, A1S_ _0071, A1S_ _0073, A1S_ _0076, A1S_ _0077, A1S_ _0079, A1S_ _0087, A1S_ _0090,
A1S_ _0091, A1S_ _0095, A1S_ _0096, A1S_ _0097, A1S_ _0098, A1S_ _0099, A1S_ _0103, A1S_ _0104,
A1S_ _0105, A1S_ _0106, A1S_ _0107, A1S_ _0108, A1S_ _0109, A1S_ _0121, A1S_ _0128, A1S_ _0129,
A1S_ _0141, A1S_ _0148, A1S_ _0150, A1S_ _0151, A1S_ _0152, A1S_ _0153, A1S_ _0154, A1S_ _0155,
A1S_ _0156, A1S_ _0157, A1S_ _0177, A1S_ _0184, A1S_ _0200, A1S_ _0201, A1S_ _0209, A1S_ _0218,
A1S_ _0238, A1S_ _0239, A1S_ _0253, A1S_ _0257, A1S_ _0258, A1S_ _0269, A1S_ _0270, A1S_ _0279,
A1S_ _0286, A1S_ _0292, A1S_ _0302, A1S_ _0303, A1S_ _0304, A1S_ _0321, A1S_ _0322, A1S_ _0323,
A1S_ _0347, A1S_ _0365, A1S_ _0369, A1S_ _0370, A1S_ _0388, A1S_ _0408, A1S_ _0410, A1S_ _0427,
A1S_ _0429, A1S_ _0447, A1S_ _0448, A1S_ _0474, A1S_ _0480, A1S_ _0481, A1S_ _0482, A1S_ _0486,
A1S_ _0490, A1S_ _0491, A1S_ _0498, A1S_ _0526, A1S_ _0533, A1S_ _0534, A1S_ _0548, A1S_ _0549,
A1S_ _0566, A1S_ _0567, A1S_ _0568, A1S_ _0591, A1S_ _0594, A1S_ _0624, A1S_ _0625, A1S_ _0626,
A1S_ _0627, A1S_ _0629, A1S_ _0630, A1S_ _0631, A1S_ _0632, A1S_ _0633, A1S_ _0634, A1S_ _0635,
A1S_ _0639, A1S_ _0640, A1S_ _0641, A1S_ _0642, A1S_ _0643, A1S_ _0644, A1S_ _0645, A1S_ _0646,
A1S_ _0647, A1S_ _0649, A1S_ _0650, A1S_ _0651, A1S_ _0690, A1S_ _0691, A1S_ _0692, A1S_ _0695,
A1S_ _0698, A1S_ _0721, A1S_ _0731, A1S_ _0732, A1S_ _0771, A1S_ _0785, A1S_ _0786, A1S_ _0787,
A1S_ _0788, A1S_ _0818, A1S_ _0822, A1S_ _0846, A1S_ _0849, A1S_ _0850, A1S_ _0851, A1S_ _0852,
A1S_ _0853, A1S_ _0854, A1S_ _0855, A1S_ _0869, A1S_ _0877, A1S_ _0882, A1S_ _0883, A1S_ _0890,
A1S_ _0891, A1S_ _0901, A1S_ _0910, A1S_ _0911, A1S_ _0912, A1S_ _0913, A1S_ _0960, A1S_ _0965,
A1S_ _0973, A1S_ _0984, A1S_ _0996, A1S_ _0997, A1S_ _0999, A1S_ _1000, A1S_ _1004, A1S_ _1008,
A1S_ _1021, A1S_ _1026, A1S_ _1044, A1S_ _1063, A1S_ _1072, A1S_ _1079, A1S_ _1080, A1S_ _1088,
A1S_ _1089, A1S_ _1091, A1S_ _1092, A1S_ _1093, A1S_ _1094, A1S_ _1109, A1S_ _1113, A1S_ _1139,
A1S_ _1142, A1S_ _1182, A1S_ _1193, A1S_ _1195, A1S_ _1199, A1S_ _1227, A1S_ _1257, A1S_ _1258,
A1S_ _1261, A1S_ _1264, A1S_ _1265, A1S_ _1266, A1S_ _1267, A1S_ _1268, A1S_ _1269, A1S_ _1270,
A1S_ _1281, A1S_ _1317, A1S_ _1318, A1S_ _1319, A1S_ _1327, A1S_ _1334, A1S_ _1335, A1S_ _1336,
A1S_ _1337, A1S_ _1338, A1S_ _1339, A1S_ _1340, A1S_ _1341, A1S_ _1342, A1S_ _1343, A1S_ _1344,
A1S_ _1345, A1S_ _1346, A1S_ _1347, A1S_ _1348, A1S_ _1349, A1S_ _1356, A1S_ _1366, A1S_ _1367, A1S_ _1368, A1S_ _1369 A1S_ _1370, A1S_ _1372 A1S_ _1373, A1S_ _1374 A1S_ _1375, A1S_ _1376,
A1S_ _1377, A1S_ _1378 A1S_ _1379, A1S_ _1380 A1S_ _1396, A1S_ _1397 A1S_ _1428, A1S_ _1442,
A1S_ _1443, A1S_ _1450 A1S_ _1466, A1S_ _1467 A1S_ _1469, A1S_ _1470 A1S_ _1476, A1S_ _1490,
A1S_ _1491, A1S_ _1492 A1S_ _1493, A1S_ _1498 A1S_ _1499, A1S_ _1505 A1S_ _1510, A1S_ _1523,
A1S_ _1528, A1S_ _1530 A1S_ _1532, A1S_ _1543 A1S_ _1579, A1S_ _1583 A1S_ _1601, A1S_ _1608,
A1S_ _1609, A1S_ _1610 A1S_ _1611, A1S_ _1612 A1S_ _1613, A1S_ _1637 A1S_ _1638, A1S_ _1639,
A1S_ _1655, A1S_ _1692 A1S_ _1698, A1S_ _1699 A1S_ _1700, A1S_ _1701 A1S_ _1703, A1S_ _1705,
A1S_ _nn, A1S_ _1719 A1S_ _1724, A1S_ _1729 A1S_ _1730, A1S_ _1731 A1S_ _1732, A1S_ _1734,
A1S_ _1735, A1S_ _1736 A1S_ _1737, A1S_ _1738 A1S_ _1742, A1S_ _1745 A1S_ _1754, A1S_ _1756,
A1S_ _1758, A1S_ _1775 A1S_ _1776, A1S_ _1790 A1S_ _1791, A1S_ _1792 A1S_ _1794, A1S_ _1795,
A1S_ _1796, A1S_ _1797 A1S_ _1805, A1S_ _1806 A1S_ _1811, A1S_ _1830 A1S_ _1834, A1S_ _1835,
A1S_ _1836, A1S_ _1837 A1S_ _1838, A1S_ _1839 A1S_ _1840, A1S_ _1841 A1S_ _1854, A1S_ _1855,
A1S_ _1856, A1S_ _1857 A1S_ _1858, A1S_ _1859 A1S_ .I860, A1S_ _1861 A1S_ _1862, A1S_ _1863,
A1S_ _1864, A1S_ _1865 A1S_ _1866, A1S_ _1879 A1S_ _1880, A1S_ _1887 A1S_ _1908, A1S_ _1924,
A1S_ _1925, A1S_ _1926 A1S_ _1935, A1S_ _1940 A1S_ _1 42, A1S_ _1948 A1S_ _1951, A1S_ _1984,
A1S_ _1996, A1S_ _2041 A1S_ _2042, A1S_ _2052 A1S_ _2053, A1S_ _2068 A1S_ _2072, A1S_ _2081,
A1S_ _2084, A1S_ _2098 A1S_ _2100, A1S_ _2101 A1S_ _2102, A1S_ _2148 A1S_ _2149, A1S_ _2150,
A1S_ _2163, A1S_ _2166 A1S_ _2167, A1S_ _2190 A1S_ _2191, A1S_ _2202 A1S_ _2203, A1S_ _2207,
A1S_ _2209, A1S_ _2218 A1S_ _2221, A1S_ _2225 A1S_ _2232, A1S_ _2234 A1S_ _2248, A1S_ _2279,
A1S_ _2280, A1S_ _2288 A1S_ _2289, A1S_ _2340 A1S_ _2341, A1S_ _2342 A1S_ _2348, A1S_ _2353,
A1S_ _2354, A1S_ _2415 A1S_ _2416, A1S_ _2417 A1S_ _2418, A1S_ _2419 A1S_ _2424, A1S_ _2425,
A1S_ _2431, A1S_ _2435 A1S_ _2443, A1S_ _2449 A1S_ _2450, A1S_ _2451 A1S_ _2452, A1S_ _2475,
A1S_ _2501, A1S_ _2509 A1S_ _2510, A1S_ _2514 A1S_ _2531, A1S_ _2532 A1S_ _2533, A1S_ _2535,
A1S_ _2601, A1S_ _2602 A1S_ _2633, A1S_ _2662 A1S_ _2670, A1S_ _2671 A1S_ _2672, A1S_ _2688,
A1S_ _2692, A1S_ _2694 A1S_ _2695, A1S_ _2696 A1S_ _2701, A1S_ _2711 A1S_ _2722, A1S_ _2724,
A1S_ _2738, A1S_ _2740 A1S_ _2741, A1S_ _2748 A1S_ _2753, A1S_ _2755 A1S_ _2758, A1S_ _2761,
A1S_ _2762, A1S_ _2769 A1S_ _2773, A1S_ _2774 A1S_ _2785, A1S_ _2788 A1S_ _2789, A1S_ _2793,
A1S_ _2809, A1S_ _2814 A1S_ _2815, A1S_ _2820 A1S_ _2823, A1S_ _2847 A1S_ _2848, A1S_ _2849,
A1S_ _2852, A1S_ _2860 A1S_ _2904, A1S_ _2905 A1S_ _2906, A1S_ _2911 A1S_ _2913, A1S_ _2919,
A1S_ _2924, A1S_ _2928 A1S_ _2939, A1S_ _2946 A1S_ _2956, A1S_ _3013 A1S_ _3014, A1S_ _3025,
A1S_ _3040, A1S_ _3043 A1S_ _3049, A1S_ _3050 A1S_ _3051, A1S_ _3074 A1S_ _3084, A1S_ _3110, A1S_ _3120, A1S_ _3121, A1S_ _3122, A1S_ _3128, A1S_ _3129, A1S_ _3130, A1S_ _3131, A1S_ _3132,
A1S_ _3133, A1S_ _3134, A1S_ _3135, A1S_ _3144, A1S_ _3174, A1S_ _3180, A1S_ _3195, A1S_ _3207,
A1S_ _3222, A1S_ _3224, A1S_ _3225, A1S_ _3231, A1S_ _3232, A1S_ _3236, A1S_ _3238, A1S_ _3248,
A1S_ _3250, A1S_ _3268, A1S_ _3269, A1S_ _3273, A1S_ _3278, A1S_ _3290, A1S_ _3297, A1S_ _3298,
A1S_ _3300, A1S_ _3301, A1S_ _3309, A1S_ _3338, A1S_ _3342, A1S_ _3355, A1S_ _3364, A1S_ _3377,
A1S_ _3397, A1S_ _3398, A1S_ _3402, A1S_ _3403, A1S_ _3404, A1S_ _3405, A1S_ _3406, A1S_ _3407,
A1S_ _3410, A1S_ _3413, A1S_ _3414, A1S_ _3415, A1S_ _3416, A1S_ _3418, A1S_ _3431, A1S_ _3450,
A1S_ _3451, A1S_ _3458, A1S_ _3460, A1S_ _3481, A1S_ _3487, A1S_ _3491, A1S_ _3494, A1S_ _3498,
A1S_ _3506, A1S_ _3508, A1S_ _3509, A1S_ _3514, A1S_ _3518, A1S_ _3519, A1S_ _3520, A1S_ _3521,
A1S_ _3522, A1S_ _3523, A1S_ _3524, A1S_ _3526, A1S_ _3528, A1S_ _3530, A1S_ J3531, A1S_ _3532,
A1S_ _3533, A1S_ _3534, A1S_ _3535, A1S_ _3537, A1S_ _3538, A1S_ _3539, A1S_ _3540, A1S_ _3541,
A1S_ _3542, A1S_ _3543, A1S_ _3544, A1S_ _3545, A1S_ _3546, A1S_ _3547, A1S_ _3548, A1S_ _3549,
A1S_ _3550, A1S_ _3552, A1S_ _3553, A1S_ _3554, A1S_ _3568, A1S_ _3569, A1S_ _3578, A1S_ _3582,
A1S_ _3586, A1S_ _3587, A1S_ _3591, A1S_ _3597, A1S_ _3599, A1S_ _3600, A1S_ _3602, A1S_ _3611,
A1S_ _3619, A1S_ _3621, A1S_ _3624, A1S_ _3629, A1S_ _3633, A1S_ _3640, A1S_ _3641, A1S_ _3644,
A1S_ _3647, A1S_ _3651, A1S_ _3652, A1S_ _3659, A1S_ _3663, A1S_ _3667, A1S_ _3673, A1S_ _3679,
A1S_ _3701, A1S_ _3707, A1S_ _3709, A1S_ _3713, A1S_ _3715, A1S_ _3717, A1S_ _3732, A1S_ _3735,
A1S_ _3738, A1S_ _3740, A1S_ _3741, A1S_ _3742, A1S_ _3759, A1S_ _3774, A1S_ _3779, A1S_ _3787,
A1S_ _3788, A1S_ _3794, A1S_ _3801, A1S_ _3802, A1S_ _3806, A1S_ _3809, A1S_ _3811, A1S_ _3813,
A1S_ _3814, A1S_ _3816, A1S_ _3817, A1S_ _3823, A1S_ _3829, A1S_ _3831, A1S_ _3832, A1S_ _3836,
A1S_ _3840, A1S_ _3846, A1S_ _3857, A1S_ _3862, A1S_ _3868, A1S_ _3870, A1S_ _3880, A1S_ _3884,
A1S_ _3886, A1S_ _3887, A1S_ _3891, A1S_ _3894, A1S_ _3898, A1S_ _3907, A1S_ _3908, A1S_ _3909,
A1S_ _3912, A1S_ _3914, or A1S_3915.
[0058] In certain embodiments of the methods, such as in methods in which the OM-stress responsive gene is downregulated in response to an outer membrane stress, the OM stress- responsive gene is A1S_0103, A1S_0645, A1S_1266, A1S_1268, A1S_1335, A1S_1336, A1S_1337, A1S_1338, A1S_1339, A1S_1340, A1S_1341, A1S_1342, A1S_1343, A1S_1344, A1S_1345, A1S_1791, A1S_1792, A1S_1794, A1S_1796, A1S_1835, A1S_1836, A1S_1837, A1S_1838, A1S_1839, A1S_2449, A1S_2450, A1S_2452, A1S_3540, A1S_3541, A1S_3542, A1S_3543, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, A1S_3809, or A1S_3908. In certain embodiments, the OM stress-responsive gene is A1S_1336, A1S_1836, A1S_1838, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, or A1S_3809.
[0059] In certain embodiments, the OM-stress responsive gene that is operably linked to the reporter molecule in the reporter vector comprised in the reporter microorganism is upregulated in response to an outer membrane stress and the change in the detectable signal is an increase in the detectable signal. In certain embodiments, the OM-stress responsive gene that is operably linked to the reporter molecule in the reporter vector comprised in the reporter microorganism is upregulated in response to an outer membrane stress and the level of the detectable signal increases.
[0060] In certain embodiments of the methods, such as in methods in which the OM-stress responsive gene is upregulated in response to an outer membrane stress, the OM stress- responsive gene is A1S_0012, A1S_0023, A1S_0027, A1S_0028, A1S_0029, A1S_0030,
A1S_ _0031, A1S_ _0032, A1S_ _0033, A1S_ _0037, A1S_ _0040, A1S_ _0041, A1S_ _0044, A1S_ _0066,
A1S_ _0092, A1S_ _0093, A1S_ _0109, A1S_ _0110, A1S_ _0112, A1S_ _0113, A1S_ _0114, A1S_ _0115,
A1S_ _0116, A1S_ _0117, A1S_ _0118, A1S_ _0126, A1S_ _0158, A1S_ _0170, A1S_ _0175, A1S_ _0178,
A1S_ _0189, A1S_ _0224, A1S_ _0245, A1S_ _0256, A1S_ _0276, A1S_ _0293, A1S_ _0301, A1S_ _0309,
A1S_ _0310, A1S_ _0332, A1S_ _0333, A1S_ _0363, A1S_ _0372, A1S_ _0376, A1S_ _0391, A1S_ _0392,
A1S_ _0401, A1S_ _0441, A1S_ _0462, A1S_ _0463, A1S_ _0464, A1S_ _0465, A1S_ _0466, A1S_ _0494,
A1S_ _0508, A1S_ _0509, A1S_ _0510, A1S_ _0511, A1S_ _0512, A1S_ _0514, A1S_ _0516, A1S_ _0518,
A1S_ _0519, A1S_ _0520, A1S_ _0521, A1S_ _0522, A1S_ _0523, A1S_ _0527, A1S_ _0535, A1S_ _0536,
A1S_ _0537, A1S_ _0538, A1S_ _0547, A1S_ _0559, A1S_ _0561, A1S_ _0562, A1S_ _0563, A1S_ _0564,
A1S_ _0566, A1S_ _0567, A1S_ _0568, A1S_ _0570, A1S_ _0624, A1S_ _0630, A1S_ _0631, A1S_ _0633,
A1S_ _0634, A1S_ _0640, A1S_ _0641, A1S_ _0642, A1S_ _0643, A1S_ _0644, A1S_ _0645, A1S_ _0646,
A1S_ _0647, A1S_ _0650, A1S_ _0663, A1S_ _0664, A1S_ _0665, A1S_ _0666, A1S_ _0667, A1S_ _0669,
A1S_ _0670, A1S_ _0671, A1S_ _0673, A1S_ _0677, A1S_ _0680, A1S_ _0683, A1S_ _0714, A1S_ _0717,
A1S_ _0718, A1S_ _0719, A1S_ _0736, A1S_ _0738, A1S_ _0739, A1S_ _0749, A1S_ _0770, A1S_ _0772,
A1S_ _0779, A1S_ _0780, A1S_ _0781, A1S_ _0800, A1S_ _0804, A1S_ _0830, A1S_ _0831, A1S_ _0832,
A1S_ _0834, A1S_ _0835, A1S_ _0861, A1S_ _0884, A1S_ _0889, A1S_ _0929, A1S_ _0930, A1S_ _0931,
A1S_ _0932, A1S_ _0935, A1S_ _0945, A1S_ _0959, A1S_ _0980, A1S_ _1003, A1S_ _1009, A1S_ _1010,
A1S_ _1027, A1S_ _1028, A1S_ _1030, A1S_ _1031, A1S_ _1049, A1S_ _1081, A1S_ _1106, A1S_ _1107,
A1S_ _1120, A1S_ _1121, A1S_ _1123, A1S_ _1132, A1S_ _1133, A1S_ _1134, A1S_ _1139, A1S_ _1143, A1S_ _1145, A1S_ _1146 A1S_ _1148, A1S_ _1149 A1S_ _1150, A1S_ _1151 A1S_ _1152, A1S_ _1153,
A1S_ _1155, A1S_ _1156 A1S_ _1157, A1S_ _1158 A1S_ _1159, A1S_ _1160 A1S_ _1161, A1S_ _1162,
A1S_ _1163, A1S_ _1164 A1S_ _1165, A1S_ _1167 A1S_ _i m, A1S_ _1172 A1S_ _1173, A1S_ _1180,
A1S_ _1184, A1S_ _1186 A1S_ _1198, A1S_ _1202 A1S_ _1203, A1S_ _1223 A1S_ _1224, A1S_ _1225,
A1S_ _1230, A1S_ _1236 A1S_ _1237, A1S_ _1248 A1S_ _1255, A1S_ _1274 A1S_ _1286, A1S_ _1359,
A1S_ _1360, A1S_ _1361 A1S_ _1362, A1S_ _1363 A1S_ _1383, A1S_ _1384 A1S_ _1385, A1S_ _1386,
A1S_ _1387, A1S_ _1393 A1S_ _1404, A1S_ _1407 A1S_ _1422, A1S_ _1454 A1S_ _1472, A1S_ _1481,
A1S_ _1494, A1S_ _1512 A1S_ _1515, A1S_ _1526 A1S_ _1535, A1S_ _1539 A1S_ _1566, A1S_ _1567,
A1S_ _1569, A1S_ _1583 A1S_ _1584, A1S_ _1585 A1S_ _1589, A1S_ _1590 A1S_ _1593, A1S_ _1595,
A1S_ _1596, A1S_ _1617 A1S_ _1630, A1S_ _1644 A1S_ _1645, A1S_ _1647 A1S_ _1648, A1S_ _1649,
A1S_ _1651, A1S_ _1655 A1S_ _1658, A1S_ _1662 A1S_ _1666, A1S_ _1667 A1S_ _1669, A1S_ _1677,
A1S_ _1680, A1S_ _1681 A1S_ _1687, A1S_ _1735 A1S_ _1741, A1S_ _1743 A1S_ _1744, A1S_ _1750,
A1S_ _1751, A1S_ _1752 A1S_ _1760, A1S_ _1762 A1S_ _1767, A1S_ _1778 A1S_ _1813, A1S_ _1827,
A1S_ _1829, A1S_ _1831 A1S_ _1843, A1S_ _1876 A1S_ _1909, A1S_ _1928 A1S_ _1929, A1S_ _1934,
A1S_ _1952, A1S_ _1955 A1S_ _1956, A1S_ _1957 A1S_ _1959, A1S_ _1960 A1S_ _1961, A1S_ _1962,
A1S_ _1963, A1S_ _1979 A1S_ _1986, A1S_ _1987 A1S_ _1988, A1S_ _2006 A1S_ _2026, A1S_ _2033,
A1S_ _2034, A1S_ _2035 A1S_ _2036, A1S_ _2038 A1S_ _2039, A1S_ _2061 A1S_ _2074, A1S_ _2079,
A1S_ _2082, A1S_ _2092 A1S_ _2093, A1S_ _2106 A1S_ _2139, A1S_ _2140 A1S_ _2141, A1S_ _2142,
A1S_ _2146, A1S_ _2157 A1S_ _2158, A1S_ _2160 A1S_ _2161, A1S_ _2162 A1S_ _2178, A1S_ _2179,
A1S_ _2183, A1S_ _2186 A1S_ _2195, A1S_ _2230 A1S_ _2247, A1S_ _2252 A1S_ _2257, A1S_ _2258,
A1S_ _2259, A1S_ _2262 A1S_ _2271, A1S_ _2272 A1S_ _2273, A1S_ _2283 A1S_ _2285, A1S_ _2298,
A1S_ _2311, A1S_ _2315 A1S_ _2325, A1S_ _2326 A1S_ _2330, A1S_ _2331 A1S_ _2366, A1S_ _2367,
A1S_ _2382, A1S_ _2387 A1S_ _2389, A1S_ _2395 A1S_ _2396, A1S_ _2414 A1S_ _2434, A1S_ _2445,
A1S_ _2446, A1S_ _2447 A1S_ _2448, A1S_ _2454 A1S_ _2455, A1S_ _2456 A1S_ _2458, A1S_ _2459,
A1S_ _2463, A1S_ _2480 A1S_ _2489, A1S_ _2503 A1S_ _2504, A1S_ _2508 A1S_ _2542, A1S_ _2543,
A1S_ _2552, A1S_ _2553 A1S_ _2555, A1S_ _2557 A1S_ _2558, A1S_ _2573 A1S_ _2577, A1S_ _2578,
A1S_ _2580, A1S_ _2586 A1S_ _2588, A1S_ _2593 A1S_ _2611, A1S_ _2612 A1S_ _2613, A1S_ _2624,
A1S_ _2650, A1S_ _2651 A1S_ _2654, A1S_ _2656 A1S_ _2660, A1S_ _2664 A1S_ _2668, A1S_ _2675,
A1S_ _2676, A1S_ _2677 A1S_ _2678, A1S_ _2684 A1S_ _2705, A1S_ _2729 A1S_ _2734, A1S_ _2756,
A1S_ _2768, A1S_ _2786 A1S_ _2798, A1S_ _2801 A1S_ _2807, A1S_ _2826 A1S_ _2827, A1S_ _2828,
A1S_ _2839, A1S_ _2863 A1S_ _2882, A1S_ _2883 A1S_ _2884, A1S_ _2885 A1S_ _2889, A1S_ _2892, A1S_ _2893, A1S_ _2942, A1S_ _2943, A1S_ _2953, A1S_ _2959, A1S_ _2960, A1S_ _2968, A1S_ _2976,
A1S_ _2992, A1S_ _3011, A1S_ _3026, A1S_ _3027, A1S_ _3034, A1S_ _3035, A1S_ _3047, A1S_ _3048,
A1S_ _3099, A1S_ _3100, A1S_ _3101, A1S_ _3104, A1S_ _3105, A1S_ _3114, A1S_ _3115, A1S_ _3116,
A1S_ _3117, A1S_ _3124, A1S_ _3125, A1S_ _3126, A1S_ _3127, A1S_ _3139, A1S_ _3146, A1S_ _3147,
A1S_ _3175, A1S_ _3206, A1S_ _3224, A1S_ _3253, A1S_ _3259, A1S_ _3280, A1S_ _3281, A1S_ _3295,
A1S_ _3317, A1S_ _3326, A1S_ _3339, A1S_ _3360, A1S_ _3361, A1S_ _3367, A1S_ _3368, A1S_ _3371,
A1S_ _3375, A1S_ _3376, A1S_ _3392, A1S_ _3411, A1S_ _3412, A1S_ _3463, A1S_ _3466, A1S_ _3468,
A1S_ _3469, A1S_ _3471, A1S_ _3479, A1S_ _3480, A1S_ _3486, A1S_ _3492, A1S_ _3493, A1S_ _3494,
A1S_ _3499, A1S_ _3510, A1S_ _3512, A1S_ _3518, A1S_ _3522, A1S_ _3523, A1S_ _3533, A1S_ _3534,
A1S_ _3535, A1S_ _3539, A1S_ _3540, A1S_ _3541, A1S_ _3542, A1S_ _3543, A1S_ _3544, A1S_ _3545,
A1S_ _3546, A1S_ _3548, A1S_ _3552, A1S_ _3553, A1S_ _3558, A1S_ _3559, A1S_ _3562, A1S_ _3563,
A1S_ _3567, A1S_ _3570, A1S_ _3577, A1S_ _3580, A1S_ _3580, A1S_ _3585, A1S_ _3586, A1S_ _3594,
A1S_ _3595, A1S_ _3596, A1S_ _3601, A1S_ _3602, A1S_ _3603, A1S_ _3604, A1S_ _3605, A1S_ _3606,
A1S_ _3607, A1S_ _3608, A1S_ _3609, A1S_ _3610, A1S_ _3611, A1S_ _3612, A1S_ _3613, A1S_ _3614,
A1S_ _3617, A1S_ _3618, A1S_ _3621, A1S_ _3630, A1S_ _3632, A1S_ _3634, A1S_ _3635, A1S_ _3636,
A1S_ _3637, A1S_ _3642, A1S_ _3645, A1S_ _3649, A1S_ _3654, A1S_ _3658, A1S_ _3661, A1S_ _3662,
A1S_ _3666, A1S_ _3682, A1S_ _3686, A1S_ _3687, A1S_ _3688, A1S_ _3694, A1S_ _3695, A1S_ _3697,
A1S_ _3704, A1S_ _3707, A1S_ _3708, A1S_ _3712, A1S_ _3716, A1S_ _3725, A1S_ _3726, A1S_ _3727,
A1S_ _3728, A1S_ _3736, A1S_ _3738, A1S_ _3739, A1S_ _3740, A1S_ _3750, A1S_ _3752, A1S_ _3760,
A1S_ _3768, A1S_ _3769, A1S_ _3770, A1S_ _3771, A1S_ _3772, A1S_ _3773, A1S_ _3776, A1S_ _3777,
A1S_ _3778, A1S_ _3782, A1S_ _3783, A1S_ _3786, A1S_ _3789, A1S_ _3790, A1S_ _3791, A1S_ _3792,
A1S_ _3797, A1S_ _3810, A1S_ _3818, A1S_ _3820, A1S_ _3835, A1S_ _3837, A1S_ _3840, A1S_ _3842,
A1S_ _3844, A1S_ _3862, A1S_ _3864, A1S_ _3865, A1S_ _3866, A1S_ _3867, A1S_ _3868, A1S_ _3873,
A1S_ _3875, A1S_ _3879, A1S_ _3889, A1S_ _3900, A1S_ _3901, A1S_ _3902, A1S_ _3908, or
A1S_3911.
[0061] In certain embodiments of the methods, such as in methods in which the OM-stress responsive gene is upregulated in response to an outer membrane stress, the OM stress- responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_0189, A1S_0516, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, A1S_3127, A1S_3492, or AIS_3791. In certain embodiments, the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or A1S_3127. In certain embodiments, the OM stress-responsive gene is A1S_0032, A1S_2885, A1S_2889, A1S_3127, A1S_3492, A1S_3791. In certain embodiments, the OM stress-responsive gene is A1S_0032, A1S_2885, or A1S_2889. In certain embodiments, the OM stress-responsive gene is A1S_0113 or A1S_1751. In certain embodiments, the OM stress-responsive gene is A1S_0189, A1S_0516, A1S_1224, A1S_2093, or A1S_2271.
[0062] Provided herein are methods of assessing OM stress, the method comprising: (a) contacting a reporter microorganism described herein or a composition described herein with an agent; and (b) identifying a reporter microorganism producing a detectable signal from the reporter molecule, thereby assessing OM stress.
[0063] Provided herein are methods of screening an agent, comprising: (a) contacting a reporter microorganism described herein or a composition described herein with an agent; and (b) identifying a reporter microorganism producing a detectable signal from the reporter molecule, thereby identifying an agent that causes OM stress to the reporter microorganism. In certain embodiments, step (a) is carried out with a plurality of agents, wherein the reporter microorganism is contacted with each of the plurality of agents.
[0064] Provided herein are methods for identifying an agent that modulates an activity or property of a microorganism comprising: (a) encapsulating in a microdroplet: (i) a reporter microorganism comprising a reporter polynucleotide described herein or a reporter vector described herein; and (ii) a cell, wherein the cell produces an agent; and (b) identifying a reporter microorganism producing a detectable signal from the reporter molecule.
[0065] Provided herein are methods of determining the drug resistance of a reporter microorganism, the method comprising: (a) contacting a reporter microorganism comprising a reporter polynucleotide described herein or a reporter vector described herein with a drug; and (b) identifying a reporter microorganism producing a detectable signal from the reporter molecule, wherein if the reporter microorganism produces a detectable signal, the reporter microorganism is not resistant to the drug and if the reporter microorganism does not produce a detectable signal, the reporter microorganism is identified as potentially being resistant to the drug. [0066] In certain embodiments, the OM-stress responsive gene that is operably linked to the reporter molecule in the reporter vector comprised in the reporter microorganism is downregulated in response to an outer membrane stress.
[0067] In certain embodiments of the methods, such as in methods in which the OM-stress responsive gene is downregulated in response to an outer membrane stress, the OM stress- responsive gene is A1S_0009, A1S_0010, A1S_0025, A1S_0027, A1S_0038, A1S_0067,
A1S_ _0070, A1S_ _0071, A1S_ _0073, A1S_ _0076, A1S_ _0077, A1S_ _0079, A1S_ _0087, A1S_ _0090,
A1S_ _0091, A1S_ _0095, A1S_ _0096, A1S_ _0097, A1S_ _0098, A1S_ _0099, A1S_ _0103, A1S_ _0104,
A1S_ _0105, A1S_ _0106, A1S_ _0107, A1S_ _0108, A1S_ _0109, A1S_ _0121, A1S_ _0128, A1S_ _0129,
A1S_ _0141, A1S_ _0148, A1S_ _0150, A1S_ _0151, A1S_ _0152, A1S_ _0153, A1S_ _0154, A1S_ _0155,
A1S_ _0156, A1S_ _0157, A1S_ _0177, A1S_ _0184, A1S_ _0200, A1S_ _0201, A1S_ _0209, A1S_ _0218,
A1S_ _0238, A1S_ _0239, A1S_ _0253, A1S_ _0257, A1S_ _0258, A1S_ _0269, A1S_ _0270, A1S_ _0279,
A1S_ _0286, A1S_ _0292, A1S_ _0302, A1S_ _0303, A1S_ _0304, A1S_ _0321, A1S_ _0322, A1S_ _0323,
A1S_ _0347, A1S_ _0365, A1S_ _0369, A1S_ _0370, A1S_ _0388, A1S_ _0408, A1S_ _0410, A1S_ _0427,
A1S_ _0429, A1S_ _0447, A1S_ _0448, A1S_ _0474, A1S_ _0480, A1S_ _0481, A1S_ _0482, A1S_ _0486,
A1S_ _0490, A1S_ _0491, A1S_ _0498, A1S_ _0526, A1S_ _0533, A1S_ _0534, A1S_ _0548, A1S_ _0549,
A1S_ _0566, A1S_ _0567, A1S_ _0568, A1S_ _0591, A1S_ _0594, A1S_ _0624, A1S_ _0625, A1S_ _0626,
A1S_ _0627, A1S_ _0629, A1S_ _0630, A1S_ _0631, A1S_ _0632, A1S_ _0633, A1S_ _0634, A1S_ _0635,
A1S_ _0639, A1S_ _0640, A1S_ _0641, A1S_ _0642, A1S_ _0643, A1S_ _0644, A1S_ _0645, A1S_ _0646,
A1S_ _0647, A1S_ _0649, A1S_ _0650, A1S_ _0651, A1S_ _0690, A1S_ _0691, A1S_ _0692, A1S_ _0695,
A1S_ _0698, A1S_ _0721, A1S_ _0731, A1S_ _0732, A1S_ _0771, A1S_ _0785, A1S_ _0786, A1S_ _0787,
A1S_ _0788, A1S_ _0818, A1S_ _0822, A1S_ _0846, A1S_ _0849, A1S_ _0850, A1S_ _0851, A1S_ _0852,
A1S_ _0853, A1S_ _0854, A1S_ _0855, A1S_ _0869, A1S_ _0877, A1S_ _0882, A1S_ _0883, A1S_ _0890,
A1S_ _0891, A1S_ _0901, A1S_ _0910, A1S_ _0911, A1S_ _0912, A1S_ _0913, A1S_ _0960, A1S_ _0965,
A1S_ _0973, A1S_ _0984, A1S_ _0996, A1S_ _0997, A1S_ _0999, A1S_ _1000, A1S_ _1004, A1S_ _1008,
A1S_ _1021, A1S_ _1026, A1S_ _1044, A1S_ _1063, A1S_ _1072, A1S_ _1079, A1S_ _1080, A1S_ _1088,
A1S_ _1089, A1S_ _1091, A1S_ _1092, A1S_ _1093, A1S_ _1094, A1S_ _1109, A1S_ _1113, A1S_ _1139,
A1S_ _1142, A1S_ _1182, A1S_ _1193, A1S_ _1195, A1S_ _1199, A1S_ _1227, A1S_ _1257, A1S_ _1258,
A1S_ _1261, A1S_ _1264, A1S_ _1265, A1S_ _1266, A1S_ _1267, A1S_ _1268, A1S_ _1269, A1S_ _1270,
A1S_ _1281, A1S_ _1317, A1S_ _1318, A1S_ _1319, A1S_ _1327, A1S_ _1334, A1S_ _1335, A1S_ _1336,
A1S_ _1337, A1S_ _1338, A1S_ _1339, A1S_ _1340, A1S_ _1341, A1S_ _1342, A1S_ _1343, A1S_ _1344, A1S_ _1345, A1S_ _1346 A1S_ _1347, A1S_ _1348 A1S_ _1349, A1S_ _1356 A1S_ _1366, A1S_ _1367,
A1S_ _1368, A1S_ _1369 A1S_ _1370, A1S_ _1372 A1S_ _1373, A1S_ _1374 A1S_ _1375, A1S_ _1376,
A1S_ _1377, A1S_ _1378 A1S_ _1379, A1S_ _1380 A1S_ _1396, A1S_ _1397 A1S_ _1428, A1S_ _1442,
A1S_ _1443, A1S_ _1450 A1S_ _1466, A1S_ _1467 A1S_ _1469, A1S_ _1470 A1S_ _1476, A1S_ _1490,
A1S_ _1491 , A1S_ _1492 A1S_ _1493, A1S_ _1498 A1S_ _1499, A1S_ _1505 A1S_ _1510, A1S_ _1523,
A1S_ _1528, A1S_ _1530 A1S_ _1532, A1S_ _1543 A1S_ _1579, A1S_ _1583 A1S_ _1601 , A1S_ _1608,
A1S_ _1609, A1S_ _1610 A1S_ _161 1 , A1S_ _1612 A1S_ _1613, A1S_ _1637 A1S_ _1638, A1S_ _1639,
A1S_ _1655, A1S_ _1692 A1S_ _1698, A1S_ _1699 A1S_ _1700, A1S_ _1701 A1S_ _1703, A1S_ _1705,
A1S_ ΙΊ ΙΊ, A1S_ _1719 A1S_ _1724, A1S_ _1729 A1S_ _1730, A1S_ ΪΊ31 A1S_ _1732, A1S_ _1734,
A1S_ _1735, A1S_ _1736 A1S_ _1737, A1S_ _1738 A1S_ _1742, A1S_ _1745 A1S_ _1754, A1S_ _1756,
A1S_ _1758, A1S_ _1775 A1S_ _1776, A1S_ _1790 A1S_ _1791 , A1S_ _1792 A1S_ _1794, A1S_ _1795,
A1S_ _1796, A1S_ _1797 A1S_ _1805, A1S_ _1806 A1S_ _181 1 , A1S_ _1830 A1S_ _1834, A1S_ _1835,
A1S_ _1836, A1S_ _1837 A1S_ _1838, A1S_ _1839 A1S_ _1840, A1S_ _1841 A1S_ _1854, A1S_ _1855,
A1S_ _1856, A1S_ _1857 A1S_ _1858, A1S_ _1859 A1S_ _1860, A1S_ _1861 A1S_ _1862, A1S_ _1863,
A1S_ _1864, A1S_ _1865 A1S_ _1866, A1S_ _1879 A1S_ _1880, A1S_ _1887 A1S_ _1908, A1S_ _1924,
A1S_ _1925, A1S_ _1926 A1S_ _1935, A1S_ _1940 A1S_ _1942, A1S_ _1948 A1S_ _1951 , A1S_ _1984,
A1S_ _1996, A1S_ _2041 A1S_ _2042, A1S_ _2052 A1S_ _2053, A1S_ _2068 A1S_ _2072, A1S_ _2081 ,
A1S_ _2084, A1S_ _2098 A1S_ _2100, A1S_ _2101 A1S_ _2102, A1S_ _2148 A1S_ _2149, A1S_ _2150,
A1S_ _2163, A1S_ _2166 A1S_ _2167, A1S_ _2190 A1S_ _2191 , A1S_ _2202 A1S_ _2203, A1S_ _2207,
A1S_ _2209, A1S_ _2218 A1S_ _2221 , A1S_ _2225 A1S_ _2232, A1S_ _2234 A1S_ _2248, A1S_ _2279,
A1S_ _2280, A1S_ _2288 A1S_ _2289, A1S_ _2340 A1S_ _2341 , A1S_ _2342 A1S_ _2348, A1S_ _2353,
A1S_ _2354, A1S_ _2415 A1S_ _2416, A1S_ _2417 A1S_ _2418, A1S_ _2419 A1S_ _2424, A1S_ _2425,
A1S_ _2431 , A1S_ _2435 A1S_ _2443, A1S_ _2449 A1S_ _2450, A1S_ _2451 A1S_ _2452, A1S_ _2475,
A1S_ _2501 , A1S_ _2509 A1S_ _2510, A1S_ _2514 A1S_ _2531 , A1S_ _2532 A1S_ _2533, A1S_ _2535,
A1S_ _2601 , A1S_ _2602 A1S_ _2633, A1S_ _2662 A1S_ _2670, A1S_ _2671 A1S_ _2672, A1S_ _2688,
A1S_ _2692, A1S_ _2694 A1S_ _2695, A1S_ _2696 A1S_ _2701 , A1S_ _271 1 A1S_ _2722, A1S_ _2724,
A1S_ _2738, A1S_ _2740 A1S_ _2741 , A1S_ _2748 A1S_ _2753, A1S_ _2755 A1S_ _2758, A1S_ _2761 ,
A1S_ _2762, A1S_ _2769 A1S_ _2773, A1S_ _2774 A1S_ _2785, A1S_ _2788 A1S_ _2789, A1S_ _2793,
A1S_ _2809, A1S_ _2814 A1S_ _2815, A1S_ _2820 A1S_ _2823, A1S_ _2847 A1S_ _2848, A1S_ _2849,
A1S_ _2852, A1S_ _2860 A1S_ _2904, A1S_ _2905 A1S_ _2906, A1S_ _291 1 A1S_ _2913, A1S_ _2919,
A1S_ _2924, A1S_ _2928 A1S_ _2939, A1S_ _2946 A1S_ _2956, A1S_ _3013 A1S_ _3014, A1S_ _3025, A1S_ _3040, A1S_ _3043, A1S_ _3049, A1S_ _3050, A1S_ _3051, A1S_ _3074, A1S_ _3084, A1S_ _3110,
A1S_ _3120, A1S_ _3121, A1S_ _3122, A1S_ _3128, A1S_ _3129, A1S_ _3130, A1S_ _3131, A1S_ _3132,
A1S_ _3133, A1S_ _3134, A1S_ _3135, A1S_ _3144, A1S_ _3174, A1S_ _3180, A1S_ _3195, A1S_ _3207,
A1S_ _3222, A1S_ _3224, A1S_ _3225, A1S_ _3231, A1S_ _3232, A1S_ _3236, A1S_ _3238, A1S_ _3248,
A1S_ _3250, A1S_ _3268, A1S_ _3269, A1S_ _3273, A1S_ _3278, A1S_ _3290, A1S_ _3297, A1S_ _3298,
A1S_ _3300, A1S_ _3301, A1S_ _3309, A1S_ _3338, A1S_ _3342, A1S_ _3355, A1S_ _3364, A1S_ _3377,
A1S_ _3397, A1S_ _3398, A1S_ _3402, A1S_ _3403, A1S_ _3404, A1S_ _3405, A1S_ _3406, A1S_ _3407,
A1S_ _3410, A1S_ _3413, A1S_ _3414, A1S_ _3415, A1S_ _3416, A1S_ _3418, A1S_ _3431, A1S_ _3450,
A1S_ _3451, A1S_ _3458, A1S_ _3460, A1S_ _3481, A1S_ _3487, A1S_ _3491, A1S_ _3494, A1S_ _3498,
A1S_ _3506, A1S_ _3508, A1S_ _3509, A1S_ _3514, A1S_ _3518, A1S_ _3519, A1S_ _3520, A1S_ _3521,
A1S_ _3522, A1S_ _3523, A1S_ _3524, A1S_ _3526, A1S_ _3528, A1S_ _3530, A1S_ _3531, A1S_ _3532,
A1S_ _3533, A1S_ _3534, A1S_ _3535, A1S_ _3537, A1S_ _3538, A1S_ _3539, A1S_ _3540, A1S_ _3541,
A1S_ _3542, A1S_ _3543, A1S_ _3544, A1S_ _3545, A1S_ _3546, A1S_ _3547, A1S_ _3548, A1S_ _3549,
A1S_ _3550, A1S_ _3552, A1S_ _3553, A1S_ _3554, A1S_ _3568, A1S_ _3569, A1S_ _3578, A1S_ _3582,
A1S_ _3586, A1S_ _3587, A1S_ _3591, A1S_ _3597, A1S_ _3599, A1S_ _3600, A1S_ _3602, A1S_ _3611,
A1S_ _3619, A1S_ _3621, A1S_ _3624, A1S_ _3629, A1S_ _3633, A1S_ _3640, A1S_ J3641, A1S_ _3644,
A1S_ _3647, A1S_ _3651, A1S_ _3652, A1S_ _3659, A1S_ _3663, A1S_ _3667, A1S_ _3673, A1S_ _3679,
A1S_ _3701, A1S_ _3707, A1S_ _3709, A1S_ _3713, A1S_ _3715, A1S_ _3717, A1S_ _3732, A1S_ _3735,
A1S_ _3738, A1S_ _3740, A1S_ _3741, A1S_ _3742, A1S_ _3759, A1S_ _3774, A1S_ _3779, A1S_ _3787,
A1S_ _3788, A1S_ _3794, A1S_ _3801, A1S_ _3802, A1S_ _3806, A1S_ _3809, A1S_ _3811, A1S_ _3813,
A1S_ _3814, A1S_ _3816, A1S_ _3817, A1S_ _3823, A1S_ _3829, A1S_ _3831, A1S_ _3832, A1S_ _3836,
A1S_ _3840, A1S_ _3846, A1S_ _3857, A1S_ _3862, A1S_ _3868, A1S_ _3870, A1S_ _3880, A1S_ _3884,
A1S_ _3886, A1S_ _3887, A1S_ _3891, A1S_ _3894, A1S_ _3898, A1S_ _3907, A1S_ _3908, A1S_ _3909,
A1S_ _3912, A1S_ _3914, or A1S_3915.
[0068] In certain embodiments of the methods, such as in methods in which the OM-stress responsive gene is downregulated in response to an outer membrane stress, the OM stress- responsive gene is A1S_0103, A1S_0645, A1S_1266, A1S_1268, A1S_1335, A1S_1336, A1S_1337, A1S_1338, A1S_1339, A1S_1340, A1S_1341, A1S_1342, A1S_1343, A1S_1344, A1S_1345, A1S_1791, A1S_1792, A1S_1794, A1S_1796, A1S_1835, A1S_1836, A1S_1837, A1S_1838, A1S_1839, A1S_2449, A1S_2450, A1S_2452, A1S_3540, A1S_3541, A1S_3542, A1S_3543, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, A1S_3809, or A1S_3908. In certain embodiments, the OM stress-responsive gene is A1S_1336, A1S_1836, A1S_1838, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, or A1S_3809.
[0069] In certain embodiments, the OM-stress responsive gene that is operably linked to the reporter molecule in the reporter vector comprised in the reporter microorganism is upregulated in response to an outer membrane stress.
[0070] In certain embodiments of the methods, such as in methods in which the OM-stress responsive gene is upregulated in response to an outer membrane stress, the OM stress- responsive gene is A1S_0012, A1S_0023, A1S_0027, A1S_0028, A1S_0029, A1S_0030,
A1S_ _0031, A1S_ _0032, A1S_ _0033, A1S_ _0037, A1S_ _0040, A1S_ _0041, A1S_ _0044, A1S_ _0066,
A1S_ _0092, A1S_ _0093, A1S_ _0109, A1S_ _0110, A1S_ _0112, A1S_ _0113, A1S_ _0114, A1S_ _0115,
A1S_ _0116, A1S_ _0117, A1S_ _0118, A1S_ _0126, A1S_ _0158, A1S_ _0170, A1S_ _0175, A1S_ _0178,
A1S_ _0189, A1S_ _0224, A1S_ _0245, A1S_ _0256, A1S_ _0276, A1S_ _0293, A1S_ _0301, A1S_ _0309,
A1S_ _0310, A1S_ _0332, A1S_ _0333, A1S_ _0363, A1S_ _0372, A1S_ _0376, A1S_ _0391, A1S_ _0392,
A1S_ _0401, A1S_ _0441, A1S_ _0462, A1S_ _0463, A1S_ _0464, A1S_ _0465, A1S_ _0466, A1S_ _0494,
A1S_ _0508, A1S_ _0509, A1S_ _0510, A1S_ _0511, A1S_ _0512, A1S_ _0514, A1S_ _0516, A1S_ _0518,
A1S_ _0519, A1S_ _0520, A1S_ _0521, A1S_ _0522, A1S_ _0523, A1S_ _0527, A1S_ _0535, A1S_ _0536,
A1S_ _0537, A1S_ _0538, A1S_ _0547, A1S_ _0559, A1S_ _0561, A1S_ _0562, A1S_ _0563, A1S_ _0564,
A1S_ _0566, A1S_ _0567, A1S_ _0568, A1S_ _0570, A1S_ _0624, A1S_ _0630, A1S_ _0631, A1S_ _0633,
A1S_ _0634, A1S_ _0640, A1S_ _0641, A1S_ _0642, A1S_ _0643, A1S_ _0644, A1S_ _0645, A1S_ _0646,
A1S_ _0647, A1S_ _0650, A1S_ _0663, A1S_ _0664, A1S_ _0665, A1S_ _0666, A1S_ _0667, A1S_ _0669,
A1S_ _0670, A1S_ _0671, A1S_ _0673, A1S_ _0677, A1S_ _0680, A1S_ _0683, A1S_ _0714, A1S_ _0717,
A1S_ _0718, A1S_ _0719, A1S_ _0736, A1S_ _0738, A1S_ _0739, A1S_ _0749, A1S_ _0770, A1S_ _0772,
A1S_ _0779, A1S_ _0780, A1S_ _0781, A1S_ _0800, A1S_ _0804, A1S_ _0830, A1S_ _0831, A1S_ _0832,
A1S_ _0834, A1S_ _0835, A1S_ _0861, A1S_ _0884, A1S_ _0889, A1S_ _0929, A1S_ _0930, A1S_ _0931,
A1S_ _0932, A1S_ _0935, A1S_ _0945, A1S_ _0959, A1S_ _0980, A1S_ _1003, A1S_ _1009, A1S_ _1010,
A1S_ _1027, A1S_ _1028, A1S_ _1030, A1S_ _1031, A1S_ _1049, A1S_ _1081, A1S_ _1106, A1S_ _1107,
A1S_ _1120, A1S_ _1121, A1S_ _1123, A1S_ _1132, A1S_ _1133, A1S_ _1134, A1S_ _H39, A1S_ _1143,
A1S_ _1145, A1S_ _1146, A1S_ _1148, A1S_ _1149, A1S_ _1150, A1S_ _1151, A1S_ _1152, A1S_ _1153,
A1S_ _1155, A1S_ _1156, A1S_ _1157, A1S_ _1158, A1S_ _1159, A1S_ _1160, A1S_ _1161, A1S_ _1162,
A1S_ _1163, A1S_ _1164, A1S_ _1165, A1S_ _1167, A1S_ _1171, A1S_ _1172, A1S_ _1173, A1S_ _1180,
A1S_ _1184, A1S_ _1186, A1S_ _1198, A1S_ _1202, A1S_ _1203, A1S_ _1223, A1S_ _1224, A1S_ _1225, A1S_ _1230, A1S_ _1236 A1S_ _1237, A1S_ _1248 A1S_ _1255, A1S_ _1274 A1S_ _1286, A1S_ _1359,
A1S_ _1360, A1S_ _1361 A1S_ _1362, A1S_ _1363 A1S_ _1383, A1S_ _1384 A1S_ _1385, A1S_ _1386,
A1S_ _1387, A1S_ _1393 A1S_ _1404, A1S_ _1407 A1S_ _1422, A1S_ _1454 A1S_ _1472, A1S_ _1481,
A1S_ _1494, A1S_ _1512 A1S_ _1515, A1S_ _1526 A1S_ _1535, A1S_ _1539 A1S_ _1566, A1S_ _1567,
A1S_ _1569, A1S_ _1583 A1S_ _1584, A1S_ _1585 A1S_ _1589, A1S_ _1590 A1S_ _1593, A1S_ _1595,
A1S_ _1596, A1S_ _1617 A1S_ _1630, A1S_ _1644 A1S_ _1645, A1S_ _1647 A1S_ _1648, A1S_ _1649,
A1S_ _1651, A1S_ _1655 A1S_ _1658, A1S_ _1662 A1S_ _1666, A1S_ _1667 A1S_ _1669, A1S_ _1677,
A1S_ _1680, A1S_ _1681 A1S_ _1687, A1S_ _1735 A1S_ _1741, A1S_ _1743 A1S_ _1744, A1S_ _1750,
A1S_ _1751, A1S_ _1752 A1S_ _1760, A1S_ _1762 A1S_ _1767, A1S_ _1778 A1S_ _1813, A1S_ _1827,
A1S_ _1829, A1S_ _1831 A1S_ _1843, A1S_ _1876 A1S_ _1909, A1S_ _1928 A1S_ _1929, A1S_ _1934,
A1S_ _1952, A1S_ _1955 A1S_ _1956, A1S_ _1957 A1S_ _1959, A1S_ _1960 A1S_ _1961, A1S_ _1962,
A1S_ _1963, A1S_ _1979 A1S_ _1986, A1S_ _1987 A1S_ _1988, A1S_ _2006 A1S_ _2026, A1S_ _2033,
A1S_ _2034, A1S_ _2035 A1S_ _2036, A1S_ _2038 A1S_ _2039, A1S_ _2061 A1S_ _2074, A1S_ _2079,
A1S_ _2082, A1S_ _2092 A1S_ _2093, A1S_ _2106 A1S_ _2139, A1S_ _2140 A1S_ _2141, A1S_ _2142,
A1S_ _2146, A1S_ _2157 A1S_ _2158, A1S_ _2160 A1S_ _2161, A1S_ _2162 A1S_ _2178, A1S_ _2179,
A1S_ _2183, A1S_ _2186 A1S_ _2195, A1S_ _2230 A1S_ _2247, A1S_ _2252 A1S_ _2257, A1S_ _2258,
A1S_ _2259, A1S_ _2262 A1S_ _2271, A1S_ _2272 A1S_ _2273, A1S_ _2283 A1S_ _2285, A1S_ _2298,
A1S_ _2311, A1S_ _2315 A1S_ _2325, A1S_ _2326 A1S_ _2330, A1S_ _2331 A1S_ _2366, A1S_ _2367,
A1S_ _2382, A1S_ _2387 A1S_ _2389, A1S_ _2395 A1S_ _2396, A1S_ _2414 A1S_ _2434, A1S_ _2445,
A1S_ _2446, A1S_ _2447 A1S_ _2448, A1S_ _2454 A1S_ _2455, A1S_ _2456 A1S_ _2458, A1S_ _2459,
A1S_ _2463, A1S_ _2480 A1S_ _2489, A1S_ _2503 A1S_ _2504, A1S_ _2508 A1S_ _2542, A1S_ _2543,
A1S_ _2552, A1S_ _2553 A1S_ _2555, A1S_ _2557 A1S_ _2558, A1S_ _2573 A1S_ _2577, A1S_ _2578,
A1S_ _2580, A1S_ _2586 A1S_ _2588, A1S_ _2593 A1S_ _2611, A1S_ _2612 A1S_ _2613, A1S_ _2624,
A1S_ _2650, A1S_ _2651 A1S_ _2654, A1S_ _2656 A1S_ _2660, A1S_ _2664 A1S_ _2668, A1S_ _2675,
A1S_ _2676, A1S_ _2677 A1S_ _2678, A1S_ _2684 A1S_ _2705, A1S_ _2729 A1S_ _2734, A1S_ _2756,
A1S_ _2768, A1S_ _2786 A1S_ _2798, A1S_ _2801 A1S_ _2807, A1S_ _2826 A1S_ _2827, A1S_ _2828,
A1S_ _2839, A1S_ _2863 A1S_ _2882, A1S_ _2883 A1S_ _2884, A1S_ _2885 A1S_ _2889, A1S_ _2892,
A1S_ _2893, A1S_ _2942 A1S_ _2943, A1S_ _2953 A1S_ _2959, A1S_ _2960 A1S_ _2968, A1S_ _2976,
A1S_ _2992, A1S_ _3011 A1S_ _3026, A1S_ _3027 A1S_ _3034, A1S_ _3035 A1S_ _3047, A1S_ _3048,
A1S_ _3099, A1S_ _3100 A1S_ _3101, A1S_ _3104 A1S_ _3105, A1S_ _3114 A1S_ _3115, A1S_ _3116,
A1S_ _3117, A1S_ _3124 A1S_ _3125, A1S_ _3126 A1S_ _3127, A1S_ _3139 A1S_ _3146, A1S_ _3147, A1S_ _3175, A1S_ _3206, A1S_ _3224, A1S_ _3253, A1S_ _3259, A1S_ _3280, A1S_ _3281, A1S_ _3295,
A1S_ _3317, A1S_ _3326, A1S_ _3339, A1S_ _3360, A1S_ _3361, A1S_ _3367, A1S_ _3368, A1S_ _3371,
A1S_ _3375, A1S_ _3376, A1S_ _3392, A1S_ _3411, A1S_ _3412, A1S_ _3463, A1S_ _3466, A1S_ _3468,
A1S_ _3469, A1S_ _3471, A1S_ _3479, A1S_ _3480, A1S_ _3486, A1S_ _3492, A1S_ _3493, A1S_ _3494,
A1S_ _3499, A1S_ _3510, A1S_ _3512, A1S_ _3518, A1S_ _3522, A1S_ _3523, A1S_ _3533, A1S_ _3534,
A1S_ _3535, A1S_ _3539, A1S_ _3540, A1S_ _3541, A1S_ _3542, A1S_ _3543, A1S_ _3544, A1S_ _3545,
A1S_ _3546, A1S_ _3548, A1S_ _3552, A1S_ _3553, A1S_ _3558, A1S_ _3559, A1S_ _3562, A1S_ _3563,
A1S_ _3567, A1S_ _3570, A1S_ _3577, A1S_ _3580, A1S_ _3580, A1S_ _3585, A1S_ _3586, A1S_ _3594,
A1S_ _3595, A1S_ _3596, A1S_ _3601, A1S_ _3602, A1S_ _3603, A1S_ _3604, A1S_ _3605, A1S_ _3606,
A1S_ _3607, A1S_ _3608, A1S_ _3609, A1S_ _3610, A1S_ _3611, A1S_ _3612, A1S_ _3613, A1S_ _3614,
A1S_ _3617, A1S_ _3618, A1S_ _3621, A1S_ _3630, A1S_ _3632, A1S_ _3634, A1S_ _3635, A1S_ _3636,
A1S_ _3637, A1S_ _3642, A1S_ _3645, A1S_ _3649, A1S_ _3654, A1S_ _3658, A1S_ _3661, A1S_ _3662,
A1S_ _3666, A1S_ _3682, A1S_ _3686, A1S_ _3687, A1S_ _3688, A1S_ _3694, A1S_ _3695, A1S_ _3697,
A1S_ _3704, A1S_ _3707, A1S_ _3708, A1S_ _3712, A1S_ _3716, A1S_ _3725, A1S_ _3726, A1S_ _3727,
A1S_ _3728, A1S_ _3736, A1S_ _3738, A1S_ _3739, A1S_ _3740, A1S_ _3750, A1S_ _3752, A1S_ _3760,
A1S_ _3768, A1S_ _3769, A1S_ _3770, A1S_ _3771, A1S_ _3772, A1S_ _3773, A1S_ _3776, A1S_ _3777,
A1S_ _3778, A1S_ _3782, A1S_ _3783, A1S_ _3786, A1S_ _3789, A1S_ _3790, A1S_ _3791, A1S_ _3792,
A1S_ _3797, A1S_ _3810, A1S_ _3818, A1S_ _3820, A1S_ _3835, A1S_ _3837, A1S_ _3840, A1S_ _3842,
A1S_ _3844, A1S_ _3862, A1S_ _3864, A1S_ _3865, A1S_ _3866, A1S_ _3867, A1S_ _3868, A1S_ _3873,
A1S_ _3875, A1S_ _3879, A1S_ _3889, A1S_ _3900, A1S_ _3901, A1S_ _3902, A1S_ _3908, or
A1S_3911.
[0071] In certain embodiments, the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or A1S_3127. In certain embodiments, the OM stress-responsive gene is
A1S_0032, A1S_2885, or A1S_2889.
[0072] In certain embodiments of any of the methods, the contacting is carried out in suspension, in an array, or in a microdroplet. In certain embodiments, the reporter
microorganism of (a) is encapsulated in a microdroplet with a cell.
[0073] In some embodiments of any of the provided methods, the agent is a candidate antibacterial agent, such as an agent known to cause or suspected of causing OM stress to the reporter microorganism and/or that may impact the integrity or biogenesis of the outer membrane of the reporter microorganism. In certain embodiments, the agent is a small molecule compound, a peptide or a protein. In certain embodiments, the agent is an antibiotic. In certain embodiments, the agent is an antibody or antigen-binding fragment thereof. In certain embodiments, the agent is an antibody and the cell is an antibody-producing cell.
[0074] In certain embodiments, the cell is a microorganism. In certain embodiments, the cell is a fungal or bacterial cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a B cell. In certain embodiments, the cell is a plasma cell or a plasmablast.
[0075] In certain embodiments, the cell is obtained from a donor that has been exposed to a target microorganism or an epitope-comprising fragment of a target microorganism or a variant thereof. In certain embodiments, the donor has been immunized or infected with a target microorganism or an epitope-comprising fragment of a target microorganism or a variant thereof. In certain embodiments, the donor is an immunized animal or an infected animal. In certain embodiments, the donor is a mammal or a bird. In certain embodiments, the donor is a human, a mouse or a chicken. In certain embodiments, the donor is a human donor who was infected by the target microorganism. In certain embodiments, the donor is a genetically modified non-human animal that produces partially human or fully human antibodies.
[0076] In certain embodiments, the microdroplet is generated by a microfluidics-based method. In certain embodiments, the microdroplet comprises agarose, carrageenan, alginate, alginate-polylysine, collagen, cellulose, methylcellulose, gelatin, chitosan, extracellular matrix, dextran, starch, inulin, heparin, hyaluronan, fibrin, polyvinyl alcohol, poly(N-vinyl-2- pyrrolidone), polyethylene glycol, poly(hydroxyethyl methacrylate), acrylate polymers and sodium polyacrylate, polydimethyl siloxane, cis- polyisoprene, Puramatrix™, poly- divenylbenzene, polyurethane, polyacrylamide, or combinations thereof. In certain
embodiments, the microdroplet comprises agarose.
[0077] In certain embodiments, the method further comprises: (c) isolating the cell from the microdroplet in which there is a change in the level of the detectable signal from the reporter molecule compared to in the absence of exposing the reporter microorganism to the agent. In certain embodiments, the isolating is carried out using a micromanipulator or an automated sorter. In certain embodiments, the method further comprises identifying the agent produced by the cell. In certain embodiments, the identifying comprises determining the sequence of the agent, optionally using single cell PCR and nucleic acid sequencing.
[0078] In certain embodiments, the method further comprises: (c) isolating the microdroplet comprising the cell producing the identified agent. In certain embodiments, the method further comprises: (d) isolating polynucleotides encoding the agent, wherein the agent is a polypeptide. In certain embodiments, the method further comprises: (e) determining the sequence of the nucleic acids encoding the polypeptide. In certain embodiments, the method further comprises prior to (a) introducing the reporter polynucleotide or reporter vector described herein into a host microorganism.
[0079] In certain embodiments, the host microorganism is a bacterium. In certain embodiments, the host microorganism is a Gram negative bacterium. In certain embodiments, the host microorganism is a proteobacterium. In certain embodiments, the host microorganism is a species of Acinetobacter, Bdellovibrio, Burkholderia, Chlamydia, Enterobacter,
Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella, Neisseria, Pantoea, Pseudomonas, Salmonella, Shigella, Stenotrophomonas, Vibrio, or Yersinia.
[0080] In certain embodiments, the bacterium Acinetobacter apis, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii,
Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter gerneri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus, Acinetobacter harbinensis, Acinetobacter indicus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter kookii, Acinetobacter Iwoj ii, Acinetobacter nectaris, Acinetobacter nosocomialis, Acinetobacter pakistanensis, Acinetobacter parvus, Acinetobacter pitii, Acinetobacter pittii, Acinetobacter puyangensis, Acinetobacter qingfengensis,
Acinetobacter radioresistans, Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii, Acinetobacter soli, Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis,
Acinetobacter venetianus, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Vibrio cholera, or Yersinia pestis. In certain embodiments, the bacterium is Acinetobacter baumannii. [0081] In certain embodiments, the agent is a drug. In certain embodiments, the drug is a small molecule compound, a peptide or a protein. In certain embodiments, the drug is an antibiotic. In certain embodiments, the drug is an antibody or antigen-binding fragment thereof. In certain embodiments, the drug is an antibody and the cell is an antibody-producing cell.
[0082] Provided herein are agents identified by any of the methods described herein.
Brief Description of the Drawings
[0083] Fig. 1 shows growth curves of a modified A. baumannii strain that is depleted for BamA unless exogenous arabinose is added. The growth curves compare growth in the presence or absence of added arabinose for up to 9 hours. Circles indicate the time points at which the cells were back-diluted to maintain cells in an exponential growth phase. Rectangles indicate the time points at which samples were taken for analysis by RNA-Seq for comparing differences in gene expression caused by outer membrane stress induced by BamA depletion as described in subsequent Examples and Figs.
[0084] Fig. 2A-2C show differential gene expression in A. baumannii with or without BamA depletion as determined by RNA-Seq with counts normalized as fragments per kilobase per million reads (FPKM). Fig. 2A depicts results for replicate transcript datasets from A.
baumannii cells without BamA depletion cultured in the presence of arabinose for 6 hours. Fig. 2B depicts results of replicate transcript datasets from A. baumannii cells with BamA depletion cultured in the absence of added arabinose for 6 hours. Fig. 2C shows differences in gene expression when comparing BamA-depleted (+Ara) and non-BamA-depleted (-Ara) cells.
[0085] Fig. 3A-3C depicts log fold differences in gene expression in A. baumannii over time in BamA depleted cell cultures (incubated without arabinose; -Ara) compared to non-BamA- depleted cell cultures (incubated in the presence of arabinose; +Ara). Fig. 3 A compares the abundance of gene transcripts after 2 hours +Ara or -Ara. Fig. 3B compares the abundance of transcripts after 4 hours -i-Ara or - Ara. Fig. 3C compares the abundance of transcripts after 6 hours -i-Ara or -Ara.
[0086] Fig. 4 shows the log fold change in gene expression of exemplary transcripts after 2, 4, and 6 hours of BamA depletion in A. baumannii compared to expression of the transcript in A. baumannii expressing BamA. [0087] Fig. 5A-5B show A. baumannii gene transcripts whose expression was increased by at least 10-fold after 6 hrs of BamA depletion. A heatmap reflecting the relative change in expression for each gene transcript at 2, 4, or 6 hours of BamA depletion is shown. Predicted functions for encoded protein products are given on the right.
[0088] Fig. 6A-6D show A. baumannii gene transcripts whose expression decreased by at least 10-fold after 6 hrs of BamA depletion. A heatmap reflecting the relative change in expression for each gene transcript at 2, 4 or 6 hours of BamA depletion is shown. Predicted functions for encoded protein products are given on the right.
[0089] Fig. 7A-7D show differences in gene expression when comparing non-PMBN-treated cells to cells treated with PMBN for various concentrations and times. FIG. 7A shows gene expression in non-PMBN-treated cells compared to cells treated with 25 μg/mL for 10 minutes. FIG. 7B shows gene expression in non-PMBN-treated cells compared to cells treated with 250 μg/mL for 10 minutes. FIG. 7C shows gene expression in non-PMBN-treated cells compared to cells treated with 25 μg/mL for 30 minutes. FIG. 7D shows gene expression in non-PMBN- treated cells compared to cells treated with or 250 μg/mL for 30 minutes. Transcripts (genes) whose expression changed by at least 10-fold after treatment with PMBN at the indicated concentration and time are shown in black.
[0090] Fig. 8 shows the log fold change after treatment of A. baumannii cells with 25 μg/mL PMBN (gray) or 250 μg/mL PMBN (black) in exemplary gene transcripts.
[0091] Fig. 9 shows the backbone vector pACH106 which encodes replication factors and selective markers necessary for autonomous replication in Acinetobacter baumannii and maintenance by antibiotic selection.
[0092] Fig. 10A shows a histogram of fluorescence intensities induced in PAis_2885-sfGFP reporter cells after treatment with PMBN. Fig. 10B shows fluorescent micrographs of PAIS_28S9- sfGFP reporter cells after treatment in the presence or absence of PMBN.
[0093] Fig. 11A-11B shows fluorescent FACs histograms of PAis_28S5-sfGFP reporter cells after treatment with PMBN. In FIG. 11 A, cells were grown in microtiter dishes without shaking or aeration. In Fig. 1 IB, cells were grown in culture flasks with aeration.
[0094] Fig. 12A-12B depict fluorescent intensities of A. baumannii PAisj»32-sfGFP and PAis_2889-sfGFP reporter cells that are depleted for BamA, an OM biogenesis factor, unless grown in the presence of arabinose. Fig. 12A depicts mean fluorescent intensities of the reporter cells in the presence or absence of arabinose. Fig. 12B shows a representative flow cytometry fluorescence histogram of PAis_2889-sfGFP reporter cells after growth with or without arabinose for 3 hrs.
[0095] Fig. 13A-13B show flow cytometry fluorescence histograms of a multi-drug resistant strain of A. baumannii expressing the OM stress transcriptional reporter constructs after culture in the presence of PMBN or vancomycin (vane). Fig. 13A depicts flow cytometry fluorescent histograms of PAis_2885-sfGFP reporter cells. Fig. 13B depicts flow cytometry fluorescent histograms of PAis_2889-sfGFP reporter cells.
[0096] Fig. 14A-14B show flow cytometry fluorescence histoGrams of A. baumannii reporter cells containing either PAisjxm-sfGFP (Fig. 14A) or PAis_2885-sfGFP (Fig. 14B) after culture in the presence of ACHN-975, an antibiotic that inhibits synthesis of lipopolysaccharide (LPS) by inhibiting the activity of the LpxC enzyme.
[0097] Fig. 15A shows flow cytometry fluorescence histograms of A. baumannii reporter cells containing PAis_oo32-sfGFP after culture in the presence of ACHN-975, an antibiotic that inhibits synthesis of lipopolysaccharide (LPS) by inhibiting the activity of the LpxC enzyme; colistin, a broad-spectrum antibiotic that binds and perturbs the LPS leaflet of the OM; and EDTA, a chelating agent that disrupts LPS layer by stripping away divalent cations that stabilize the OM by neutralizing the negative charges in LPS.
[0098] Fig. 15B shows flow cytometry fluorescence histograms of A. baumannii reporter cells containing PAis_2885-sfGFP after culture in the presence of PMBN, EDTA, and EDTA with SDS, a detergent that acts synergistically with EDTA to permeabilize the destabilized OM.
[0099] Fig. 16 depicts one embodiment of a screening assay for identifying anti-bacterial molecules that induce outer membrane stress. In the screening assay, animals are immunized with Acinetobacter cells to generate antibody- secreting B cells, isolated B cells and reporter A. baumannii bacterial cells (containing an outer membrane stress transcriptional reporter construct in which a regulatory region of a gene induced by outer membrane stress is fused to a detectable moiety, such as GFP, as described) are mixed and co-encapsulated to form microparticles using microfluidics, and the microparticles are screened for activated reporter cells expressing a detectable signal (e.g. fluorescence). [0100] Fig. 17A-17B show fluorescent micrograph images of A. baumannii reporter cells containing PAis_28S9-sfGFP encapsulated in agarose microparticles after treatment of the reporter cells either with colistin (Fig. 17B) or without colistin (Fig. 17A).
[0101] Fig. 18A-18C show micrographs of co-encapsulated A. baumannii reporter cells and antibody- secreting murine B cells (plasmablasts) after a one-hour incubation. Fig. 18A is a brightfield image of the co-encapsulated agarose microparticles. Fig. 18B is a fluorescent (FITC) image of the co-encapsulated agarose microparticles. Fig. 18C is an overlay of the brightfield and fluorescent images.
[0102] Fig. 19A-19D show micrographs of a mixed population of A. baumannii cells expressing inducible mRuby2 or mClover3. Fig. 19A is a brightfield image of the mixed population of A. baumannii cells. Fig. 19B is a fluorescent (RFP) image of the mixed population of A. baumannii cells. Cells expressing mRuby2 can be seen. Fig. 19C is a fluorescent (GFP) image of the mixed population of A. baumannii cells. Cells expressing mClover3 can be seen. Fig. 19D is an overlay of the brightfield and fluorescent images.
[0103] Fig. 20A depicts mean fluorescent intensities of PAis_oo32-sfGFP, PAis_2889-sfGFP, or PAIS 2885-sfGFP reporter cells in the presence of various concentrations of PMBN. Fig. 20B depicts mean fluorescent intensities of cells expressing Ptac-sfGFP (an IPTG-inducible reporter) in the presence of various concentrations of IPTG.
[0104] Fig. 21A-F shows flow cytometry fluorescence histograms of A. baumannii reporter cells containing PAis_2889-sfGFP after culture without serum (Fig. 21A-C) or with serum (Fig. 21D-F) and in media only (Fig. 21 A, D) or in the presence of an IgG isotype control (Fig. 2 IB, E) or a monoclonal antibody that binds the OM (Fig. 21C, F). In Fig. 21F, the dashed circle highlights a population of fluorescent cells.
Detailed Description
[0105] Provided herein are reporter polynucleotides containing a sequence comprising a regulatory region of an outer membrane (OM) stress-responsive gene of an Acinetobacter bacterium operably linked to a sequence encoding a reporter molecule. Also provided are reporter vectors, genetically engineered reporter microorganisms, and uses thereof. The provided reporter polynucleotides, reporter vectors and engineered reporter microorganisms are useful for detecting outer membrane (OM) stress, including in connection with screening a plurality of candidate molecules or agents to identify molecules or agents that induce and/or modulate outer membrane stress and/or for determining or assessing if a microorganism is drug resistant.
[0106] Drug resistance for pathogenic bacteria is a growing problem and researchers need new tools and methods for identifying specific new treatments. This problem is especially difficult of Gram-negative bacteria which have a tough outer membrane which can block access of conventional antibiotics.
[0107] The provided reporter polynucleotides, reporter vectors and reporter microorganisms can be used in methods for the rapid identification or characterization of agents, including drug or other therapeutic candidates, to address a multitude of infectious disease threats.
I. DEFINITIONS
[0108] Unless defined otherwise, all terms of art, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art.
[0109] As used herein, the term "effective amount" refers to at least an amount effective, at dosages and for periods of time necessary, to achieve the desired result, e.g., an enhanced immune response to an antigen, a decrease in tumor growth or metastasis, or a reduction in tumor size. An effective amount can be provided in one or more administrations.
[0110] As used herein, the singular form "a", "an", and "the" includes plural references unless indicated otherwise.
[0111] Reference to "about" a value or parameter herein refers to the usual error range for the respective value readily known to the skilled person in this technical field. In particular embodiments, reference to about refers to a range within 10% higher or lower than the value or parameter, while in other embodiments, it refers to a range within 5% or 20% higher or lower than the value or parameter. Reference to "about" a value or parameter herein includes (and describes) aspects that are directed to that value or parameter per se. For example, description referring to "about X" includes description of "X." [0112] As used herein, the term "modulating" means changing, and includes positive modulating, such as "increasing," "enhancing," "inducing" or "stimulating," as well as negative modulating such as "decreasing," "inhibiting" or "reducing," typically in a statistically significant or a physiologically significant amount as compared to a control. An "increased," "stimulated" or "enhanced" amount is typically a "statistically significant" amount, and may include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the amount produced by no treatment as described herein or by a control treatment, including all integers in between. A "decreased," "inhibited" or "reduced" amount is typically a "statistically significant" amount, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18% , 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease in the amount produced by no treatment as described herein or by a control treatment, including all integers in between.
[0113] By "statistically significant," it is meant that the result was unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art.
[0114] Commonly used measures of significance include the p-value, which is the frequency or probability with which the observed event would occur, if the null hypothesis were true. If the obtained p-value is smaller than the significance level, then the null hypothesis is rejected. In simple cases, the significance level is defined at a p-value of 0.05 or less.
[0115] It is understood that aspects and embodiments of the invention described herein include "comprising," "consisting," and "consisting essentially of" aspects and embodiments.
[0116] The terms "antibodies" and "immunoglobulin" include antibodies or
immunoglobulins of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single- chain antibodies, and fusion proteins comprising an antigen- binding portion of an antibody and a non-antibody protein. Antibodies may exist in a variety of other forms including, for example, Fv, Fab, and (Fab')2, as well as bi-functional (i.e. bi- specific) hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in single chains (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426 (1988)). (See, generally, Hood et al., "Immunology", Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller and Hood, Nature, 323, 15- 16 (1986),).
[0117] As used herein, vector (or plasmid) refers to a nucleic acid construct, typically a circular DNA vector, that contains discrete elements that are used to introduce heterologous nucleic acid into cells for either expression of the nucleic acid or replication thereof. The vectors typically remain episomal, but can be designed to effect stable integration of a gene or portion thereof into a chromosome of the genome. In some cases, vectors contain an origin of replication that allows many copies of the plasmid to be produced in a bacterial or eukaryotic cell without integration of the plasmid into the host cell DNA. Selection and use of such vectors are well known to those of skill in the art.
[0118] The terms "polynucleotide" and "nucleic acid molecule" are used interchangeably to refer to a single-stranded and/or double- stranded polynucleotides, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), as well as analogs or derivatives of either RNA or DNA. The length of a polynucleotide molecule is given herein in terms of nucleotides
(abbreviated "nt") or base pairs (abbreviated "bp"). Also included in the term "nucleic acid" are analogs of nucleic acids such as peptide nucleic acid (PNA), phosphorothioate DNA, and other such analogs and derivatives. Nucleic acids can encode gene products, such as, for example, polypeptides, regulatory RNAs, microRNAs, siRNAs and functional RNAs. Hence, nucleic acid molecule is meant to include all types and sizes of DNA molecules including cDNA, plasmids or vectors and DNA including modified nucleotides and nucleotide analogs.
[0119] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Polypeptides may include amino acid residues including natural and/or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
[0120] As used herein, the term "gene" refers to any and all discrete coding regions of a host genome, or regions that code for a functional RNA only (e.g. , tRNA, rRNA, regulatory RNAs such as ribozymes etc.) as well as associated non-coding regions and optionally regulatory regions. In certain embodiments, the term "gene" includes within its scope the open reading frame encoding specific polypeptides, introns, and adjacent 5' and 3' non- coding nucleotide sequences involved in the regulation of expression. In this regard, the gene can further contain control signals such as promoters, enhancers, termination and/or polyadenylation signals that are naturally associated with a given gene, or heterologous control signals. The gene sequences can be cDNA or genomic DNA or a fragment thereof. The gene can be introduced into an appropriate vector for extrachromosomal maintenance or for integration into the host.
[0121] As used herein, an outer membrane (OM) stress-responsive gene is a gene whose expression or activity is modulated (e.g. increased or decreased) in a microorganism (e.g. an Acinetobacter spp. ) in response to a stress condition to the outer membrane of the
microorganism, in which the stress condition modulates the biogenesis and/or integrity of the OM, including that disrupts the OM; destabilizes the OM, for example, by inhibiting the synthesis of lipopolysaccharide (LPS) or the peptidoglycan cell wall; or permeabilizes the OM. In some embodiments, the expression or activity of the gene is increased or decreased by greater than or greater than about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold or more.
[0122] As used herein, "regulatory sequence" or "regulatory region" as used in reference to a specific gene, refers to the coding or non-coding nucleic acid expression control sequences within that gene that are necessary or sufficient to provide for the regulated expression of the coding region of a gene. Thus, the term encompasses promoter sequences, regulatory protein binding sites, upstream activator sequences and the like. Specific nucleotides within a regulatory region may serve multiple functions. For example, a specific nucleotide may be part of a promoter and participate in the binding of a transcriptional activator protein.
[0123] As used herein, "open reading frame" (ORF) refers to a DNA sequence starting with a start codon and ending with a stop codon, and therefore signaling a coding sequence that is translated into a functional product RNA or polypeptide. Hence, an ORF is synonymous with coding sequence.
[0124] By "operably linked" is meant a functional linkage between a nucleic acid expression control sequence (such as a promoter) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
[0125] Percent "identical" or "identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences that are the same or have a specified percentage of nucleic acid residues or amino acid residues, respectively, that are the same, when compared and aligned for maximum similarity, as determined using a sequence comparison algorithm or by visual inspection. "Percent sequence identity" or "% identity" or "% sequence identity or "% amino acid sequence identity" of a subject amino acid sequence to a reference amino acid sequence means that the subject amino acid sequence is identical (i.e., on an amino acid-by-amino acid basis) by a specified percentage to the reference amino acid sequence over a comparison length when the sequences are optimally aligned. Thus, 80% amino acid sequence identity or 80% identity with respect to two amino acid sequences means that 80% of the amino acid residues in two optimally aligned amino acid sequences are identical.
[0126] As used herein, a "reporter molecule" refers to a molecule that is directly or indirectly detectable or whose presence is otherwise capable of being measured. In some aspects, reporter molecules include proteins that can emit a detectable signal such as a fluorescence signal, and enzymes that can catalyze a detectable reaction or catalyze formation of a detectable product. Reporter molecules also can include detectable nucleic acids. In some embodiments, a reporter molecule is a polypeptide which can be detected when it is expressed in the cell. In some cases, expression of the detectable reporter may lead to the production of a signal, for example a fluorescent, bio luminescent or colorimetric signal, which can be detected using routine techniques. The signal may be produced directly from the reporter, after expression, or indirectly through a secondary molecule, such as a labelled antibody.
[0127] The terms "reporter cell" and "reporter microorganism" are used interchangeably to refer to an engineered microorganism into which an exogenous or heterologous polynucleotide, such as a cDNA or gene, encoding a reporter molecule has been introduced. Therefore, reporter cells are distinguishable from naturally occurring microorganisms which do not contain a recombinantly introduced exogenous polynucleotide. Reporter cells are thus cells having a gene or genes introduced through human intervention and that express an exogenous reporter molecule. [0128] As used herein, heterologous with reference to a polynucleotide or gene (also referred to as exogenous or foreign) refers to a nucleotide sequence that is not native to the organism or a gene contained therein or not normally produced in vivo by an organism, such as bacteria, from which it is expressed.
[0129] As used herein, a kit is a packaged combination that optionally includes other elements, such as additional reagents and instructions for use of the combination or elements thereof. Kits optionally include instructions for use.
[0130] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell culturing, molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual, third edition (Sambrook et al., 2001) Cold Spring Harbor Press;
Oligonucleotide Synthesis (P. Herdewijn, ed., 2004); Animal Cell Culture (R. I. Freshney), ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental
Immunology (D. M. Weir & C. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M.
Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Manual of Clinical Laboratory Immunology (B. Detrick, N. R. Rose, and J. D. Folds eds., 2006); Immunochemical Protocols (J. Pound, ed., 2003); Lab Manual in Biochemistry: Immunology and Biotechnology (A. Nigam and A. Ayyagari, eds. 2007); Immunology Methods Manual: The Comprehensive Sourcebook of Techniques (Ivan Lefkovits, ed., 1996); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, eds., 1988); and others.
[0131] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference. [0132] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
II. REPORTER CONSTRUCTS
[0133] In certain embodiments, there is provided a reporter polynucleotide comprising a sequence comprising a regulatory region of an OM stress-responsive gene of an Acinetobacter species (spp). operably linked to a sequence encoding a reporter molecule, wherein the OM stress-responsive gene is modulated in response to a stress to the outer membrane (hereinafter referred to as "OM-stress) of he Acinetobacter spp.
[0134] In some embodiments, the sequence comprising the regulatory region is directly or indirectly linked to the sequence encoding the reporter molecule. In certain embodiments, the sequence comprising the regulatory region is directly linked to the 5' end of the open reading frame (ORF) of the reporter molecule. In certain embodiments, the sequence comprising the regulatory region is indirectly linked to the 5' end of the ORF of the reporter molecule.
[0135] The provided reporter polynucleotides and reporter vectors can be produced or generated by standard DNA techniques. Genes or nucleotide sequences of interest can be obtained by amplification of nucleic acids, such as by polymerase chain reaction methods; generated de novo by synthetic construction (e.g. overlapping PCR and/or oligonucleotide hybridization); or desired sequences can be removed from already existing sources such as commercially available vectors. Synthesized or harvested sequences can be further modified by site directed mutagenesis, PCR, or other methods known to those of skill in the art. Such modifications include, but are not limited to optimize codon usage for a host microorganism, modification of a coding sequence to reduce or enhance a desired activity, and addition of restriction sequences for cloning purposes. In some embodiments, codon optimization can be employed to optimize expression in a host microorganism, such as by modification of certain codons to reflect particular codon usage of the reporter polynucleotide containing the reporter molecule to that which is found more frequently in particular species in which it is being expressed.
[0136] Nucleic acids molecules can be synthesized by methods known to one of skill in the art using synthetic gene synthesis. In some embodiments, individual oligonucleotides corresponding to fragments of a polynucleotide sequence vector are synthesized by standard automated methods and mixed together in an annealing or hybridization reaction. In some strategies, synthetic genes are assembled from a large number of short partially overlapping DNA oligonucleotides or fragments, generally each independently about or about at least 20, 30, 40, 50, 60, 70, 80, 90 100, 200, 300, 400, 500 or more nucleotides in length. In some cases, DNA fragments with overlapping ends can be generated by restriction digest, by PCR or by gene synthesis methods. In some cases, such oligonucleotides can be commercially obtained, such as from Integrated DNA Technologies (Coralville, IA). In some aspects, overlapping segments are allowed to anneal and then are assembled into longer double- stranded DNA, for example, by ligation and/or polymerase extension reactions, either alone or in combination. Single nucleotide "nicks" in the duplex DNA are sealed using ligation, for example with bacteriophage T4 DNA ligase. Such strategies are variously referred to as "assembly PCR," "splicing by overlap extension," "polymerase chain assembly" and others. In some
embodiments, Gibson Assembly methods can be employed.
[0137] A description of the regulatory region and reporter molecule of the provided reporter polynucleotides is provided below. It is within the level of one of skill in the art to generate and design a reporter polynucleotide, and reporter vectors containing such reporter polynucleotides, as described herein. Exemplary reporter polynucleotides and reporter vectors are provided.
A. Regulatory Region
[0138] In some embodiments, the provided reporter polynucleotides contain a regulatory region of an Acinetobacter spp. gene that is responsive to and/or whose expression is modulated in response to stress to the outer membrane (hereinafter called "OM stress-responsive gene").
[0139] In certain embodiments, the stress to the outer membrane (OM) is caused by altering, mutating, depleting, or removing a component from the OM. For example, the stress to the OM can be caused by genetically altering the Acinetobacter spp. to reduce expression of an OM lipid, polysaccharide, or protein. In certain embodiments, the OM stress responsive gene is responsive to stress caused by altering, mutating, depleting, or removing BamA, LptD, FhuA, PldA, OmpT, PagP, OstA, OmpA, OmpF, Omp200, Ompl21, Omp71, Ompl l7, OprF, PhoE, OmpC, OmpF, NmpC; PorA, PorB, OprA, OprM, OprN, OprJ, OprB, NspA, PagL, OmpW, OpcA, NalP, NupA, OmpG, FadL, PhoE, LamB, FhaC, SucY, FepA, FecA, BtuB, TolC, Porin P, Porin D, SmeC, SmeF, MepC, SrpC, TtgC, TtgF, AdeC, AdeK, or MexA. In particular embodiments, the stress to the OM of the Acinetobacter bacterium is or is caused by depletion of BamA. [0140] In some embodiments, the genetic alteration (e.g. deletion) can be constitutively active or require induction. In some aspects, an outer membrane gene can be deleted from a microorganism and, instead, can be exogenously expressed under the control of an inducer. In such an example, the outer membrane gene is expressed only in the presence of the inducer but is not expressed, such as is not expressed on the cell surface, in the absence of the inducer. In some embodiments, the inducer is arabinose and the genetic alteration is induced by the addition of an inducer. In some embodiments, the inducer is arabinose. In some embodiments, the concentration of the arabinose is about 0.0004 -0.2%, such as about 0.0006%-0.02%, 0.0008- 0.02%, or about 0.001%-0.02%. Exemplary of such an Acinetobacter spp. is AABA046, which carries a deletion of the bamA gene by replacement with a selective marker conferring resistance to kanamycin and contains an exogenous polynucleotide encoding bamA fused to the araBAD promoter from E. coli which allows regulation of transcription by addition of the inducing sugar arabinose. Also exemplary of such an Acinetobacter spp. is AABA041, which carries a deletion of the bamA gene by replacement with a selective marker conferring resistance to carbenicillin and contains an exogenous polynucleotide encoding bamA fused to the araBAD promoter from E. coli which allows regulation of transcription by addition of the inducing sugar arabinose.
[0141] In certain embodiments, the stress to the outer membrane (OM) can be caused by treatment with a molecule or combination of molecules that causes temporary or permanent damage to the OM. In certain embodiments, the stress to the OM is or is caused by treatment with polymyxin B nonapeptide (PMBN), vancomycin, ACHN-975, colistin,
ethylenediaminetetraacetic acid (EDTA), Sodium dodecyl sulfate (SDS), or combinations thereof. In certain embodiments, the stress to the OM is or is caused by treatment with PMBN. In certain embodiments the concentration of the molecule is a sub-lethal concentration. In certain embodiments the concentration of the molecule is below the MIC of the molecule. In certain embodiments the concentration of the molecule is about 0.1 μg/mL - 1000 μg/mL, such as about 1 μg/mL - 750 μg/mL, 10 μg/mL - 500 μg/mL, 25 μg/mL - 500 μg/mL, or 25 μg/mL - 250 μg/mL. In certain embodiments, the concentration of the molecule is about 0.1 mM- 500 mM, such as about 0.1 mM - 500 mM, 1 mM - 50 mM, or 1 mM - 10 mM. In certain embodiments, the concentration of the molecule is about 0.1% - 5%, such as about 0.5% - 2.5% or 0.75% - 2%. [0142] In some embodiments, the OM stress-responsive gene is modulated in response to more than 1, 2, 3, 4, 5, or 6 stresses. In certain embodiments, the OM stress-responsive gene is modulated in response to depletion of BamA and treatment with PMBN.
[0143] In certain embodiments, the OM stress-responsive gene is identified using a method that includes a) inducing OM stress in Acinetobacter spp. by exposing or subjecting an
Acinetobacter spp. to one or more stress conditions, such as any described above and b) identifying one or more genes that is modulated in response to the OM stress (e.g. using RNA- Seq or other method).
[0144] One of skill in the art can readily determine whether expression of a gene is modulated. For example, one can compare transcription levels of A. bacterium genes, such as Acinetobacter spp. genes in the presence of one or more stress conditions, such as described above. In some embodiments, expression levels can be assessed or determined using any method known to a skilled artisan, such as by using quantitative PCR, microarrays, RNA-Seq, northern blotting, or SAGE. In some embodiments, genes whose sequences, or portions or fragments of sequences, have been identified as having been modulated (e.g. increased or decreased) can be identified using a reference sequence of the A. bacterium genome. Exemplary A. bacterium genome sequences are known and readily available online on the world wide web at tigr.org, kegg.jp, or ncbi.nlm.nih.gov/genbank. Exemplary genomic sequences of A.
baumannii species or isolates include GENBANK™ Accession Nos. CP00521.1 or
NC 011595.1.
[0145] In some embodiments, the Acinetobacter spp. is an Acinetobacter apis, Acinetobacter baumannii, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii, Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter gerneri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus,
Acinetobacter harbinensis, Acinetobacter indicus, Acinetobacter junii, Acinetobacter kookii, Acinetobacter Iwoffii, Acinetobacter nectaris, Acinetobacter nosocomialis, Acinetobacter parvus, Acinetobacter pakistanensis, Acinetobacter pittii, Acinetobacter puyangensis,
Acinetobacter qingfengensis, Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii, Acinetobacter soli, Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis, or Acinetobacter venetianus.
[0146] In particular embodiments, the Acinetobacter spp. is Acinetobacter baumannii. In certain embodiments, the Acinetobacter spp. is ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA- 1605, ATCC BAA-1709, ATCC BAA-1710, ATCC BAA-1789, ATCC BAA-1790, ATCC BAA- 1791, ATCC BAA- 1792, ATCC BAA-1793, ATCC BAA-1794, ATCC BAA-1795, ATCC BAA- 1796, ATCC BAA-1797, ATCC BAA-1798, ATCC BAA- 1799, ATCC BAA-1800, ATCC BAA-1878, ATCC BAA-2093, ATCC BAA-747, SDF, AYE, ACICU, AB0057, AB307-0294, 1656-2, MDR-ZJ06, TCDC-AB0715, MDR-TJ, TYTH-1, D1279779, BJAB07104, BJAB0715, BJAB0868, ZW85-1, AbH120-A2, AB030, AB031, AC29, LAC-4, Ab307-0294, 1656-2, ID 403, AABA041, AABA046, or a modified strain thereof. In particular embodiments, the Acinetobacter spp. is ATCC 17978, AABA041, AABA046, or Ab307-0294.
[0147] In some embodiments, the Acinetobacter spp. is a multi-drug resistant bacterium.
[0148] In some embodiments, the OM stress-responsive gene is one that is upregulated or downregulated in response to the stress to the outer membrane of Acinetobacter spp.
[0149] In certain embodiments, the OM stress-responsive gene is downregulated in response to the stress. In certain embodiments, the OM stress-responsive gene is downregulated by at least or at least about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20- fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold or more in response to the stress compared to the expression level of the gene in the absence of the stress condition.
[0150] In certain embodiments, the OM stress-responsive gene, such as an OM-stress responsive gene that is downregulated in response to OM stress, is A1S_0009, A1S_0010, A1S_0025, A1S_0027, A1S_0038, A1S_0067, A1S_0070, A1S_0071, A1S_0073, A1S_0076, A1SJ3077, A1SJ3079, A1S_0087, A1S_0090, A1S_0091, A1S_0095, A1S_0096, A1S_0097, A1S_0098, A1S_0099, A1S_0103, A1S_0104, A1S_0105, A1S_0106, A1S_0107, A1S_0108, A1S_0109, A1S_0121, A1S_0128, A1S_0129, A1S_0141, A1S_0148, A1S_0150, A1S_0151, A1SJ3152, A1SJ3153, A1S_0154, A1S_0155, A1S_0156, A1S_0157, A1S_0177, A1S_0184, A1S_ _0200, A1S_ _0201 A1S_ _0209, A1S_ 0218, A1S_ 0238, A1S_ 0239, A1S_ 0253, A1S_ 0257,
A1S_ _0258, A1S_ _0269 A1S_ _0270, A1S_ 0279, A1S_ 0286, A1S_ 0292, A1S_ 0302, A1S_ 0303,
A1S_ _0304, A1S_ _0321 A1S_ _0322, A1S_ 0323, A1S_ _0347, A1S_ 0365, A1S_ 0369, A1S_ 0370,
A1S_ _0388, A1S_ _0408 A1S_ _0410, A1S_ 0427, A1S_ 0429, A1S_ 0447, A1S_ 0448, A1S_ 0474,
A1S_ _0480, A1S_ _0481 A1S_ _0482, A1S_ 0486, A1S_ 0490, A1S_ 0491, A1S_ 0498, A1S_ 0526,
A1S_ _0533, A1S_ _0534 A1S_ _0548, A1S_ 0549, A1S_ 0566, A1S_ 0567, A1S_ 0568, A1S_ 0591,
A1S_ _0594, A1S_ _0624 A1S_ _0625, A1S_ 0626, A1S_ 0627, A1S_ 0629, A1S_ 0630, A1S_ 0631,
A1S_ _0632, A1S_ _0633 A1S_ _0634, A1S_ 0635, A1S_ 0639, A1S_ 0640, A1S_ 0641, A1S_ 0642,
A1S_ _0643, A1S_ _0644 A1S_ _0645, A1S_ 0646, A1S_ 0647, A1S_ 0649, A1S_ 0650, A1S_ 0651,
A1S_ _0690, A1S_ _0691 A1S_ _0692, A1S_ 0695, A1S_ 0698, A1S_ 0721, A1S_ 0731, A1S_ 0732,
A1S_ _0771, A1S_ _0785 A1S_ _0786, A1S_ _0787, A1S_ 0788, A1S_ 0818, A1S_ _0822, A1S_ 0846,
A1S_ _0849, A1S_ _0850 A1S_ _0851, A1S_ 0852, A1S_ 0853, A1S_ 0854, A1S_ 0855, A1S_ 0869,
A1S_ _0877, A1S_ _0882 A1S_ _0883, A1S_ 0890, A1S_ 0891, A1S_ 0901, A1S_ 0910, A1S_ 0911,
A1S_ _0912, A1S_ _0913 A1S_ _0960, A1S_ 0965, A1S_ 0973, A1S_ 0984, A1S_ 0996, A1S_ 0997,
A1S_ _0999, A1S_ _1000 A1S_ _1004, A1S_ _1008, A1S_ .1021, A1S_ .1026, A1S_ .1044, A1S_ _1063,
A1S_ _1072, A1S_ _1079 A1S_ _1080, A1S_ _1088, A1S_ _1089, A1S_ .1091, A1S_ .1092, A1S_ .1093,
A1S_ _1094, A1S_ _1109 A1S_ _1113, A1S_ .1139, A1S_ .1142, A1S_ .1182, A1S_ .1193, A1S_ .1195,
A1S_ _H99, A1S_ _1227 A1S_ _1257, A1S_ _1258, A1S_ .1261, A1S_ .1264, A1S_ .1265, A1S_ .1266,
A1S_ _1267, A1S_ _1268 A1S_ _1269, A1S_ _1270, A1S_ .1281, A1S_ .1317, A1S_ .1318, A1S_ .1319,
A1S_ _1327, A1S_ _1334 A1S_ _1335, A1S_ _1336, A1S_ .1337, A1S_ _1338, A1S_ .1339, A1S_ .1340,
A1S_ _1341, A1S_ _1342 A1S_ _1343, A1S_ _1344, A1S_ .1345, A1S_ .1346, A1S_ .1347, A1S_ .1348,
A1S_ _1349, A1S_ _1356 A1S_ _1366, A1S_ _1367, A1S_ _1368, A1S_ .1369, A1S_ .1370, A1S_ .1372,
A1S_ _1373, A1S_ _1374 A1S_ _1375, A1S_ _1376, A1S_ .1377, A1S_ .1378, A1S_ .1379, A1S_ _1380,
A1S_ _1396, A1S_ _1397 A1S_ _1428, A1S_ _1442, A1S_ .1443, A1S_ .1450, A1S_ .1466, A1S_ .1467,
A1S_ _1469, A1S_ _1470 A1S_ _1476, A1S_ _1490, A1S_ .1491, A1S_ .1492, A1S_ .1493, A1S_ .1498,
A1S_ _1499, A1S_ _1505 A1S_ _1510, A1S_ _1523, A1S_ .1528, A1S_ _1530, A1S_ .1532, A1S_ .1543,
A1S_ _1579, A1S_ _1583 A1S_ _1601, A1S_ _1608, A1S_ .1609, A1S_ .1610, A1S_ .1611, A1S_ .1612,
A1S_ _1613, A1S_ _1637 A1S_ _1638, A1S_ _1639, A1S_ _1655, A1S_ .1692, A1S_ .1698, A1S_ .1699,
A1S_ _1700, A1S_ _1701 A1S_ _1703, A1S_ _1705, A1S_ .1717, A1S_ .1719, A1S_ .1724, A1S_ .1729,
A1S_ _1730, A1S_ _1731 A1S_ _1732, A1S _1734 A1S _1735, A1S _1736 A1S _1737, A1S _1738,
A1S_ _1742, A1S_ _1745 A1S_ _1754, A1S_ _1756, A1S_ _1758, A1S_ .1775, A1S_ _1776, A1S_ .1790, A1S_ _1791, A1S_ _1792 A1S_ _1794, A1S_ _1795 A1S_ _1796, A1S_ _1797 A1S_ _1805, A1S_ _1806,
A1S_ _1811, A1S_ _1830 A1S_ _1834, A1S_ _1835 A1S_ _1836, A1S_ _1837 A1S_ _1838, A1S_ _1839,
A1S_ _1840, A1S_ _1841 A1S_ _1854, A1S_ _1855 A1S_ _1856, A1S_ _1857 A1S_ _1858, A1S_ _1859,
A1S_ .I860, A1S_ _1861 A1S_ _1862, A1S_ _1863 A1S_ _1864, A1S_ _1865 A1S_ _1866, A1S_ _1879,
A1S_ _1880, A1S_ _1887 A1S_ _1908, A1S_ _1924 A1S_ _1925, A1S_ _1926 A1S_ _1935, A1S_ _1940,
A1S_ _1942, A1S_ _1948 A1S_ _1951, A1S_ _1984 A1S_ _1996, A1S_ _2041 A1S_ _2042, A1S_ _2052,
A1S_ _2053, A1S_ _2068 A1S_ _2072, A1S_ _2081 A1S_ _2084, A1S_ _2098 A1S_ _2100, A1S_ _2101,
A1S_ _2102, A1S_ _2148 A1S_ _2149, A1S_ _2150 A1S_ _2163, A1S_ _2166 A1S_ _2167, A1S_ _2190,
A1S_ _2191, A1S_ _2202 A1S_ _2203, A1S_ _2207 A1S_ _2209, A1S_ _2218 A1S_ _2221, A1S_ _2225,
A1S_ _2232, A1S_ _2234 A1S_ _2248, A1S_ _2279 A1S_ _2280, A1S_ _2288 A1S_ _2289, A1S_ _2340,
A1S_ _2341, A1S_ _2342 A1S_ _2348, A1S_ _2353 A1S_ _2354, A1S_ _2415 A1S_ _2416, A1S_ _2417,
A1S_ _2418, A1S_ _2419 A1S_ _2424, A1S_ _2425 A1S_ _2431, A1S_ _2435 A1S_ _2443, A1S_ _2449,
A1S_ _2450, A1S_ _2451 A1S_ _2452, A1S_ _2475 A1S_ _2501, A1S_ _2509 A1S_ _2510, A1S_ _2514,
A1S_ _2531, A1S_ _2532 A1S_ _2533, A1S_ _2535 A1S_ _2601, A1S_ _2602 A1S_ _2633, A1S_ _2662,
A1S_ _2670, A1S_ _2671 A1S_ _2672, A1S_ _2688 A1S_ _2692, A1S_ _2694 A1S_ _2695, A1S_ _2696,
A1S_ _2701, A1S_ _21\ \ A1S_ _2722, A1S_ _2724 A1S_ _2738, A1S_ _2740 A1S_ _2741, A1S_ _2748,
A1S_ _2753, A1S_ _2755 A1S_ _2758, A1S_ _2761 A1S_ _2762, A1S_ _2769 A1S_ _2773, A1S_ _2774,
A1S_ _2785, A1S_ _2788 A1S_ _2789, A1S_ _2793 A1S_ _2809, A1S_ _2814 A1S_ _2815, A1S_ _2820,
A1S_ _2823, A1S_ _2847 A1S_ _2848, A1S_ _2849 A1S_ _2852, A1S_ _2860 A1S_ _2904, A1S_ _2905,
A1S_ _2906, A1S_ _2911 A1S_ _2913, A1S_ _2919 A1S_ _2924, A1S_ _2928 A1S_ _2939, A1S_ _2946,
A1S_ _2956, A1S_ _3013 A1S_ _3014, A1S_ _3025 A1S_ _3040, A1S_ _3043 A1S_ _3049, A1S_ _3050,
A1S_ _3051, A1S_ _3074 A1S_ _3084, A1S_ _3110 A1S_ _3120, A1S_ _3121 A1S_ _3122, A1S_ _3128,
A1S_ _3129, A1S_ _3130 A1S_ _3131, A1S_ _3132 A1S_ _3133, A1S_ _3134 A1S_ _3135, A1S_ _3144,
A1S_ _3174, A1S_ _3180 A1S_ _3195, A1S_ _3207 A1S_ _3222, A1S_ _3224 A1S_ _3225, A1S_ _3231,
A1S_ _3232, A1S_ _3236 A1S_ _3238, A1S_ _3248 A1S_ _3250, A1S_ _3268 A1S_ _3269, A1S_ _3273,
A1S_ _3278, A1S_ _3290 A1S_ _3297, A1S_ _3298 A1S_ _3300, A1S_ _3301 A1S_ _3309, A1S_ _3338,
A1S_ _3342, A1S_ _3355 A1S_ _3364, A1S_ _3377 A1S_ _3397, A1S_ _3398 A1S_ _3402, A1S_ _3403,
A1S_ _3404, A1S_ _3405 A1S_ _3406, A1S_ _3407 A1S_ _3410, A1S_ _3413 A1S_ _3414, A1S_ _3415,
A1S_ _3416, A1S_ _3418 A1S_ _3431, A1S_ _3450 A1S_ _3451, A1S_ _3458 A1S_ _3460, A1S_ _3481,
A1S_ _3487, A1S_ _3491 A1S_ _3494, A1S_ _3498 A1S_ _3506, A1S_ _3508 A1S_ _3509, A1S_ _3514,
A1S_ _3518, A1S_ _3519 A1S_ _3520, A1S_ _3521 A1S_ _3522, A1S_ _3523 A1S_ _3524, A1S_ _3526, A1S_3528, A1S_3530, A1S_3531, A1S_3532, A1S_3533, A1S_3534, A1S_3535, A1S_3537, A1S_3538, A1S_3539, A1S_3540, A1S_3541, A1S_3542, A1S_3543, A1S_3544, A1S_3545, A1S_3546, A1S_3547, A1S_3548, A1S_3549, A1S_3550, A1S_3552, A1S_3553, A1S_3554, A1S_3568, A1S_3569, A1S_3578, A1S_3582, A1S_3586, A1S_3587, A1S_3591, A1S_3597, A1S_3599, A1S_3600, A1S_3602, A1S_3611, A1S_3619, A1S_3621, A1S_3624, A1S_3629, A1S_3633, A1S_3640, A1S_3641, A1S_3644, A1S_3647, A1S_3651, A1S_3652, A1S_3659, A1S_3663, A1S_3667, A1S_3673, A1S_3679, A1S_3701, A1S_3707, A1S_3709, A1S_3713, A1S_3715, A1S_3717, A1S_3732, A1S_3735, A1S_3738, A1S_3740, A1S_3741, A1S_3742, A1S_3759, A1S_3774, A1S_3779, A1S_3787, A1S_3788, A1S_3794, A1S_3801, A1S_3802, A1S_3806, A1S_3809, A1S_3811, A1S_3813, A1S_3814, A1S_3816, A1S_3817, A1S_3823, A1S_3829, A1S_3831, A1S_3832, A1S_3836, A1S_3840, A1S_3846, A1S_3857, A1S_3862, A1S_3868, A1S_3870, A1S_3880, A1S_3884, A1S_3886, A1S_3887, A1S_3891, A1S_3894, A1S_3898, A1S_3907, A1S_3908, A1S_3909, A1S_3912, A1S_3914, or A1S_3915.
[0151] In some embodiments, the OM stress-responsive gene, such as an OM-stress responsive gene that is downregulated in response to OM stress, is A1S_0103, A1S_0645, A1S_1266, A1S_1268, A1S_1335, A1S_1336, A1S_1337, A1S_1338, A1S_1339, A1S_1340, A1S_1341, A1S_1342, A1S_1343, A1S_1344, A1S_1345, A1S_1791, A1S_1792, A1S_1794, A1S_1796, A1S_1835, A1S_1836, A1S_1837, A1S_1838, A1S_1839, A1S_2449, A1S_2450, A1S_2452, A1S_3540, A1S_3541, A1S_3542, A1S_3543, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, A1S_3809, or A1S_3908. In some embodiments, such an OM stress- responsive gene is A1S_1336, A1S_1836, A1S_1838, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, or A1S_3809.
[0152] In some embodiments, the OM stress-responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any of the gene described herein. In some embodiments, the OM stress- responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with A1S_0103, A1S_0645, A1S_1266, A1S_1268, A1S_1335, A1S_1336, A1S_1337, A1S_1338, A1S_1339, A1S_1340, A1S_1341, A1S_1342, A1S_1343, A1S_1344, A1S_1345, A1S_1791, A1S_1792, A1S_1794, A1S_1796, A1S_1835, A1S_1836, A1S_1837, A1S_1838, A1S_1839, A1S_2449, A1S_2450, A1S_2452, A1S_3540, A1S_3541, A1S_3542, A1S_3543, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, A1S_3809, or A1S_3908. In certain embodiments, the OM stress-responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with A1S_1336, A1S_1836, A1S_1838, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, or A1S_3809.
[0153] In some embodiments, the OM stress-responsive gene is one that is upregulated in response to the stress to the outer membrane of Acinetobacter spp. In some embodiments, the OM stress-responsive gene is upregulated in response to the stress by at least or at least about 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50- fold, 60-fold, 70-fold, 80, 90-fold, 100-fold, 200-fold, 300-fold, 400-fold or more in response to the stress compared to the expression level of the gene in the absence of the stress condition.
[0154] In some embodiments, the OM stress-responsive gene, such as an OM-stress responsive gene that is upregulated in response to OM stress, is A1S_0012, A1S_0023,
A1S_ _0027, A1S_ _0028, A1S_ _0029, A1S_ _0030, A1S_ _0031, A1S_ _0032, A1S_ _0033, A1S_ _0037,
A1S_ _0040, A1S_ _0041, A1S_ _0044, A1S_ _0066, A1S_ _0092, A1S_ _0093, A1S_ _0109, A1S_ _0110,
A1S_ _0112, A1S_ _0113, A1S_ _0114, A1S_ _0115, A1S_ _0116, A1S_ _0117, A1S_ _0118, A1S_ _0126,
A1S_ _0158, A1S_ _0170, A1S_ _0175, A1S_ _0178, A1S_ _0189, A1S_ _0224, A1S_ _0245, A1S_ _0256,
A1S_ _0276, A1S_ _0293, A1S_ _0301, A1S_ _0309, A1S_ _0310, A1S_ _0332, A1S_ _0333, A1S_ _0363,
A1S_ _0372, A1S_ _0376, A1S_ _0391, A1S_ _0392, A1S_ _0401, A1S_ _0441, A1S_ _0462, A1S_ _0463,
A1S_ _0464, A1S_ _0465, A1S_ _0466, A1S_ _0494, A1S_ _0508, A1S_ _0509, A1S_ _0510, A1S_ _0511,
A1S_ _0512, A1S_ _0514, A1S_ _0516, A1S_ _0518, A1S_ _0519, A1S_ _0520, A1S_ _0521, A1S_ _0522,
A1S_ _0523, A1S_ _0527, A1S_ _0535, A1S_ _0536, A1S_ _0537, A1S_ _0538, A1S_ _0547, A1S_ _0559,
A1S_ _0561, A1S_ _0562, A1S_ _0563, A1S_ _0564, A1S_ _0566, A1S_ _0567, A1S_ _0568, A1S_ _0570,
A1S_ _0624, A1S_ _0630, A1S_ _0631, A1S_ _0633, A1S_ _0634, A1S_ _0640, A1S_ _0641, A1S_ _0642,
A1S_ _0643, A1S_ _0644, A1S_ _0645, A1S_ _0646, A1S_ _0647, A1S_ _0650, A1S_ _0663, A1S_ _0664,
A1S_ _0665, A1S_ _0666, A1S_ _0667, A1S_ _0669, A1S_ _0670, A1S_ _0671, A1S_ _0673, A1S_ _0677,
A1S_ _0680, A1S_ _0683, A1S_ _0714, A1S_ _0717, A1S_ _0718, A1S_ _0719, A1S_ _0736, A1S_ _0738,
A1S_ _0739, A1S_ _0749, A1S_ _0770, A1S_ _0772, A1S_ _0779, A1S_ _0780, A1S_ _0781, A1S_ _0800,
A1S_ _0804, A1S_ _0830, A1S_ _0831, A1S_ _0832, A1S_ _0834, A1S_ _0835, A1S_ _0861, A1S_ _0884,
A1S_ _0889, A1S_ _0929, A1S_ _0930, A1S_ _0931, A1S_ _0932, A1S_ _0935, A1S_ _0945, A1S_ _0959,
A1S_ _0980, A1S_ _1003, A1S_ _1009, A1S_ _1010, A1S_ _1027, A1S_ _1028, A1S_ _1030, A1S_ _1031,
A1S_ _1049, A1S_ _1081, A1S_ _1106, A1S_ _1107, A1S_ _1120, A1S_ _1121, A1S_ _1123, A1S_ _1132, A1S_ _1133, A1S_ _1134 A1S_ _1139, A1S_ _1143 A1S_ _1145, A1S_ M46 A1S_ _1148, A1S_ _1149,
A1S_ _1150, A1S_ _1151 A1S_ _1152, A1S_ _1153 A1S_ _1155, A1S_ _1156 A1S_ _1157, A1S_ _1158,
A1S_ _1159, A1S_ _1160 A1S_ _1161, A1S_ _1162 A1S_ _1163, A1S_ _1164 A1S_ _1165, A1S_ _1167,
A1S_ _1171, A1S_ _1172 A1S_ _1173, A1S_ _1180 A1S_ _1184, A1S_ _1186 A1S_ _1198, A1S_ _1202,
A1S_ _1203, A1S_ _1223 A1S_ _1224, A1S_ _1225 A1S_ _1230, A1S_ _1236 A1S_ _1237, A1S_ _1248,
A1S_ _1255, A1S_ _1274 A1S_ _1286, A1S_ _1359 A1S_ _1360, A1S_ _1361 A1S_ _1362, A1S_ _1363,
A1S_ _1383, A1S_ _1384 A1S_ _1385, A1S_ _1386 A1S_ _1387, A1S_ _1393 A1S_ _1404, A1S_ _1407,
A1S_ _1422, A1S_ _1454 A1S_ _1472, A1S_ _1481 A1S_ _1494, A1S_ _1512 A1S_ _1515, A1S_ _1526,
A1S_ _1535, A1S_ _1539 A1S_ _1566, A1S_ _1567 A1S_ _1569, A1S_ _1583 A1S_ _1584, A1S_ _1585,
A1S_ _1589, A1S_ _1590 A1S_ _1593, A1S_ _1595 A1S_ _1596, A1S_ _1617 A1S_ _1630, A1S_ _1644,
A1S_ _1645, A1S_ _1647 A1S_ _1648, A1S_ _1649 A1S_ _1651, A1S_ _1655 A1S_ _1658, A1S_ _1662,
A1S_ _1666, A1S_ _1667 A1S_ _1669, A1S_ _1677 A1S_ _1680, A1S_ _1681 A1S_ _1687, A1S_ _1735,
A1S_ _1741, A1S_ _1743 A1S_ _1744, A1S_ _1750 A1S_ _1751, A1S_ _1752 A1S_ _1760, A1S_ _1762,
A1S_ _1767, A1S_ _1778 A1S_ _1813, A1S_ _1827 A1S_ _1829, A1S_ _1831 A1S_ _1843, A1S_ _1876,
A1S_ _1909, A1S_ _1928 A1S_ _1929, A1S_ _1934 A1S_ _1952, A1S_ _1955 A1S_ _1956, A1S_ _1957,
A1S_ _1959, A1S_ _1960 A1S_ _1961, A1S_ _1962 A1S_ _1963, A1S_ _1979 A1S_ _1986, A1S_ _1987,
A1S_ _1988, A1S_ _2006 A1S_ _2026, A1S_ _2033 A1S_ _2034, A1S_ _2035 A1S_ _2036, A1S_ _2038,
A1S_ _2039, A1S_ _2061 A1S_ _2074, A1S_ _2079 A1S_ _2082, A1S_ _2092 A1S_ _2093, A1S_ _2106,
A1S_ _2139, A1S_ _2140 A1S_ _2141, A1S_ _2142 A1S_ _2146, A1S_ _2157 A1S_ _2158, A1S_ _2160,
A1S_ _2161, A1S_ _2162 A1S_ _2178, A1S_ _2179 A1S_ _2183, A1S_ _2186 A1S_ _2195, A1S_ _2230,
A1S_ _2247, A1S_ _2252 A1S_ _2257, A1S_ _2258 A1S_ _2259, A1S_ _2262 A1S_ _2271, A1S_ _2272,
A1S_ _2273, A1S_ _2283 A1S_ _2285, A1S_ _2298 A1S_ _2311, A1S_ _2315 A1S_ _2325, A1S_ _2326,
A1S_ _2330, A1S_ _2331 A1S_ _2366, A1S_ _2367 A1S_ _2382, A1S_ _2387 A1S_ _2389, A1S_ _2395,
A1S_ _2396, A1S_ _2414 A1S_ _2434, A1S_ _2445 A1S_ _2446, A1S_ _2447 A1S_ _2448, A1S_ _2454,
A1S_ _2455, A1S_ _2456 A1S_ _2458, A1S_ _2459 A1S_ _2463, A1S_ _2480 A1S_ _2489, A1S_ _2503,
A1S_ _2504, A1S_ _2508 A1S_ _2542, A1S_ _2543 A1S_ _2552, A1S_ _2553 A1S_ _2555, A1S_ _2557,
A1S_ _2558, A1S_ _2573 A1S_ _2577, A1S_ _2578 A1S_ _2580, A1S_ _2586 A1S_ _2588, A1S_ _2593,
A1S_ _2611, A1S_ _2612 A1S_ _2613, A1S_ _2624 A1S_ _2650, A1S_ _2651 A1S_ _2654, A1S_ _2656,
A1S_ _2660, A1S_ _2664 A1S_ _2668, A1S_ _2675 A1S_ _2676, A1S_ _2677 A1S_ _2678, A1S_ _2684,
A1S_ _2705, A1S_ _2729 A1S_ _2734, A1S_ _2756 A1S_ _2768, A1S_ _2786 A1S_ _2798, A1S_ _2801,
A1S_ _2807, A1S_ _2826 A1S_ _2827, A1S_ _2828 A1S_ _2839, A1S_ _2863 A1S_ _2882, A1S_ _2883, A1S_ _2884, A1S_ _2885, A1S_ _2889, A1S_ _2892, A1S_ _2893, A1S_ _2942, A1S_ _2943, A1S_ _2953,
A1S_ _2959, A1S_ _2960, A1S_ _2968, A1S_ _2976, A1S_ _2992, A1S_ _3011, A1S_ _3026, A1S_ _3027,
A1S_ _3034, A1S_ _3035, A1S_ _3047, A1S_ _3048, A1S_ _3099, A1S_ _3100, A1S_ _3101, A1S_ _3104,
A1S_ _3105, A1S_ _3114, A1S_ _3115, A1S_ _3116, A1S_ _3117, A1S_ _3124, A1S_ _3125, A1S_ _3126,
A1S_ _3127, A1S_ _3139, A1S_ _3146, A1S_ _3147, A1S_ _3175, A1S_ _3206, A1S_ _3224, A1S_ _3253,
A1S_ _3259, A1S_ _3280, A1S_ _3281, A1S_ _3295, A1S_ _3317, A1S_ _3326, A1S_ _3339, A1S_ _3360,
A1S_ _3361, A1S_ _3367, A1S_ _3368, A1S_ _3371, A1S_ _3375, A1S_ _3376, A1S_ _3392, A1S_ _3411,
A1S_ _3412, A1S_ _3463, A1S_ _3466, A1S_ _3468, A1S_ _3469, A1S_ _3471, A1S_ _3479, A1S_ _3480,
A1S_ _3486, A1S_ _3492, A1S_ _3493, A1S_ _3494, A1S_ _3499, A1S_ _3510, A1S_ _3512, A1S_ _3518,
A1S_ _3522, A1S_ _3523, A1S_ _3533, A1S_ _3534, A1S_ _3535, A1S_ _3539, A1S_ _3540, A1S_ _3541,
A1S_ _3542, A1S_ _3543, A1S_ _3544, A1S_ _3545, A1S_ _3546, A1S_ _3548, A1S_ _3552, A1S_ _3553,
A1S_ _3558, A1S_ _3559, A1S_ _3562, A1S_ _3563, A1S_ _3567, A1S_ _3570, A1S_ _3577, A1S_ _3580,
A1S_ _3580, A1S_ _3585, A1S_ _3586, A1S_ _3594, A1S_ _3595, A1S_ _3596, A1S_ _3601, A1S_ _3602,
A1S_ _3603, A1S_ _3604, A1S_ _3605, A1S_ _3606, A1S_ _3607, A1S_ _3608, A1S_ _3609, A1S_ _3610,
A1S_ _3611, A1S_ _3612, A1S_ _3613, A1S_ _3614, A1S_ _3617, A1S_ _3618, A1S_ _3621, A1S_ _3630,
A1S_ _3632, A1S_ _3634, A1S_ _3635, A1S_ _3636, A1S_ _3637, A1S_ _3642, A1S_ _3645, A1S_ _3649,
A1S_ _3654, A1S_ _3658, A1S_ _3661, A1S_ _3662, A1S_ _3666, A1S_ _3682, A1S_ _3686, A1S_ _3687,
A1S_ _3688, A1S_ _3694, A1S_ _3695, A1S_ _3697, A1S_ _3704, A1S_ _3707, A1S_ _3708, A1S_ _3712,
A1S_ _3 16, A1S_ _3725, A1S_ _3726, A1S_ _3727, A1S_ _3728, A1S_ _3736, A1S_ _3738, A1S_ _3739,
A1S_ _3740, A1S_ _3750, A1S_ _3752, A1S_ _3760, A1S_ _3768, A1S_ _3769, A1S_ _3770, A1S_ _3771,
A1S_ _3772, A1S_ _3773, A1S_ _3776, A1S_ _3777, A1S_ _3778, A1S_ _3782, A1S_ _3783, A1S_ _3786,
A1S_ _3789, A1S_ _3790, A1S_ _3791, A1S_ _3792, A1S_ _3797, A1S_ _3810, A1S_ _3818, A1S_ _3820,
A1S_ _3835, A1S_ _3837, A1S_ _3840, A1S_ _3842, A1S_ _3844, A1S_ _3862, A1S_ _3864, A1S_ _3865,
A1S_ _3866, A1S_ _3867, A1S_ _3868, A1S_ _3873, A1S_ _3875, A1S_ _3879, A1S_ _3889, A1S_ _3900,
A1S_ _3901, A1S_ _3902, A1S_ _3908, or A1S_3911.
[0155] In certain embodiments, the OM stress-responsive gene, such as an OM-stress responsive gene that is upregulated in response to OM stress, is A1S_0032, A1S_0033, A1S_0113, A1S_0189, A1S_0516, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, A1S_3127, A1S_3492, or AIS_3791. In some
embodiments, the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or A1S_3127. In certain embodiments, the OM stress-responsive gene is A1S_0032, A1S_2885, A1S_2889, A1S_3127, A1S_3492, or A1S_3791. In some embodiments, the OM stress- responsive gene is A1S_0032, A1S_2885, or A1S_2889. In certain embodiments, the OM stress-responsive gene is A1S_0113 or A1S_1751. In certain embodiments, the OM stress- responsive gene is A1S_0189, A1S_0516, A1S_1224, A1S_2093, or A1S_2271.
[0156] In certain embodiments, the OM stress-responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any of the genes described herein. In some embodiments, the OM stress- responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with A1SJ3032, A1S_0033, A1S_0113, A1S_0189, A1SJ3516, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, A1S_3127, A1S_3492, or AIS_3791. In some embodiments, the OM stress-responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or A1S_3127. In some embodiments, the OM stress-responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with A1S_0032, A1S_2885, A1S_2889, A1S_3127, A1S_3492, AIS_3791. In some embodiments, the OM stress-responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with A1S_0032, A1S_2885, or A1S_2889. In some embodiments, the OM stress-responsive gene has at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with A1S_0113 or
A1S_1751. In certain embodiments, the OM stress-responsive gene is A1S_0189, A1S_0516, A1S_1224, A1S_2093, or A1S_2271.
[0157] In some embodiments, the provided reporter polynucleotides contain a regulatory region or portion thereof of an OM stress-responsive gene, such as any described above, operatively linked to a reporter molecule. In some embodiments, the regulatory region or portion thereof comprises a sequence upstream or 5' of the open reading frame (ORF) of the OM stress- responsive gene, such as any described above. In some embodiments, the sequence of the regulatory region or portion thereof is sufficient to provide for regulated expression of the coding region of the reporter molecule operatively linked thereto, such as upon induction or in the presence of an OM stress condition. It is within the level of a skilled artisan to carry out recombinant DNA techniques, including deletional analysis, to determine or identify regulatory region sequences, or portions thereof, sufficient to induce expression of the reporter molecule under different conditions, such as OM stress. In some embodiments, the regulatory region is or comprises a native promoter of the OM-responsive gene.
[0158] One of skill in the art can identify a regulatory region through standard techniques. For example, one could identify a regulatory region by fusing a putative regulatory region or sequence upstream of an OM stress-responsive gene to a sequence encoding a reporter molecule, introducing the construct using standard techniques into an Acinetobacter spp., inducing the putative regulatory region or upstream sequence by causing OM stress, and determining if the reporter molecule is induced. Putative regulatory regions can often be shortened or lengthened without influencing activity or inducibility. One of skill in the art can systematically test the effect of removing nucleotides from putative regulatory region sequence to determine what putative regulatory elements are critical or required for the OM stress-responsive behavior.
[0159] In certain embodiments, the regulatory region comprises a sequence to further promote translation of the encoded reporter molecule. In certain embodiments, the sequence to further promote translation of the encoded reporter molecule is directly linked to the 5' end of the open reading frame (ORF) of the reporter molecule. In certain embodiments, the sequence to further promote translation of the encoded reporter molecule is indirectly linked to the 5' end of the ORF of the reporter molecule. In certain embodiments, the sequence further promoting translation is or comprises a bacterial ribosome binding site.
[0160] In some embodiments, the ribosome binding site is a Shine-Dalgarno (SD) sequence, which is a sequence usually found in natural prokaryotic genes 5' of the ATG translation start codon that acts as a binding site on the mRNA molecule for the ribosome, thereby facilitating translation of the mRNA. In some embodiments, the regulatory region is one that contains a SD sequence in which, when operatively linked to the sequence encoding the reporter molecule, such SD sequence is about 2 to about 15 nucleotides 5' of the ATG of the sequence encoding the reporter molecule, such as about 5 to about 7 nucleotides upstream of the ATG.
[0161] In some embodiments, the SD sequence contained in the regulatory region can be any variant of the consensus sequence AGGAGG that retains the characteristic of facilitating translation of the sequence encoding the reporter molecule. In some embodiments, the SD is from about 3 nucleotides to about 9 nucleotides in length. In some embodiments, the Shine- Dalgarno sequence is native to the regulatory region of the OM stress-responsive gene. In certain embodiments, the Shine-Dalgarno sequence is synthetic and/or heterologous to the regulatory region of the OM stress-responsive gene. In certain embodiments, the Shine- Dalgarno sequence comprises a sequence with at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 14.
[0162] In some embodiments, the regulatory region, such as the promoter, is modified in its sequence compared to the native sequence of an OM stress-responsive gene, such as any described above. In some embodiments, the regulatory region is modified or altered in its sequence, for example to alter the configuration of regulatory binding regions, such as for transcription factors and/or can be made to have specific regions deleted. Such mutational and/or deletional analysis can be rationally or empirically performed and the resulting constructs tested by one of skill in the art. In some examples, the various constructs containing a modified regulatory region can be tested in a construct in which expression of an operatively linked reporter molecule (e.g. GFP) can be used to determine the activity of the modified regulatory region under different conditions, such as one or more different OM stress conditions. In some cases, this approach can be used to identify the smallest region capable of conferring or regulating expression of an operatively linked reporter molecule.
[0163] In some embodiments, the regulatory region includes a contiguous sequence of nucleotides within or within about 1000, 750, 500, 450, 400, 350, 300, or 250 base pairs upstream or 5' of the open reading frame (ORF) of the OM stress-responsive gene. In certain embodiments, the regulatory region comprises a contiguous sequence of nucleotides within about 500 base pairs upstream or 5' of the ORF of the OM stress-responsive gene. In certain embodiments, the regulatory region comprises a contiguous sequence of nucleotides within about 250 base pairs upstream or 5' of the ORF of the OM stress-responsive gene. In certain embodiments, the contiguous sequence of nucleotides comprises at least or at least about 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400 or more base pairs including any ranges between these values. [0164] In some embodiments, the regulatory region comprises a sequence with at least or at least about 50%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 1-13. In some embodiments, the regulatory region is or comprises the sequence of nucleotides set forth in any of SEQ ID NOs: 1-13.
[0165] In some embodiments, the regulatory region does not only contain the promoter sequence of the A1S_2889 OM-responsive stress gene. In some embodiments, the regulatory region does not contain a regulatory region of the A1S_2889 OM-responsive stress gene. In some embodiments, the regulatory region does not include the sequence set forth in SEQ ID NO: 11.
B. Reporter Molecules
[0166] In some embodiments, the regulatory region of an OM stress-responsive gene, such as any described above, is operatively linked to a sequence encoding a reporter molecule.
Reporter molecules include molecules that express or that are capable of expressing or producing a detectable signal that is assayable or can be detected. In some aspects, the extent of detection of the assayable product indicates the presence, absence or quantity of the reporter molecule which, in turn, indicates the presence, absence, or degree of an OM-stress on a reporter microorganism bearing the reporter molecule. In some embodiments, the reporter molecule is any that is capable of producing a detectable signal when expressed in a reporter microorganism, such as Acinetobacter spp. (e.g. Acinetobacter baumannii). Such signals, including a fluorescent, bio luminescent or colorimetric signal, can be detected using routine techniques.
[0167] In certain embodiments, the reporter molecule is a fluorescent protein, a luminescent protein, a chromoprotein, or an enzyme.
[0168] In certain embodiments, the reporter molecule is a fluorescent protein. In certain embodiments, the fluorescent protein is a red, green, blue, or yellow fluorescent protein. In certain embodiments, the fluorescent protein is Sirius, SBFP2, Azurite, mAzurite, EBFP2, moxBFP, mKalamal, mTagBFP2, Aquamarine, ECFP, Cerulean, mCerulean, mCerulean3, moxCerulean3, SCFP3A, mTurquoise2, CyPet, AmCyanl, MiCy (Midoriishi-Cyan), iLOV, AcGFPl, sfGFP, moxGFP, mEmerald, EGFP, mEGFP, AzamiGreen, cfSGFP2, ZsGreen, SGFP2, Clover, mClover2, mClover3, EYFP, Topaz, mTopaz, mVenus, mox Venus, SYFP2, mCitrine, YPet, ZsYellowl, mPapayal, mKusabira-Orange (mKO), mOrange, mOrange2, mK02, TurboRFP, tdTomato, mScarlet-H, mNectarine, mRuby2, eqFP611, DsRed2, mApple, mScarlet, mStrawberry, FusionRed, mRFPl, mCherry, mCherry2, mCrimson3, HcRedl, dKatushka, mKatel.3, mPlum, niRaspberry, TagRFP675, mNeptune, mCardinal, mMaroon, TagRFP657, smURFP, miRFP670, iRFP670, iRFP682, miRFP703, iRFP702, miRFP709, mIFP 683, IFP2.0, iRFP, iSplit, iRFP720, T-sapphire, mT-sapphire, mAmetrine, LSSmOrange, mKeima Red, dKeima Red, LSSmKatel, LSSmKate2, Phamret, PA-sfGFP, mPA-Emerald, PA- GFP, PATagRFP, PAmCherryl, PAmCherry2, PAmCherry3, PAmKate, PAiRFPl, PAiRFP2, Dendra2, mEos3.2, mEos4a, dEos 505, tdEos 505, mKikGR, Kaede 508, PSmOrange, PSmOrange2, rsTagRFP, rsEmerald, rsGFPl, Dronpa3, mGeos-M, amilGFP, amilCP, or a modified version thereof. In some embodiments, the fluorescent protein is sfGFP, mClover3, mRuby2, Ypet, mCerulean, or mTagBFP2. In some embodiments, the fluorescent protein is sfGFP, mClover3, or mRuby2. In some embodiments, the fluorescent protein is sfGFP.
[0169] Suitable fluorescent reporters are available commercially (Clontech Labs Inc. USA, Evrogen Moscow, RU; MBL Int MA USA; Addgene Inc. MA USA).
[0170] In some embodiments, the sequence encoding the reporter molecule comprises a sequence with at least or at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOs: 15, 33, or 40. In some embodiments, the sequence encoding the reporter molecule comprises a sequence with at least or at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 15. In some
embodiments, the sequence encoding the reporter molecule comprises a sequence with at least or at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 33. In some embodiments, the sequence encoding the reporter molecule comprises a sequence with at least or at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 40.
[0171] In some embodiments, the reporter molecule is a luminescent protein. In certain embodiments, the luminescent protein is North American firefly luciferase, Genji-botaru luciferase, Italian firefly luciferase, Heike luciferase, East European firefly luciferase,
Pennsylvania firefly luciferase, Click beetle luciferase, Railroad worm luciferase, Renilla luciferase, Rluc8, Green Renilla luciferase, Gaussia luciferase, Gaussia-Dura luciferase, Cypridina luciferase, Vargula luciferase, Metridia luciferase, OLuc, bacterial luciferase (LuxAB), or a modified version thereof.
[0172] In certain embodiments, the reporter molecule is a chromoprotein. In certain embodiments the chromoprotein comprises a heme, riboflavin, or retinal. In certain
embodiments the chromoprotein is a hemoglobin, hemocyanin, chlorophyll, cytochrome, carotenoid, flavoprotein, or rhodopsin.
[0173] In certain embodiments, the reporter molecule is an enzyme. In some embodiments, the enzyme is one whose expression in a cell can be detected with a live cell assay (i.e. an assay which does not require cell fixation or lysis). In certain embodiments, the enzyme is chloramphenicol acetyltransferase (CAT), β-galactosidase, alkaline phosphatase, β- glucuronidase, β -lactamase, neomycin phosphotransferase, or a modified version thereof.
[0174] In certain embodiments, the reporter molecule is a gene necessary for the production of essential metabolites such as tryptophan, leucine, uracil, histidine, or methionine.
III. REPORTER VECTORS
[0175] In some embodiments, the provided reporter polynucleotides, such as any described in Section II, are comprised within a vector (hereinafter called a "reporter vector"). Suitable vectors can be used for any of the embodiments described herein. In some embodiments, a suitable vector can be used that is capable of replicating in a microorganism to which it is introduced. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences for driving transcription of the coding nucleotide sequence, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate, for expression in a bacterial cell as described herein.
[0176] In some embodiments, the vector comprises a reporter polynucleotide described herein. In some embodiments, the vector comprises a first reporter polynucleotide described herein and a second reporter polynucleotide described herein. In certain embodiments, the vector comprises (i) a first reporter polynucleotide comprising a first sequence comprising a regulatory region of a first OM stress-responsive gene of an Acinetobacter species (spp).
operably linked to a first sequence encoding a first reporter molecule, and (ii) a second reporter polynucleotide comprising a second sequence comprising a regulatory region of a second OM stress-responsive gene of an Acinetobacter species (spp). operably linked to a second sequence encoding a second reporter molecule, wherein the first and second OM stress-responsive genes are modulated in response to a stress to the outer membrane (OM-stress) of the Acinetobacter spp.
[0177] In certain embodiments, the first and second reporter polynucleotides share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second reporter polynucleotides are the same. In certain embodiments, the first and second reporter polynucleotides are different.
[0178] In some embodiments, the regulatory region of the first reporter polynucleotide and the regulatory region of the second reporter polynucleotide are from different OM stress- responsive genes. Thus, in some aspects, the first OM stress-responsive gene and the second OM stress-responsive gene are different. In some aspects, operatively linking a reporter molecule to different regulatory regions that are both responsive to OM stress can minimize false positives. In other aspects, the different regulatory regions may be responsive to different types of stress, such that the presence of a detectable signal induced from a microorganism carrying both reporter polynucleotides, or a first microorganism comprising a first reporter polynucleotide and a second microorganism comprising a second reporter polynucleotide, can assess different parameters or features of outer membrane stress. In some embodiments, in accord with the provided methods below, an agent is identified as causing or inducing OM stress only if a change in a detectable signal by or from both reporter molecules is observed. In some embodiments, the different regulatory regions are each operatively linked to the same reporter molecule. In some embodiments, the different regulatory regions are each operatively linked to different reporter molecules.
[0179] In certain embodiments, the first and second OM stress-responsive genes are the same or are functional variants. In certain embodiments, the first and second OM stress- responsive genes share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second OM stress-responsive genes are the same. In certain embodiments, the first and second OM stress-responsive genes are different.
[0180] In certain embodiments, the first and second sequences comprising the regulatory regions are the same or are functional variants. In certain embodiments, the first and second sequences comprising the regulatory regions share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second sequences comprising the regulatory regions are the same. In certain embodiments, the first and second sequences comprising the regulatory regions are different.
[0181] In some embodiments, the reporter molecule encoded by the first reporter polynucleotide, i.e. first reporter molecule, and the reporter molecule encoded by the second reporter polynucleotide, i.e. second reporter molecule, are different. Thus, in some aspects, the first reporter molecule and the second reporter molecule are different. In some embodiments, the first reporter molecule and the second reporter molecule do not exhibit an overlapping emission and absorption spectra. Reporter molecules capable of producing different, such as non-overlapping signals, can be separately or distinguishably detected. The use of several reporters can provide a multiplexed system to simultaneously measure or assess the presence or absence of a microorganism to one or more stress condition or potential stress condition and/or ensure that a response to a condition, e.g. caused by a physical condition or by an agent, is real or not likely to be a false positive. In one example, the first and second reporter molecule is independently sfGFP and mRuby2.
[0182] In certain embodiments, the first and second sequences encoding the first and second reporter molecules are the same or are functional variants. In certain embodiments, the first and second sequences encoding the reporter molecules share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second sequences encoding the first and second reporter molecules are the same. In certain
embodiments, the first and second sequences encoding the first and second reporter molecules are different. In certain embodiments, the first and second reporter molecules are the same.
[0183] In some embodiments, provided is a combination of two reporter vectors, such as a first and second reporter vector, as described herein. In some aspects, the first reporter vector includes a first reporter polynucleotide comprising a first sequence comprising a regulatory region of a first OM stress-responsive gene of an Acinetobacter species (spp). operably linked to a first sequence encoding a first reporter molecule. In some embodiments, the second reporter vector includes a second reporter polynucleotide comprising a second sequence comprising a regulatory region of a second OM stress-responsive gene of an Acinetobacter species (spp). operably linked to a second sequence encoding a second reporter molecule, wherein the first and second OM stress-responsive genes are modulated in response to a stress to the outer membrane (OM-stress) of the Acinetobacter spp. In some embodiments, the first and second reporter vector can be introduced into the same microorganism. In some embodiments, the first and second reporter vector can be introduced into different microorganisms.
[0184] One of skill in the art will recognize that reporter vectors are designed to contain certain components which optimize gene expression for certain microorganisms. Such optimization components include, but are not limited to, origins of replication, promoters, and enhancers.
[0185] In some embodiments, the vector contains an origin of replication in the IncQ incompatibility group that is proficient for replication in a broad host range, including in Acinetobacter spp. In some embodiments, the plasmid is a low-copy plasmid. In some embodiments, the vector is a high-copy plasmid.
[0186] In some aspects, the reporter vector contains a selective marker. Examples of selectable markers include, but are not limited to, antibiotic resistance nucleic acids (e.g., kanamycin, ampicillin, carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin, neomycin, or chloramphenicol) and/or nucleic acids that confer a metabolic advantage, such as a nutritional advantage on the reporter microorganism.
[0187] In certain embodiments, the provided reporter vector is capable of being expressed in any microorganism or progeny thereof that can be used to heterologously express genes. In certain embodiments, the reporter vector is capable of being expressed in any microorganisms described herein.
[0188] In certain embodiments, the reporter vector is capable of being expressed in a Gram- negative bacterium. In certain embodiments, the reporter vector is capable of being expressed in a multi-drug resistant bacterium. In certain embodiments, the vector is capable of being expressed in Acinetobacter, Bdellovibrio, Burkholderia, Chlamydia, Enterobacter, Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella, Neisseria, Pantoea, Pseudomonas, Salmonella, Shigella, Stenotrophomonas, Vibrio, or Yersinia. In particular embodiments, the reporter vector is capable of being expressed in Acinetobacter. In certain embodiments, the reporter vector is capable of being expressed in Acinetobacter apis,
Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii,
Acinetobacter brisouii, Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter gerneri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus, Acinetobacter harbinensis, Acinetobacter indicus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter kookii, Acinetobacter Iwoffii, Acinetobacter nectaris, Acinetobacter nosocomialis, Acinetobacter parvus, Acinetobacter pakistanensis, Acinetobacter pittii, Acinetobacter puyangensis, Acinetobacter qingfengensis, Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii,
Acinetobacter soli, Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis, or Acinetobacter venetianus.
[0189] In certain embodiments, the reporter vector is capable of being expressed in
Acinetobacter baumannii. In certain embodiments, the reporter vector is capable of being expressed in ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA-1605, ATCC BAA-1709, ATCC BAA-1710, ATCC BAA- 1789, ATCC BAA-1790, ATCC BAA-1791, ATCC BAA-1792, ATCC BAA- 1793, ATCC BAA-1794, ATCC BAA-1795, ATCC BAA- 1796, ATCC BAA-1797, ATCC BAA-1798, ATCC BAA-1799, ATCC BAA-1800, ATCC BAA-1878, ATCC BAA-2093, ATCC BAA-747, SDF, AYE, ACICU, AB0057, AB307-0294, 1656-2, MDR-ZJ06, TCDC-AB0715, MDR-TJ, TYTH-1, D1279779, BJAB07104, BJAB0715, BJAB0868, ZW85- 1, AbH120-A2, AB030, AB031, AC29, LAC-4, Ab307-0294, 1656-2, ID 403, AABA041, AABA046, or a modified strain thereof. In certain embodiments, the reporter vector is capable of being expressed in ATCC 17978, AABA041, AABA046, or Ab307-0294.
[0190] In certain embodiments, the reporter polynucleotide as described herein is comprised in a backbone vector. In some embodiments, a nucleotide sequence comprising the reporter polynucleotide is inserted into or replaces a portion of the nucleotide sequence of the backbone vector. Backbone vectors that can be expressed in specific microorganism are known in the art, for example pWH1266, or pET-RA (GenBank: M36473.1 and HM219006.1) are known to be capable of expression in Acinetobacter. In some embodiments, the backbone vector is pACH106 (SEQ ID NO: 29), pWH1266, or pET-RA. In some embodiments, the backbone vector has sequence with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:29. [0191] In some embodiments, the backbone vector comprises the sequence of nucleotides set forth in SEQ ID NO: 29 and a nucleotide sequence comprising the reporter polynucleotide replaces nucleotides 5,715-7,395 of the backbone vector. In some embodiments, the provided reporter vector comprises the sequence of nucleotides set forth in SEQ ID NO:29 in which nucleotides 5,715-7,395 thereof are replaced with a nucleotide sequence comprising a reporter polynucleotide comprising any of SEQ ID NOS: 1- 13, or a reporter polynucleotide with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOS: l-13; or a sequence with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of such reporter vectors. In some embodiments, the provided reporter vector comprises the sequence of nucleotides set forth in SEQ ID NO:29 in which nucleotides 5,715-7,395 thereof are replaced with a nucleotide sequence comprising a reporter polynucleotide comprising any of SEQ ID NOS: 16-28 or 34-36, or a reporter polynucleotide with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NOS: 16-28 or 34-36; or a sequence with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of such reporter vectors. In some embodiments, the provided reporter vector comprises the sequence of nucleotides set forth in SEQ ID NO:29 in which nucleotides 5,715-7,395 thereof are replaced with a nucleotide sequence comprising any of SEQ ID NOS: 1-13, or a sequence with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of such reporter vectors.
[0192] In some embodiments, the reporter vector is or comprises a sequence with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 30-32 or 37-39. In some embodiments, the reporter vector is or comprises the sequences set forth in any of SEQ ID NOS: 30-32 or 37-39. In some embodiments, the reporter vector is or comprises a sequence with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 30-32. In some embodiments, the reporter vector is or comprises the sequences set forth in any of SEQ ID NOS: 30-32. In some embodiments, the reporter vector is or comprises a sequence with at least or at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to any of SEQ ID NOs: 37-39. In some embodiments, the reporter vector is or comprises the sequences set forth in any of SEQ ID NOS: 37-39.
IV. REPORTER MICROORGANISMS
[0193] Provided herein are reporter microorganisms comprising any one or more reporter polynucleotide described herein or any one or more reporter vector described herein. In some embodiments, the reporter microorganism comprises 1, 2, 3, 4 or more reporter polynucleotides as described herein and/or 1, 2, 3, 4 or more reporter vectors as described herein. Thus, the reporter microorganism is a microorganism that is modified by introduction of one or more heterologous or exogenous reporter polynucleotides or reporter vectors as described into a host microorganism. For purposes of the description, where a reporter microorganism is described as being a particular microorganism it means that such reporter microorganism is derived or generated from such host microorganism by introduction of such one or more reporter polynucleotides or reporter vectors. Thus, the provided reporter microorganisms include a host microorganism comprising a reporter polynucleotide described herein or a reporter vector described herein. In some embodiments, the host microorganism comprises a first reporter polynucleotide and a second reporter polynucleotide. In some embodiment, the reporter polynucleotide is integrated into the genome of the host microorganism.
[0194] In some aspects, one or more reporter polynucleotides present in the provided reporter microorganisms includes as least two different reporter polynucleotides, i.e. a first reporter polynucleotide and a second reporter polynucleotide, as described. In some embodiments, the first and second reporter polynucleotides are comprised in or are part of the same reporter vector. In some embodiments, the first and second reporter polynucleotides are comprised in or are part of different reporter vectors. Hence, in some aspects, a reporter microorganism as provided herein can include two or more reporter vectors in which, for example, at least one reporter vector is a first reporter vector comprising the first reporter polynucleotide and at least one reporter vector is a second reporter vector comprising the second reporter polynucleotide.
[0195] In some embodiments, the reporter microorganism comprises more than one reporter polynucleotide described herein. In some embodiments, the reporter microorganism comprises a first reporter polynucleotide described herein and a second reporter polynucleotide described herein. In some embodiments, the reporter microorganism comprises more than one reporter vector described herein. In some embodiments, the reporter microorganism comprises a first reporter vector comprising a first reporter polynucleotide described herein and a second vector comprising a second reporter polynucleotide described herein. In certain embodiments, multiple copies of the same polynucleotide increase the signal from the reporter microorganism.
[0196] In some embodiments, the reporter microorganism comprises a first reporter polynucleotide described herein and a second reporter polynucleotide described herein. In certain embodiments, the reporter microorganism comprises (i) a first reporter polynucleotide comprising a first sequence comprising a regulatory region of a first OM stress-responsive gene of an Acinetobacter species (spp). operably linked to a first sequence encoding a first reporter molecule, and (ii) a second reporter polynucleotide comprising a second sequence comprising a regulatory region of a second OM stress-responsive gene of an Acinetobacter species (spp). operably linked to a second sequence encoding a second reporter molecule, wherein the first and second OM stress-responsive genes are modulated in response to a stress to the outer membrane (OM-stress) of the Acinetobacter spp.
[0197] In some embodiments, a reporter vector comprises the first reporter polynucleotide and the second reporter polynucleotide. In certain embodiments, a first reporter vector comprises a first reporter polynucleotide and a second reporter vector comprises the second reporter polynucleotide.
[0198] In certain embodiments, the first and second vectors are the same or are functional variants. In certain embodiments, multiple copies of the same vector increases signal from the reporter molecule. In certain embodiments, the first and second vectors share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second vectors are the same. In certain embodiments, the first and second vectors are different.
[0199] In certain embodiments, the first and second reporter polynucleotides are the same or functional variants. In certain embodiments, the first and second reporter polynucleotides share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second reporter polynucleotides are the same. In certain
embodiments, the first and second reporter polynucleotides are different. In certain
embodiments, different reporter polynucleotides allows for assaying different genes or strains in the same assay. [0200] In certain embodiments, the first and second OM stress-responsive genes are the same or are functional variants. In certain embodiments, the first and second OM stress- responsive genes share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second OM stress-responsive genes are the same. In certain embodiments, the first and second OM stress-responsive genes are different. In certain embodiments, different OM stress-responsive genes allow for the identification of broader spectrum or more potent agents. In certain embodiments, different OM stress- responsive genes allow for identification of more selective agents. For example, if the first OM stress-responsive gene was responsive to PMBN but not BamA depletion and the second OM stress-responsive gene was responsive to BamA depletion but not PMBN, different agents may be identified as including the first and/or second OM stress-responsive gene.
[0201] In some embodiments, the reporter microorganism includes at least two different reporter polynucleotides as described in which the regulatory region of a first reporter polynucleotide and the regulatory region of a second reporter polynucleotide are from different OM stress-responsive genes. Thus, in some aspects, the first OM stress-responsive gene and the second OM stress-responsive gene are different. In some aspects, such reporter microorganisms are capable of modulating a detectable signal in response to at least two different stress conditions, e.g. a physical condition or by an agent. In some cases, the ability of a single reporter microorganism to produce or modulate a detectable signal in response to one or more stress condition or potential stress condition can provide a multiplexed system to simultaneously measure or assess the susceptibility or resistance of a microorganism to one or more stress condition or potential stress condition and/or ensure that a response to a condition, e.g. caused by a physical condition or by an agent, is real or is not likely to be a false positive. In some embodiments, the different regulatory regions are each operatively linked to the same reporter molecule, such that the reporter microorganism is capable of producing a single detectable signal. In some embodiments, the different regulatory regions are each operatively linked to different reporter molecules, such that the reporter microorganism is capable of producing different detectable signals, e.g. induced by the different regulatory regions.
[0202] In certain embodiments, the first and second sequences comprising the regulatory regions are the same or functional variants. In certain embodiments, the first and second sequences comprising the regulatory regions share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second sequences comprising the regulatory regions are the same.
[0203] In some embodiments, the reporter microorganism is capable of producing at least two different detectable signals. Thus, in some aspects, the reporter microorganism contains at least two reporter polynucleotides as described, in which, for example, a regulatory region of the first OM-responsive stress gene is operatively linked to a first reporter molecule and the regulatory region of the second OM-responsive gene is operatively linked to a second reporter molecule that is different from the first reporter molecule. In some embodiments, the first reporter molecule and the second reporter molecule do not exhibit an overlapping emission and absorption spectra. Reporter molecules capable of producing different, such as non-overlapping signals, can be separately or distinguishably detected. In one example, the reporter
microorganism is capable of producing a detectable signal from a first and second reporter molecule that is independently sfGFP and mRuby2.
[0204] In certain embodiments, the first and second sequences encoding the first and second reporter molecules are the same or functional variants. In certain embodiments, the first and second sequences encoding the reporter molecules share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second sequences encoding the first and second reporter molecules are the same. In certain
embodiments, the first and second sequences encoding the first and second reporter molecules are different. In certain embodiments, the first and second reporter molecules are the same.
[0205] Also provided is a plurality of reporter microorganisms each including any one or more reporter polynucleotides as described or any one or more reporter vectors as described. In some embodiments, each of the plurality of reporter microorganisms includes a reporter polynucleotide that includes the same regulatory region of an OM-responsive gene. In some embodiments, each of the plurality of microorganisms includes the same reporter
polynucleotide. For example, each of the plurality of microorganisms includes a reporter polynucleotide containing the same regulatory region of the same OM responsive gene operatively linked to the sequence encoding a reporter molecule. In other embodiments, each of the plurality of microorganisms includes a different reporter polynucleotide. For example, in some cases, each of the plurality of reporter microorganisms is capable of producing a different detectable signal, whereby each contains a reporter polynucleotide that includes the same regulatory region of an OM-responsive gene, but that is operatively linked to a sequence encoding different reporter molecules. In some embodiments, the different reporter molecules do not exhibit overlapping emission and absorption spectra. Reporter molecules capable of producing different, such as non-overlapping signals, can be separately or distinguishably detected. In some aspects, such a plurality of microorganisms may be useful for comparing the affect or impact of various outer membrane stress conditions capable of engaging a common regulatory region in different microorganisms. In some aspects, the plurality of microorganisms include microorganisms across different strains, isolates or subtypes, such as any as described.
[0206] In some embodiments, each of the plurality of reporter microorganisms includes a different reporter polynucleotide that includes a regulatory region of a different OM-responsive gene. In some aspects, the microorganisms are of the same strain, isolate or subtype of a microorganism, e.g. bacteria. In some aspects, the plurality of microorganisms represents different strains, isolate or subtype of a microorganism, e.g. bacteria. In some aspects, such a plurality of reporter microorganisms can be screened together in response to the same stress condition, e.g. physical or via an agent. For example, the plurality of microorganisms can be co- encapsulated in a gel microdroplet as described. In some cases, a change in a detectable signal in or from each of the plurality of microorganisms in response to the same stress condition can indicate that the microorganisms are universally responsive or susceptible to the outer membrane stress condition and/or that there is one or more feature (e.g. conserved targeting of an outer membrane protein) that is similar or the same to each of the plurality of microorganisms rendering each similarly susceptible to the outer membrane stress. On the other hand, a change in a detectable signal in only one or a few, but not all, of the plurality of different
microorganisms in response to the same stress condition can indicate that susceptibility to the stress condition is specific to one or more microorganism, such as one or more strain, isolate or subtype.
[0207] In any of the provided embodiments, once the vectors are produced, they may be used to transfect and transform a host microorganism. Any procedure able to introduce the genetic material into the host microorganism for modulation (e.g. increase or decrease) of expression of the reporter molecule in the presence of OM-stress can be employed. In some embodiments, such methods include calcium phosphate transfection, electroporation, retroviral mediated transfection, polybrene, protoplast fusion, electroporation, liposomes, microinjection, plasmid vectors, viral vectors and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host
microorganism cell (see Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, (1989)).
[0208] Exemplary host microorganisms, and thus the resulting reporter microorganisms, include yeast, bacteria, and archaea. Bacteria cells, including Gram positive or Gram negative bacteria can be used to express any of the reporter polynucleotides described above. In certain embodiments, the host or reporter microorganism is a multi-drug resistant bacterium. In some embodiments, the host or reporter microorganism is a Gram-negative bacterium. Non-limiting examples include strains of Acinetobacter, Bdellovibrio, Burkholderia, Chlamydia,
Enterobacter, Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella, Neisseria, Pantoea, Pseudomonas, Salmonella, Shigella, Stenotrophomonas, Vibrio and Yersinia. In particular, the reporter polynucleotides or reporter vectors described above can be expressed in Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae,
Pseudomonas aeruginosa, Salmonella typhimurium, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Vibrio cholera, or Yersinia pestis.
[0209] In certain embodiments, the host microorganism, and hence the resulting reporter microorganism, is Acinetobacter. In certain embodiments, the host or reporter microorganism is Acinetobacter apis, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii, Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter gerneri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus, Acinetobacter harbinensis, Acinetobacter indicus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter kookii, Acinetobacter Iwoffii, Acinetobacter nectaris, Acinetobacter nosocomialis, Acinetobacter parvus, Acinetobacter pakistanensis, Acinetobacter pittii, Acinetobacter puyangensis, Acinetobacter qingfengensis, Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii, Acinetobacter soli, Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis, or Acinetobacter venetianus.
[0210] In particular embodiments, the host or reporter microorganism is Acinetobacter baumannii. In certain embodiments, the host microorganism is or is derived from ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA-1605, ATCC BAA-1709, ATCC BAA- 1710, ATCC BAA-1789, ATCC BAA-1790, ATCC BAA-1791, ATCC BAA-1792, ATCC BAA-1793, ATCC BAA- 1794, ATCC BAA- 1795, ATCC BAA-1796, ATCC BAA-1797, ATCC BAA-1798, ATCC BAA- 1799, ATCC BAA-1800, ATCC BAA-1878, ATCC BAA-2093, ATCC BAA-747, SDF, AYE, ACICU, AB0057, AB307-0294, 1656-2, MDR-ZJ06, TCDC-AB0715, MDR-TJ, TYTH-1, D1279779, BJAB07104, BJAB0715, BJAB0868, ZW85- 1, AbH120-A2, AB030, AB031, AC29, LAC-4, Ab307-0294, 1656-2, ID 403, AABA041, AABA046, or a modified strain thereof. In certain embodiments, the host microorganism is ATCC 17978, AABA041, AABA046, or Ab307-0294. In certain embodiments, the reporter microorganism is derived from ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA- 1605, ATCC BAA-1709, ATCC BAA-1710, ATCC BAA-1789, ATCC BAA- 1790, ATCC BAA-1791, ATCC BAA-1792, ATCC BAA- 1793, ATCC BAA-1794, ATCC BAA-1795, ATCC BAA-1796, ATCC BAA-1797, ATCC BAA-1798, ATCC BAA- 1799, ATCC BAA- 1800, ATCC BAA-1878, ATCC BAA-2093, ATCC BAA-747, SDF, AYE, ACICU, AB0057, AB307-0294, 1656-2, MDR-ZJ06, TCDC-AB0715, MDR-TJ, TYTH-1, D1279779,
BJAB07104, BJAB0715, BJAB0868, ZW85- 1, AbH120-A2, AB030, AB031, AC29, LAC-4, Ab307-0294, 1656-2, ID 403, AABA041, AABA046, or a modified strain thereof. In certain embodiments, the reporter microorganism is derived from ATCC 17978, AABA041, AABA046, or Ab307-0294.
[0211] In some embodiments, once transfected, the transformed reporter strains may be selected utilizing a selectable marker present on the vector (e.g. a drug resistance gene such as a Kanamycin resistance gene) and cultured by a variety of means known to those of skill in the art (see, e.g. Good et al. Clin. Chest Med. 10: 315-322 (1984), Heifets Ann. Rev. Respir. Dis. , 137: 1217-1222 (1988), and Sommers et al. in Color Atlas and Textbook of Diagnostic Microbiology, Third .Edition, J.B. Lippincott Co., Philadelphia, PA (1988)). V. MICRODROPLETS
[0212] Provided herein are microdroplets comprising a reporter microorganism or a plurality of reporter microorganisms as described herein. In some embodiments, the microdroplet comprises (i) an agent, such as a candidate agent, which, in some cases, can be an agent- producing cell, e.g. an antibody-producing cell and (ii) a reporter microorganism or a plurality of reporter microorganisms as provided herein.
[0213] In some embodiments, the microdroplet may comprise multiple copies of reporter microorganisms, such as multiple copies of the reporter microorganism or multiple copies and/or agent or agent-producing cell. In some embodiments the microdroplets may contain a single agent or agent-producing cell (e.g. antibody-producing cell) and multiple reporter
microorganisms. In some embodiments, the average number of reporter microorganisms per microdroplet can be between about 5 and about 500, such as about 10 and about 250, about 50 and about 200, about 50 and about 150, about 50 and about 100, or about 80 and about 120, such as about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200. The number of reporter microorganisms per microdroplet may be lower on average for microorganisms that are larger in cell size, e.g., a fungus or a parasite.
[0214] In some embodiments, the agent can be any agent as described herein. In some embodiments, the agent is a candidate antibacterial agent. In some embodiments, the agent is a small molecule, a peptide, or a protein, such as an antibody or antigen-binding fragment thereof. In some embodiments, the agent is a small molecule antibiotic or peptide antibiotic. In some embodiments, the peptide is a fragment of a larger protein. In some embodiments, the peptide comprises about 50 or fewer amino acids, such as about 45, 40, 35, 30, 25, 20, or fewer amino acids.
[0215] In some embodiments, the agent is bound to a solid support, such as a bead. In some embodiments, a cell comprises the agent. In some embodiments, a cell produces or secretes the agent. In some embodiments, the cell is an antibody-producing cell. In some embodiments, the cell is a B cell, a plasma cell, or a plasmablast. In some embodiments, the cell is a mammalian cell or a microorganism, such as a fungal or bacterial cell.
[0216] In some embodiments, the microdroplet comprises (i) an agent, such as a candidate agent, which, in some cases, can be an agent-producing cell, e.g. an antibody-producing cell; (ii) a first reporter microorganism as provided herein; and (iii) a second reporter microorganism as provided herein.
[0217] In some embodiments, the microdroplet comprises (i) an agent, such as a candidate agent, which, in some cases, can be an agent-producing cell, e.g. an antibody-producing cell; (ii) a first reporter microorganism comprising a first reporter polynucleotide comprising a first sequence comprising a regulatory region of a first OM stress-responsive gene of an
Acinetobacter species (spp). operably linked to a first sequence encoding a first reporter molecule; and (iii) a second reporter microorganism comprising a second reporter
polynucleotide comprising a second sequence comprising a regulatory region of a second OM stress-responsive gene of an Acinetobacter species (spp). operably linked to a second sequence encoding a second reporter molecule, wherein the first and second OM stress-responsive genes are modulated in response to a stress to the outer membrane (OM-stress) of the Acinetobacter spp.
[0218] In certain embodiments, the first reporter microorganism and the second reporter microorganism are clonal. In certain embodiments, the first reporter microorganism and the second reporter microorganism are not clonal. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from the same host strain. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from different host strains. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from different host species. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from the same host species. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from the same host strain and are not clonal. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from the same host species and are derived from different host strains. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from the same host species and are not clonal.
[0219] In certain embodiments, a first vector comprises a first reporter polynucleotide and a second vector comprises the second reporter polynucleotide. In certain embodiments, the first and second vectors share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second vectors are the same. In certain embodiments, the first and second vectors are different.
[0220] In certain embodiments, the first and second reporter polynucleotides share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second reporter polynucleotides are the same. In certain embodiments, the first and second reporter polynucleotides are different.
[0221] In certain embodiments, the first and second OM stress-responsive genes share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second OM stress-responsive genes are the same. In certain embodiments, the first and second OM stress-responsive genes are different.
[0222] In certain embodiments, the first and second sequences comprising the regulatory regions share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second sequences comprising the regulatory regions are the same. In certain embodiments, the first and second sequences comprising the regulatory regions are different.
[0223] In certain embodiments, the first and second sequences encoding the reporter molecules share at least or at least about 75%, 80%, 85%, 90%, 95%, or 99% sequence identity. In certain embodiments, the first and second sequences encoding the first and second reporter molecules are the same. In certain embodiments, the first and second sequences encoding the first and second reporter molecules are different. In certain embodiments, the first and second reporter molecules are the same. In certain embodiments, the first and second reporter molecules are different.
[0224] In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from different host strains and the first and second reporter polynucleotides are different. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from different host strains; the first and second reporter molecules are different; and the first and second OM stress-responsive genes are the same.
[0225] In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from the same host strain and the first and second reporter polynucleotides are different. In certain embodiments, the first reporter microorganism and the second reporter microorganism are derived from the same host strain; the first and second reporter molecules are different; and the first and second OM stress-responsive genes are different.
[0226] In some embodiments, the microdroplet comprises a polymer matrix and/or a gel matrix. In certain embodiments, microdroplet comprises agarose, carrageenan, alginate, alginate -polylysine, collagen, cellulose, methylcellulose, gelatin, chitosan, extracellular matrix, dextran, starch, inulin, heparin, hyaluronan, fibrin, polyvinyl alcohol, poly(N-vinyl-2- pyrrolidone), polyethylene glycol, poly(hydroxyethyl methacrylate), acrylate polymers and sodium
polyacrylate, polydimethyl siloxane, cis-polyisoprene, Puramatrix™, poly-divenylbenzene, polyurethane, or polyacrylamide. In particular embodiments, the microdroplet comprises a polymer matrix, which may be e.g., agarose, carrageenan, alginate, alginate-polylysine, collagen, a plant-derived gum, cellulose or a derivatives thereof (e.g., methylcellulose), gelatin, chitosan or an extracellular matrix (ECM), as described by Kleinman (U.S. Pat. No. 4,829,000), or combinations thereof. Synthetic hydrogels that may be used in the microdroplet include but are not limited to polyvinyl alcohol, block copolymer of ethylene- vinylalcohol, sodium polystyrene sulfonate, vinyl-methyl-tribenzyl ammonium chloride and polyphosphazene.
[0227] In some embodiments, microdroplet comprises agarose. In some embodiments, the agarose is low gelling temperature agarose, such as an ultra-low gelling temperature agarose. In some embodiments, the low gelling temperature agarose allows for the agarose to stay liquid at lower temperatures, e.g., temperatures that permit viability of the agent-producing cells (e.g. antibody-producing cell) and the reporter microorganism and thereby allow live cell and reporter microorganism encapsulation. In some embodiments, the gelling temperature of the agarose used in encapsulation is such that the temperature of liquid agarose does not adversely affect viability of the agent-producing cells (e.g. antibody-producing cell) and/or the reporter microorganism, and encapsulation can be carried out in a liquid state. In some embodiments, the agarose has a gelling temperature of lower than about 35°C, such as about 30°C, about 25°C, about 20°C, about 15°C, about 10°C or about 5°C. In some embodiments, the agarose is an ultra- low gelling temperature agarose, such as those with a gelling temperature of lower than about 20°C, about 15°C, about 10°C or about 5°C. In some embodiments, the agarose has a gelling temperature of between about 5°C and about 30°C, about 5°C and about 20°C, about 5°C and about 15°C, about 8°C and about 17°C or about 5°C and about 10°C, such as about 8°C and about 17°C.
[0228] In some embodiments, the microdroplet comprises growth media and/or is surrounded by a non-aqueous environment. In some embodiments, the non-aqueous
environment includes an oil. In some embodiments, the oil is gas permeable.
[0229] In some embodiments, the microdroplet comprises serum. In some embodiments, the serum is human, bovine, rabbit, or mouse serum. In some aspects, the concentration of serum is at least or at least about 2.5% (vol/vol), 5.0% (vol/vol), 7.5% (vol/vol), 10.0% (vol/vol), 15.0% (vol/vol), 20% (vol/vol) or 25% (vol/vol). In some aspects, the concentration of serum is from or from about 2.5% (vol/vol) to 25% (vol/vol), 2.5% (vol/vol) to 15% (vol/vol), 2.5% (vol/vol) to 10% (vol/vol), 2.5% (vol/vol) to 5.0% (vol/vol), 5.0% (vol/vol) to 25% (vol/vol), 5.0% (vol/vol) to 15% (vol/vol), 5.0% (vol/vol) to 10% (vol/vol), 10.0% (vol/vol) to 25% (vol/vol), 10.0% (vol/vol) to 15% (vol/vol) or 15% (vol/vol) to 25% (vol/vol).
[0230] Provided herein are populations of microdroplets comprising reporter
microorganisms. In some embodiments, the microdroplets, on average, comprise one or fewer agent or agent-producing cell (e.g. antibody-producing cells). In some embodiments, the average ratio of candidate agents or candidate agent-producing cell (e.g. antibody-producing cell) per microdroplet is less than or less than about 1. In some embodiments, the average ratio of candidate agents or agent-producing cell (e.g. antibody-producing cell) per microdroplet is between about 0.05 and about 1.0, about 0.05 and about 0.5, about 0.05 and about 0.25, about 0.05 and about 0.1, about 0.1 and about 1.0, about 0.1 and about 0.5, about 0.1 and about 0.25, about 0.25 and about 1.0, about 0.25 and about 0.5 or 0.5 and about 1.0, each inclusive. In some embodiments, t the average ratio of agent or agent-producing cell (e.g. antibody-producing cells) per microdroplet is or is about 0.1.
I. COMPOSITIONS, KITS AND ARTICLES OF MANUFACTURE
[0231] Provided herein are compositions comprising a reporter polynucleotide, a reporter vector, a reporter microorganism or a microdroplet as described herein. In certain embodiments, the composition further comprises an excipient. Acceptable excipients are known in the art, such as, for example buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins;
polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In some embodiments, the composition comprises media. In some embodiments, the composition comprises an agent or a combination of agents as described herein. In some embodiments, the composition comprises one or more components capable of activating the complement pathway. In some embodiments, the composition comprises serum, such as human, bovine, rabbit, or mouse serum. In some embodiments, the composition comprises a plurality of the reporter
polynucleotides, reporter vectors, reporter microorganisms or microdroplets as described herein.
[0232] Provided herein are articles of manufacture or kits comprising a reporter
polynucleotide, a reporter vector, a reporter microorganism, a microdroplet, or a composition. In some embodiments, the kits include instructions for use in the provided methods.
[0233] In certain embodiments, kits include one or more containers containing one component, and the component is a reporter polynucleotide, a reporter vector, a reporter microorganism, or a composition described herein. In some embodiments, the kit further comprises instructions for use in accordance with any of the methods described herein.
Instructions supplied in the kits are typically written instructions on a label or package insert (e.g., a paper sheet included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable. Kits may optionally provide additional components such as buffers and interpretative information. In some embodiments, the components of the kits are contained in one or more containers.
[0234] In some embodiments, the kit contains a reporter microorganism. In some aspects, the reporter microorganism may be in a form suitable for culture or for direct use in an assay, such as a screening assay as described. In some embodiments, the kits may contain other materials suitable for the practice of the assays. For example, the kits may also contain buffers, media for culture of the reporter strains, and other components for performing the assay or culturing or maintaining the cells.
[0235] In some embodiments, the kits provide a reporter vector suitable for the production of reporter microorganisms. In some aspects, the kits contain various reagents to facilitate the production of reporter microorganisms. Exemplary of such reagents include, but are not limited to, the pathogen to be transformed, culture media, buffers, drugs for selection of transformants, and components for producing a reporter microorganism. [0236] In certain embodiments, the kits are in suitable packaging. Suitable packaging include, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like.
VII. METHODS OF USE
[0237] Provided herein are methods of using the reporter polynucleotides, reporter vectors, reporter microorganisms, and compositions described herein. The reporter microorganisms containing the reporter polynucleotides or reporter vectors can be used in any desired method in which a change (e.g. increase or decrease) in a signal from the reporter molecule provides a readout of a property or activity of a microorganism, including any property or activity that is or may be or is suspected of being modulated in a response to a condition or in the presence of one or more agents compared to in the absence of the condition or the one or more agents. In certain embodiments, provided herein are methods of assessing outer membrane (OM) stress, methods for identifying an agent that modulates an activity or property of a microorganism, methods of screening agents, including candidate antibacterial agents, and methods of determining the drug resistance of a microorganism. Also provided herein are agents identified by any method described herein.
[0238] In some embodiments, the provided methods include contacting a reporter microorganism (i) to a condition that is known to cause or suspected of causing OM stress to the reporter microorganism and/or (ii) with one or more agents, including candidate agents, that may impact the integrity or biogenesis of the outer membrane of the reporter microorganism. In some embodiments, following the contacting the reporter microorganism can be assessed or monitored for a change in a detectable signal from the reporter molecule contained therein compared to in the absence of the condition or in the absence of contacting the reporter microorganism with the one or more agents or candidate agents.
[0239] In some embodiments, the provided methods include: (a) contacting or exposing the reporter microorganism or a microorganism containing the reporter polynucleotide or reporter vector provided herein with a condition that is known to cause or suspected of causing stress to the outer membrane and (b) determining if there is a change in a detectable signal from the reporter molecule compared to in the absence of the conditions. In some such embodiments, such methods can be used to assess if the outer membrane of the reporter microorganism is stressed and/or if the integrity or biogenesis of the outer membrane of the reporter microorganism is being impacted in the presence of the condition. In some embodiments, the condition can be a physical condition. In some embodiments, the condition is caused by the addition of an exogenous agent, such as one or more candidate agents, to which the reporter microorganism is exposed.
[0240] In some embodiments, the provided methods include: (a) contacting the reporter microorganism or a microorganism containing the reporter polynucleotide or reporter vector provided herein with an agent and (b) identifying a reporter microorganism in which there is a change in a detectable signal from the reporter molecule compared to in the absence of contacting with the agent. In some such embodiments, such methods can be used to identify an agent that causes stress to the outer membrane of a microorganism and/or impacts or modulates the integrity or biogenesis of the outer membrane of a microorganism. In some embodiments, the agent is a biological molecule or drug, such as a small molecule compound, peptide, or polypeptide or protein. In some embodiments, the agent is produced or secreted from a cell, in which case the reporter microorganism is contacted with an agent-producing cell. In some embodiments, the method can be carried out a plurality of agents, such as a plurality or library of candidate agents or a plurality of agent-producing cells, such that the reporter microorganism is contacted with each of the plurality of agents. In some embodiments, the reporter
microorganism is separately contacted with each of the plurality of agents, such as each of the plurality of agent-producing cells.
[0241] In some embodiments, the provided methods include (a) contacting the reporter microorganism or a microorganism containing the reporter polynucleotide or reporter vector provided herein with a drug and (b) identifying a reporter microorganism in which there is a change in a detectable signal from the reporter molecule compared to in the absence of contacting with the drug. In some embodiments, such methods can be used to identify if a microorganism is resistant to the drug. In some embodiments, if there is a change in the detectable signal, the microorganism is not resistant to the drug and if there is not a change in the detectable signal, the microorganism is identified as potentially being resistant to the drug. In some embodiments, the drug is a small molecule antibiotic or peptide antibiotic. In some embodiments, the drug is known.
[0242] In some embodiments, the change in signal is a decrease in signal. In some embodiments, the change in signal is an increase in signal. [0243] In some embodiments, expression of the reporter molecule and/or presence of a signal therefrom is not detected or present in the reporter microorganism or is detected or present at only a low background level when cultured under conditions that optimize or maintain the health of the bacteria and/or under conditions that do not impact the integrity or biogenesis of the outer membrane. In certain embodiments, expression of the reporter molecule and/or presence of a signal therefrom is induced or increased by OM- stress of the Acinetobacter spp., such as in the presence of an OM stress condition and/or in the presence of one or more agent that causes outer membrane stress and/or impacts the integrity or biogenesis of the outer membrane. In certain embodiments, expression of the reporter molecule and/or presence of a signal therefrom is increased at least or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100 or more fold. In certain embodiments, the increased expression is compared to a reporter microorganism that was not contacted with the agent. In certain embodiments, the increased expression is compared to the reporter microorganism prior to contact with the agent. In certain embodiments, the reporter molecule is not expressed unless the reporter microorganism is contacted with the agent.
[0244] In some embodiments in which a change in signal is an increase in signal, the regulatory region present in the provided reporter polynucleotides or vectors contained in the reporter microorganism is from or derived from a non-coding regulatory region of an OM stress- responsive gene whose expression is upregulated or increased in the presence of OM stress. In some embodiments, the OM stress-responsive gene is A1S_0012, A1S_0023, A1S_0027, A1S_0028, A1S_0029, A1S_0030, A1S_0031, A1S_0032, A1S_0033, A1S_0037, A1S_0040, A1S_0041, A1SJ3044, A1S_0066, A1S_0092, A1S_0093, A1S_0109, A1S_0110, A1S_0112, A1S_0113, A1S_0114, A1S_0115, A1S_0116, A1S_0117, A1S_0118, A1S_0126, A1S_0158, A1S_0170, A1S_0175, A1S_0178, A1S_0189, A1S_0224, A1S_0245, A1S_0256, A1S_0276, A1SJ3293, A1SJ3301, A1S_0309, A1S_0310, A1S_0332, A1S_0333, A1S_0363, A1S_0372, A1S_0376, A1S_0391, A1S_0392, A1S_0401, A1S_0441, A1S_0462, A1S_0463, A1S_0464, A1S_0465, A1S_0466, A1S_0494, A1S_0508, A1S_0509, A1S_0510, A1S_0511, A1S_0512, A1SJ3514, A1SJ3516, A1S_0518, A1S_0519, A1S_0520, A1S_0521, A1S_0522, A1S_0523, A1S_0527, A1S_0535, A1S_0536, A1S_0537, A1S_0538, A1S_0547, A1S_0559, A1S_0561, A1S_0562, A1S_0563, A1S_0564, A1S_0566, A1S_0567, A1S_0568, A1S_0570, A1S_0624, A1S_0630, A1S_0631, A1S_0633, A1S_0634, A1S_0640, A1S_0641, A1S_0642, A1S_0643, A1S_ _0644, A1S_ _0645 A1S_ _0646, A1S_ _0647 A1S_ _0650, A1S_ _0663 A1S_ _0664, A1S_ _0665,
A1S_ _0666, A1S_ _0667 A1S_ _0669, A1S_ _0670 A1S_ _0671, A1S_ _0673 A1S_ _0677, A1S_ _0680,
A1S_ _0683, A1S_ _0714 A1S_ _0717, A1S_ _0718 A1S_ _0719, A1S_ _0736 A1S_ _0738, A1S_ _0739,
A1S_ _0749, A1S_ _0770 A1S_ _0772, A1S_ _0779 A1S_ _0780, A1S_ _0781 A1S_ _0800, A1S_ _0804,
A1S_ _0830, A1S_ _0831 A1S_ _0832, A1S_ _0834 A1S_ _0835, A1S_ _0861 A1S_ _0884, A1S_ _0889,
A1S_ _0929, A1S_ _0930 A1S_ _0931, A1S_ _0932 A1S_ _0935, A1S_ _0945 A1S_ _0959, A1S_ _0980,
A1S_ _1003, A1S_ _1009 A1S_ _1010, A1S_ _1027 A1S_ _1028, A1S_ _1030 A1S_ _1031, A1S_ _1049,
A1S_ _1081, A1S_ _1106 A1S_ _1107, A1S_ _1120 A1S_ _1121, A1S_ _1123 A1S_ _1132, A1S_ _1133,
A1S_ _1134, A1S_ _1139 A1S_ _1143, A1S_ _1145 A1S_ _1146, A1S_ _1148 A1S_ _1149, A1S_ _1150,
A1S_ _1151, A1S_ _1152 A1S_ _1153, A1S_ _1155 A1S_ _1156, A1S_ _1157 A1S_ _1158, A1S_ _1159,
A1S_ _1160, A1S_ _1161 A1S_ _1162, A1S_ _1163 A1S_ _1164, A1S_ _1165 A1S_ _1167, A1S_ _i m,
A1S_ _1172, A1S_ _1173 A1S_ _1180, A1S_ _1184 A1S_ _1186, A1S_ _1198 A1S_ _1202, A1S_ _1203,
A1S_ _1223, A1S_ _1224 A1S_ _1225, A1S_ _1230 A1S_ _1236, A1S_ _1237 A1S_ _1248, A1S_ _1255,
A1S_ _1274, A1S_ _1286 A1S_ _1359, A1S_ _1360 A1S_ _1361, A1S_ _1362 A1S_ _1363, A1S_ _1383,
A1S_ _1384, A1S_ _1385 A1S_ _1386, A1S_ _1387 A1S_ _1393, A1S_ _1404 A1S_ _1407, A1S_ _1422,
A1S_ _1454, A1S_ _1472 A1S_ _1481, A1S_ _1494 A1S_ _1512, A1S_ _1515 A1S_ _1526, A1S_ _1535,
A1S_ _1539, A1S_ _1566 A1S_ _1567, A1S_ _1569 A1S_ _1583, A1S_ _1584 A1S_ _1585, A1S_ _1589,
A1S_ _1590, A1S_ _1593 A1S_ _1595, A1S_ _1596 A1S_ _1617, A1S_ _1630 A1S_ _1644, A1S_ _1645,
A1S_ _1647, A1S_ _1648 A1S_ _1649, A1S_ _1651 A1S_ _1655, A1S_ _1658 A1S_ _1662, A1S_ _1666,
A1S_ _1667, A1S_ _1669 A1S_ _1677, A1S_ _1680 A1S_ _1681, A1S_ _1687 A1S_ _1735, A1S_ _1741,
A1S_ _1743, A1S_ _1744 A1S_ _1750, A1S_ _1751 A1S_ _1752, A1S_ _1760 A1S_ _1762, A1S_ _1767,
A1S_ _1778, A1S_ _1813 A1S_ _1827, A1S_ _1829 A1S_ _1831, A1S_ _1843 A1S_ _1876, A1S_ _1909,
A1S_ _1928, A1S_ _1929 A1S_ _1934, A1S_ _1952 A1S_ _1955, A1S_ _1956 A1S_ _1957, A1S_ _1959,
A1S_ _1960, A1S_ _1961 A1S_ _1962, A1S_ _1963 A1S_ _1979, A1S_ _1986 A1S_ _1987, A1S_ _1988,
A1S_ _2006, A1S_ _2026 A1S_ _2033, A1S_ _2034 A1S_ _2035, A1S_ _2036 A1S_ _2038, A1S_ _2039,
A1S_ _2061, A1S_ _2074 A1S_ _2079, A1S_ _2082 A1S_ _2092, A1S_ _2093 A1S_ _2106, A1S_ _2139,
A1S_ _2140, A1S_ _2141 A1S_ _2142, A1S_ _2146 A1S_ _2157, A1S_ _2158 A1S_ _2160, A1S_ _2161,
A1S_ _2162, A1S_ _2178 A1S_ _2179, A1S_ _2183 A1S_ _2186, A1S_ _2195 A1S_ _2230, A1S_ _2247,
A1S_ _2252, A1S_ _2257 A1S_ _2258, A1S_ _2259 A1S_ _2262, A1S_ _2271 A1S_ _2272, A1S_ _2273,
A1S_ _2283, A1S_ _2285 A1S_ _2298, A1S_ _2311 A1S_ _2315, A1S_ _2325 A1S_ _2326, A1S_ _2330,
A1S_ _2331, A1S_ _2366 A1S_ _2367, A1S_ _2382 A1S_ _2387, A1S_ _2389 A1S_ _2395, A1S_ _2396, A1S_ _2414, A1S_ _2434 A1S_ _2445, A1S_ _2446 A1S_ _2447, A1S_ _2448 A1S_ _2454, A1S_ _2455,
A1S_ _2456, A1S_ _2458 A1S_ _2459, A1S_ _2463 A1S_ _2480, A1S_ _2489 A1S_ _2503, A1S_ _2504,
A1S_ _2508, A1S_ _2542 A1S_ _2543, A1S_ _2552 A1S_ _2553, A1S_ _2555 A1S_ _2557, A1S_ _2558,
A1S_ _2573, A1S_ _2577 A1S_ _2578, A1S_ _2580 A1S_ _2586, A1S_ _2588 A1S_ _2593, A1S_ _2611,
A1S_ _2612, A1S_ _2613 A1S_ _2624, A1S_ _2650 A1S_ _2651, A1S_ _2654 A1S_ _2656, A1S_ _2660,
A1S_ _2664, A1S_ _2668 A1S_ _2675, A1S_ _2676 A1S_ _2677, A1S_ _2678 A1S_ _2684, A1S_ _2705,
A1S_ _2729, A1S_ _2734 A1S_ _2756, A1S_ _2768 A1S_ _2786, A1S_ _2798 A1S_ _2801, A1S_ _2807,
A1S_ _2826, A1S_ _2827 A1S_ _2828, A1S_ _2839 A1S_ _2863, A1S_ _2882 A1S_ _2883, A1S_ _2884,
A1S_ _2885, A1S_ _2889 A1S_ _2892, A1S_ _2893 A1S_ _2942, A1S_ _2943 A1S_ _2953, A1S_ _2959,
A1S_ _2960, A1S_ _2968 A1S_ _2976, A1S_ _2992 A1S_ _3011, A1S_ _3026 A1S_ _3027, A1S_ _3034,
A1S_ _3035, A1S_ _3047 A1S_ _3048, A1S_ _3099 A1S_ _3100, A1S_ _3101 A1S_ _3104, A1S_ _3105,
A1S_ _3114, A1S_ _3115 A1S_ _3116, A1S_ _3117 A1S_ _3124, A1S_ _3125 A1S_ _3126, A1S_ _3127,
A1S_ _3139, A1S_ _3146 A1S_ _3147, A1S_ _3175 A1S_ _3206, A1S_ _3224 A1S_ _3253, A1S_ _3259,
A1S_ _3280, A1S_ _3281 A1S_ _3295, A1S_ _3317 A1S_ _3326, A1S_ _3339 A1S_ _3360, A1S_ _3361,
A1S_ _3367, A1S_ _3368 A1S_ _3371, A1S_ _3375 A1S_ _3376, A1S_ _3392 A1S_ _3411, A1S_ _3412,
A1S_ _3463, A1S_ _3466 A1S_ _3468, A1S_ _3469 A1S_ _3471, A1S_ _3479 A1S_ _3480, A1S_ _3486,
A1S_ _3492, A1S_ _3493 A1S_ _3494, A1S_ _3499 A1S_ _3510, A1S_ _3512 A1S_ _3518, A1S_ _3522,
A1S_ _3523, A1S_ _3533 A1S_ _3534, A1S_ _3535 A1S_ _3539, A1S_ _3540 A1S_ _3541, A1S_ _3542,
A1S_ _3543, A1S_ _3544 A1S_ _3545, A1S_ _3546 A1S_ _3548, A1S_ _3552 A1S_ _3553, A1S_ _3558,
A1S_ _3559, A1S_ _3562 A1S_ _3563, A1S_ _3567 A1S_ _3570, A1S_ _3577 A1S_ _3580, A1S_ _3580,
A1S_ _3585, A1S_ _3586 A1S_ _3594, A1S_ _3595 A1S_ _3596, A1S_ _3601 A1S_ _3602, A1S_ _3603,
A1S_ _3604, A1S_ _3605 A1S_ _3606, A1S_ _3607 A1S_ _3608, A1S_ _3609 A1S_ _3610, A1S_ _3611,
A1S_ _3612, A1S_ _3613 A1S_ _3614, A1S_ _3617 A1S_ _3618, A1S_ _3621 A1S_ _3630, A1S_ _3632,
A1S_ _3634, A1S_ _3635 A1S_ _3636, A1S_ _3637 A1S_ _3642, A1S_ _3645 A1S_ _3649, A1S_ _3654,
A1S_ _3658, A1S_ _3661 A1S_ _3662, A1S_ _3666 A1S_ _3682, A1S_ _3686 A1S_ _3687, A1S_ _3688,
A1S_ _3694, A1S_ _3695 A1S_ _3697, A1S_ _3704 A1S_ _3707, A1S_ _3708 A1S_ _3712, A1S_ _3716,
A1S_ _3725, A1S_ _3726 A1S_ _3727, A1S_ _3728 A1S_ _3736, A1S_ _3738 A1S_ _3739, A1S_ _3740,
A1S_ _3750, A1S_ _3752 A1S_ _3760, A1S_ _3768 A1S_ _3769, A1S_ _3770 A1S_ _3771, A1S_ _3772,
A1S_ _3773, A1S_ _3776 A1S_ _3777, A1S_ _3778 A1S_ _3782, A1S_ _3783 A1S_ _3786, A1S_ _3789,
A1S_ _3790, A1S_ _3791 A1S_ _3792, A1S_ _3797 A1S_ _3810, A1S_ _3818 A1S_ _3820, A1S_ _3835,
A1S_ _3837, A1S_ _3840 A1S_ _3842, A1S_ _3844 A1S_ _3862, A1S_ _3864 A1S_ _3865, A1S_ _3866, A1S_3867, A1S_3868, A1S_3873, A1S_3875, A1S_3879, A1S_3889, A1S_3900, A1S_3901, A1S_3902, A1S_3908, or A1S_3911.
[0245] In certain embodiments, the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_0189, A1S_0516, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, A1S_3127, A1S_3492, or AIS_3791. In some
embodiments, the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or
A1S_3127. In certain embodiments, the OM stress-responsive gene is A1S_0032, A1S_2885, A1S_2889, A1S_3127, A1S_3492, or A1S_3791. In some embodiments, the OM stress- responsive gene is A1S_0032, A1S_2885, or A1S_2889. In certain embodiments, the OM stress-responsive gene is A1S_0113 or A1S_1751. In certain embodiments, the OM stress- responsive gene is A1S_0189, A1S_0516, A1S_1224, A1S_2093, or A1S_2271.
[0246] In some embodiments, expression of the reporter molecule and/or presence of a signal therefrom is constitutive in the reporter microorganism when cultured under conditions that optimize or maintain the health of the bacteria and/or that do not impact the integrity or biogenesis of the outer membrane. In some embodiments, expression of the reporter molecule and/or presence of a signal therefrom is decreased by OM-stress of the Acinetobacter spp., such as in the presence of an OM stress condition and/or in the presence of one or more agent that causes outer membrane stress and/or impacts the integrity or biogenesis of the outer membrane. In certain embodiments, the expression of the reporter molecule and/or presence of a signal therefrom is decreased at least or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 100 or more fold. In certain embodiments, the decreased expression is compared to a reporter microorganism that was not contacted with the agent. In certain embodiments, the decreased expression is compared to the reporter microorganism prior to contact with the agent.
[0247] In some embodiments in which a change in signal is a decrease in signal, the regulatory region present in the provided reporter polynucleotides or vectors contained in the reporter microorganism is from or derived from a non-coding regulatory region of an OM stress- responsive gene whose expression is down-regulated in the presence of OM stress. In some embodiments, the OM stress-responsive gene is A1SJ3009, A1S_0010, A1SJ3025, A1SJ3027, A1S_0038, A1S_0067, A1S_0070, A1S_0071, A1S_0073, A1S_0076, A1S_0077, A1S_0079, A1SJ3087, A1SJ3090, A1S_0091, A1S_0095, A1S_0096, A1S_0097, A1S_0098, A1S_0099, A1S_ _0103, A1S_ _0104 A1S_ _0105, A1S_ _0106 A1S_ _0107, A1S_ _0108 A1S_ _0109, A1S_ _0121,
A1S_ _0128, A1S_ _0129 A1S_ _0141, A1S_ _0148 A1S_ _0150, A1S_ _0151 A1S_ _0152, A1S_ _0153,
A1S_ _0154, A1S_ _0155 A1S_ _0156, A1S_ _0157 A1S_ _0177, A1S_ _0184 A1S_ _0200, A1S_ _0201,
A1S_ _0209, A1S_ _0218 A1S_ _0238, A1S_ _0239 A1S_ _0253, A1S_ _0257 A1S_ _0258, A1S_ _0269,
A1S_ _0270, A1S_ _0279 A1S_ _0286, A1S_ _0292 A1S_ _0302, A1S_ _0303 A1S_ _0304, A1S_ _0321,
A1S_ _0322, A1S_ _0323 A1S_ _0347, A1S_ _0365 A1S_ _0369, A1S_ _0370 A1S_ _0388, A1S_ _0408,
A1S_ _0410, A1S_ _0427 A1S_ _0429, A1S_ _0447 A1S_ _0448, A1S_ _0474 A1S_ _0480, A1S_ _0481,
A1S_ _0482, A1S_ _0486 A1S_ _0490, A1S_ _0491 A1S_ _0498, A1S_ _0526 A1S_ _0533, A1S_ _0534,
A1S_ _0548, A1S_ _0549 A1S_ _0566, A1S_ _0567 A1S_ _0568, A1S_ _0591 A1S_ _0594, A1S_ _0624,
A1S_ _0625, A1S_ _0626 A1S_ _0627, A1S_ _0629 A1S_ _0630, A1S_ _0631 A1S_ _0632, A1S_ _0633,
A1S_ _0634, A1S_ _0635 A1S_ _0639, A1S_ _0640 A1S_ _0641, A1S_ _0642 A1S_ _0643, A1S_ _0644,
A1S_ _0645, A1S_ _0646 A1S_ _0647, A1S_ _0649 A1S_ _0650, A1S_ _0651 A1S_ _0690, A1S_ _0691,
A1S_ _0692, A1S_ _0695 A1S_ _0698, A1S_ _0721 A1S_ _0731, A1S_ _0732 A1S_ _0771, A1S_ _0785,
A1S_ _0786, A1S_ _0787 A1S_ _0788, A1S_ _0818 A1S_ _0822, A1S_ _0846 A1S_ _0849, A1S_ _0850,
A1S_ _0851, A1S_ _0852 A1S_ _0853, A1S_ _0854 A1S_ _0855, A1S_ _0869 A1S_ _0877, A1S_ _0882,
A1S_ _0883, A1S_ _0890 A1S_ _0891, A1S_ _0901 A1S_ _0910, A1S_ _0911 A1S_ _0912, A1S_ _0913,
A1S_ _0960, A1S_ _0965 A1S_ _0973, A1S_ _0984 A1S_ _0996, A1S_ _0997 A1S_ _0999, A1S_ _1000,
A1S_ _1004, A1S_ _1008 A1S_ _1021, A1S_ _1026 A1S_ _1044, A1S_ _1063 A1S_ _1072, A1S_ _1079,
A1S_ _1080, A1S_ _1088 A1S_ _1089, A1S_ _1091 A1S_ _1092, A1S_ _1093 A1S_ _1094, A1S_ _1109,
A1S_ _1113, A1S_ _1139 A1S_ _1142, A1S_ _1182 A1S_ _1193, A1S_ _1195 A1S_ _1199, A1S_ _1227,
A1S_ _1257, A1S_ _1258 A1S_ _1261, A1S_ _1264 A1S_ _1265, A1S_ _1266 A1S_ _1267, A1S_ _1268,
A1S_ _1269, A1S_ _1270 A1S_ _1281, A1S_ _1317 A1S_ _1318, A1S_ _1319 A1S_ _1327, A1S_ _1334,
A1S_ _1335, A1S_ _1336 A1S_ _1337, A1S_ _1338 A1S_ _1339, A1S_ _1340 A1S_ _1341, A1S_ _1342,
A1S_ _1343, A1S_ _1344 A1S_ _1345, A1S_ _1346 A1S_ _1347, A1S_ _1348 A1S_ _1349, A1S_ _1356,
A1S_ _1366, A1S_ _1367 A1S_ _1368, A1S_ _1369 A1S_ _1370, A1S_ _1372 A1S_ _1373, A1S_ _1374,
A1S_ _1375, A1S_ _1376 A1S_ _1377, A1S_ _1378 A1S_ _1379, A1S_ _1380 A1S_ _1396, A1S_ _1397,
A1S_ _1428, A1S_ _1442 A1S_ _1443, A1S_ _1450 A1S_ _1466, A1S_ _1467 A1S_ _1469, A1S_ _1470,
A1S_ _1476, A1S_ _1490 A1S_ _1491, A1S_ _1492 A1S_ _1493, A1S_ _1498 A1S_ _1499, A1S_ _1505,
A1S_ _1510, A1S_ _1523 A1S_ _1528, A1S_ _1530 A1S_ _1532, A1S_ _1543 A1S_ _1579, A1S_ _1583,
A1S_ _1601, A1S_ _1608 A1S_ _1609, A1S_ _1610 A1S_ _1611, A1S_ _1612 A1S_ _1613, A1S_ _1637,
A1S_ _1638, A1S_ _1639 A1S_ _1655, A1S_ _1692 A1S_ _1698, A1S_ _1699 A1S_ _1700, A1S_ _1701, A1S_ .1703, A1S_ _1705, A1S_ .1717, A1S_ .1719, A1S_ _1724, A1S_ .1729, A1S_ _1730, A1S_ .1731,
A1S_ _1732, A1S _1734 A1S _1735, A1S _1736 , A1S _1737, A1S _1738 A1S _1742, A1S _1745,
A1S_ _1754, A1S_ _1756, A1S_ .1758, A1S_ .1775, A1S_ .1776, A1S_ .1790, A1S_ .1791, A1S_ .1792,
A1S_ _1794, A1S_ _1795, A1S_ .1796, A1S_ .1797, A1S_ _1805, A1S_ _1806, A1S_ .1811, A1S_ _1830,
A1S_ _1834, A1S_ _1835, A1S_ _1836, A1S_ .1837, A1S_ _1838, A1S_ .1839, A1S_ _1840, A1S_ .1841,
A1S_ _1854, A1S_ _1855, A1S_ _1856, A1S_ .1857, A1S_ _1858, A1S_ .1859, A1S_ _1860, A1S_ .1861,
A1S_ _1862, A1S_ _1863, A1S_ .1864, A1S_ _1865, A1S_ _1866, A1S_ .1879, A1S_ _1880, A1S_ .1887,
A1S_ _1908, A1S_ _1924, A1S_ .1925, A1S_ .1926, A1S_ .1935, A1S_ .1940, A1S_ .1942, A1S_ .1948,
A1S_ _1951, A1S_ _1984, A1S_ .1996, A1S_ .2041, A1S_ _2042, A1S_ .2052, A1S_ _2053, A1S_ _2068,
A1S_ _2072, A1S_ .2081, A1S_ _2084, A1S_ _2098, A1S_ .2100, A1S_ .2101, A1S_ .2102, A1S_ .2148,
A1S_ _2149, A1S_ _2150, A1S_ .2163, A1S_ .2166, A1S_ .2167, A1S_ .2190, A1S_ .2191, A1S_ _2202,
A1S_ _2203, A1S_ _2207, A1S_ _2209, A1S_ .2218, A1S_ .2221, A1S_ .2225, A1S_ _2232, A1S_ _2234,
A1S_ _2248, A1S_ _2279, A1S_ _2280, A1S_ _2288, A1S_ _2289, A1S_ _2340, A1S_ .2341, A1S_ _2342,
A1S_ _2348, A1S_ _2353, A1S_ .2354, A1S_ .2415, A1S_ .2416, A1S_ .2417, A1S_ .2418, A1S_ .2419,
A1S_ _2424, A1S_ _2425, A1S_ .2431, A1S_ .2435, A1S_ _2443, A1S_ _2449, A1S_ .2450, A1S_ .2451,
A1S_ _2452, A1S_ _2475, A1S_ .2501, A1S_ .2509, A1S_ .2510, A1S_ .2514, A1S_ .2531, A1S_ .2532,
A1S_ _2533, A1S_ _2535, A1S_ .2601, A1S_ _2602, A1S_ _2633, A1S_ _2662, A1S_ .2670, A1S_ .2671,
A1S_ _2672, A1S_ _2688, A1S_ .2692, A1S_ .2694, A1S_ .2695, A1S_ .2696, A1S_ .2701, A1S_ .2711,
A1S_ _2722, A1S_ _2724, A1S_ _2738, A1S_ _2740, A1S_ .2741, A1S_ _2748, A1S_ .2753, A1S_ .2755,
A1S_ _2758, A1S_ .2761, A1S_ .2762, A1S_ .2769, A1S_ _2773, A1S_ _2774, A1S_ .2785, A1S_ _2788,
A1S_ _2789, A1S_ _2793, A1S_ _2809, A1S_ .2814, A1S_ .2815, A1S_ _2820, A1S_ _2823, A1S_ _2847,
A1S_ _2848, A1S_ _2849, A1S_ .2852, A1S_ _2860, A1S_ _2904, A1S_ .2905, A1S_ .2906, A1S_ .2911,
A1S_ _2913, A1S_ _2919, A1S_ _2924, A1S_ _2928, A1S_ _2939, A1S_ .2946, A1S_ .2956, A1S_ _3013,
A1S_ _3014, A1S_ _3025, A1S_ _3040, A1S_ _3043, A1S_ _3049, A1S_ _3050, A1S_ .3051, A1S_ _3074,
A1S_ _3084, A1S_ .3110, A1S_ .3120, A1S_ .3121, A1S_ .3122, A1S_ .3128, A1S_ .3129, A1S_ _3130,
A1S_ _3131, A1S_ .3132, A1S_ _3133, A1S_ .3134, A1S_ _3135, A1S_ .3144, A1S_ .3174, A1S_ _3180,
A1S_ _3195, A1S_ _3207, A1S_ _3222, A1S_ _3224, A1S_ .3225, A1S_ .3231, A1S_ _3232, A1S_ _3236,
A1S_ _3238, A1S_ _3248, A1S_ _3250, A1S_ _3268, A1S_ .3269, A1S_ _3273, A1S_ _3278, A1S_ _3290,
A1S_ _3297, A1S_ _3298, A1S_ _3300, A1S_ .3301, A1S_ _3309, A1S_ _3338, A1S_ _3342, A1S_ _3355,
A1S_ _3364, A1S_ _3377, A1S_ _3397, A1S_ _3398, A1S_ _3402, A1S_ _3403, A1S_ _3404, A1S_ _3405,
A1S_ _3406, A1S_ _3407, A1S_ .3410, A1S_ .3413, A1S_ .3414, A1S_ .3415, A1S_ .3416, A1S_ .3418, A1S_ .3431, A1S_ _3450, A1S_ .3451, A1S_ _3458, A1S_ _3460, A1S_ _3481, A1S_ _3487, A1S_ .3491,
A1S_ _3494, A1S_ _3498, A1S_ _3506, A1S_ _3508, A1S_ _3509, A1S_ _3514, A1S_ _3518, A1S_ .3519,
A1S_ _3520, A1S_ _3521, A1S_ .3522, A1S_ _3523, A1S_ _3524, A1S_ _3526, A1S_ _3528, A1S_ _3530,
A1S_ .3531, A1S_ _3532, A1S_ _3533, A1S_ _3534, A1S_ _3535, A1S_ _3537, A1S_ _3538, A1S_ _3539,
A1S_ _3540, A1S_ _3541, A1S_ .3542, A1S_ _3543, A1S_ _3544, A1S_ _3545, A1S_ _3546, A1S_ .3547,
A1S_ _3548, A1S_ _3549, A1S_ _3550, A1S_ _3552, A1S_ _3553, A1S_ _3554, A1S_ _3568, A1S_ _3569,
A1S_ _3578, A1S_ _3582, A1S_ _3586, A1S_ _3587, A1S_ .3591, A1S_ _3597, A1S_ _3599, A1S_ _3600,
A1S_ _3602, A1S_ _3611, A1S_ .3619, A1S_ _3621, A1S_ _3624, A1S_ _3629, A1S_ _3633, A1S_ _3640,
A1S_ .3641, A1S_ _3644, A1S_ .3647, A1S_ _3651, A1S_ _3652, A1S_ _3659, A1S_ _3663, A1S_ _3667,
A1S_ _3673, A1S_ _3679, A1S_ .3701, A1S_ _3707, A1S_ _3709, A1S_ _3713, A1S_ _3715, A1S_ .3717,
A1S_ _3732, A1S_ _3735, A1S_ _3738, A1S_ _3740, A1S_ _3741, A1S_ _3742, A1S_ _3759, A1S_ _3774,
A1S_ _3779, A1S_ _3787, A1S_ _3788, A1S_ _3794, A1S_ _3801, A1S_ _3802, A1S_ _3806, A1S_ _3809,
A1S_ .3811, A1S_ _3813, A1S_ .3814, A1S_ _3816, A1S_ _3817, A1S_ _3823, A1S_ _3829, A1S_ .3831,
A1S_ _3832, A1S_ _3836, A1S_ _3840, A1S_ _3846, A1S_ _3857, A1S_ _3862, A1S_ _3868, A1S_ _3870,
A1S_ _3880, A1S_ _3884, A1S_ _3886, A1S_ _3887, A1S_ _3891, A1S_ _3894, A1S_ _3898, A1S_ _3907,
A1S_ _3908, A1S_ _3909, A1S_ .3912, A1S_ _3914, or AlS_3915.
[0248] In some embodiments, the OM stress-responsive gene is A1S_0103, A1S_0645,
A1S_ _1266, A1S_ _1268, A1S_ _1335, A1S_ _1336, A1S_ _1337, A1S_ _1338, A1S_ _1339, A1S_ .1340,
A1S_ .1341, A1S_ _1342, A1S_ .1343, A1S_ _1344, A1S_ _1345, A1S_ .1791, A1S_ _1792, A1S_ .1794,
A1S_ .1796, A1S_ _1835, A1S_ _1836, A1S_ _1837, A1S_ _1838, A1S_ _1839, A1S_2449, A1S_2450,
A1S_ .2452, A1S_ _3540, A1S_ .3541, A1S_ _3542, A1S_ _3543, A1S_ _3586, A1S_ _3663, A1S_ _3707,
A1S_ _3738, A1S_ _3806, A1S_ _3809, or A1S_3908. In some embodiments, the OM stress responsive gene is A1S_1336, A1S_1836, A1S_1838, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, or A1S_3809.
A. Conditions or Agents
[0249] In some embodiments, the reporter microorganism is contacted or exposed to a condition that can cause, may likely cause, or is suspected of causing stress to the outer membrane and/or can impact, may likely impact, or is suspected of impacting the biogenesis or integrity of the outer membrane is one that alters, mutates, depletes, or removes a component from the OM. In some embodiments, the stress to the OM can be caused by genetically altering the reporter microorganism, such as reporter Acinetobacter spp., to reduce expression of an OM lipid, polysaccharide, or protein. In certain embodiments, the stress is caused by altering, mutating, depleting, or removing BamA, LptD, FhuA, PldA, OmpT, PagP, OstA, OmpA, OmpF, Omp200, Ompl21, Omp71, Ompl l7, OprF, PhoE, OmpC, OmpF, NmpC; PorA, PorB, OprA, OprM, OprN, OprJ, OprB, NspA, PagL, OmpW, OpcA, NalP, NupA, OmpG, FadL, PhoE, LamB, FhaC, SucY, FepA, FecA, BtuB, TolC, Porin P, Porin D, SmeC, SmeF, MepC, SrpC, TtgC, TtgF, AdeC, AdeK, or MexA. In particular embodiments, the stress to the OM of the Acinetobacter bacterium is or is caused by depletion of BamA.
[0250] In some embodiments, the genetic alteration (e.g. deletion) can be constitutively active or require induction. In some aspects, an outer membrane gene can be deleted from a microorganism and, instead, can be exogenously expressed under the control of an inducer. In such an example, the outer membrane gene is expressed only in the presence of the inducer but is not expressed, such as is not expressed on the cell surface, in the absence of the inducer. In some embodiments, the inducer is arabinose and the genetic alteration is induced by the addition of an inducer. In some embodiments, the inducer is arabinose. Exemplary of such an
Acinetobacter spp. is AABA046, which carries a deletion of the bamA gene by replacement with a selective marker conferring resistance to kanamycin and contains an exogenous reporter polynucleotide encoding BamA fused to the araBAD promoter from E. coli which allows regulation of transcription by addition of the inducing sugar arabinose. Also exemplary of such an Acinetobacter spp. is AABA041, which carries a deletion of the bamA gene by replacement with a selective marker conferring resistance to carbenicillin and contains an exogenous polynucleotide encoding bamA fused to the araBAD promoter from E. coli which allows regulation of transcription by addition of the inducing sugar arabinose.
[0251] In certain embodiments, the stress to the outer membrane (OM) can be caused by treatment with a molecule or combination of molecules that causes temporary or permanent damage to the OM. In certain embodiments, the stress to the OM is or is caused by treatment with polymyxin B nonapeptide (PMBN), vancomycin, ACHN-975, colistin,
ethylenediaminetetraacetic acid (EDTA), Sodium dodecyl sulfate (SDS), or combinations thereof. In certain embodiments, the stress to the OM is or is caused by treatment with PMBN.
[0252] In some embodiments, the stress to the outer membrane is caused by more than 1, 2, 3, 4, 5, or 6 stresses. In certain embodiments, the stress the outer membrane is caused by depletion of BamA from the reporter microorganism and treatment with PMBN. [0253] In some embodiments, the reporter microorganism is contacted with one or more agents that can cause, may likely cause, is suspected of causing, or is a candidate for causing stress to the outer membrane and/or can impact, may likely impact, is suspected of impacting, or is a candidate for impacting the biogenesis or integrity of the outer membrane. The agent can be a biomolecule or a drug. In some embodiments, the agent is naturally occurring or synthetic. In some embodiments, the agent can have a known or unknown structure. In certain embodiments, the agent is a small molecule compound, DNA, RNA, a peptide, or a protein. In certain embodiments, the agent is an antibody or antigen-binding fragment thereof.
[0254] In some embodiments, the agent can have a known or unknown function. In certain embodiments, the agent is suspected of being able to kill, inhibit growth, or reduce the total number of a pathogen. In certain embodiments, the agent can kill, inhibit growth, or reduce the total number of a pathogen.
[0255] In certain embodiments, the agent is a candidate antimicrobial, candidate sanitizer, candidate disinfectant, candidate antiseptic, candidate antibiotic, or candidate microbial biostatic. In certain embodiments, the molecule, agent, or drug is an antimicrobial, sanitizer, disinfectant, antiseptic, antibiotic, or microbial biostatic. In certain embodiments, the agent is antibacterial. In certain embodiments, the agent is a small molecule antibiotic, peptide antibiotic, or protein antibiotic.
[0256] In certain embodiments, the agent is a candidate prophylactic or candidate therapeutic. In certain embodiments, the agent is a candidate prophylactic. In certain embodiments, the agent is a candidate therapeutic. In certain embodiments, the agent is a prophylactic or therapeutic. In certain embodiments, the agent is a prophylactic. In certain embodiments, the agent is a therapeutic.
[0257] In certain embodiments, the agent is suspected of being able to cause OM stress. In certain embodiments, the agent is suspected of being able to disturb or disrupt the OM. In certain embodiments, the agent is suspected of being able to permeablize the OM.
[0258] In certain embodiments, the agent can cause OM stress. In certain embodiments, the agent can disturb or disrupt the OM. In certain embodiments, the agent can permeablize the OM.
[0259] In certain embodiments, the agent is in a composition. In certain embodiments, the composition further comprises an excipient. Acceptable excipients are known in the art, such as, for example buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose, dextrans, or mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.
[0260] In certain embodiments, the agent is in a complex with one or more other molecules, such as for example 1, 2, 3, 4, 5, 6 or more other molecules. In certain embodiments, the agent is part of a delivery vehicle. In certain embodiments, the agent is a part of a cell, such as is part of a microorganism, such as is part of a bacteria, virus or fungi. In some embodiments, the agent is produced or secreted by the cell.
[0261] In some embodiments, an agent that is produced or secreted by a cell is native to the cell. In certain embodiments, the agent is heterologous to the cell. In certain embodiments, expression of the agent in the cell is increased compared to a wild-type cell.
[0262] In some embodiments, the cell is any cell that can produce or secrete the agent. In certain embodiments, the cell is prokaryotic or eukaryotic. In certain embodiments, the cell is a microorganism. In certain embodiments, the cell is an archaeon, bacterium, or fungus. In certain embodiments, the cell is a plant or animal cell. In certain embodiments, the cell is a mammalian cell. In certain embodiments, the cell is a human, bird, or mouse cell.
[0263] In certain embodiments, there is a plurality of cells. In certain embodiments, the plurality of cells can each individually express one or more agents from a library of agents.
[0264] In certain embodiments, the agent the cell produces is a small molecule, peptide, or protein. In certain embodiments, the cell is an antibody-producing cell. In certain
embodiments, the antibody-producing cell is a mammalian cell. In certain embodiments, the antibody-producing cell is a B cell. In certain embodiments, the antibody-producing cell is a plasma cell or a plasmablast. In certain embodiments, the antibody-producing cell is a microorganism. In certain embodiments, the antibody-producing cell is a fungal or bacterial cell.
[0265] In certain embodiments, the cell is obtained from a donor that has been exposed to pathogen or an epitope-comprising fragment of pathogen or a variant thereof. In certain embodiments, the donor has been immunized or infected with a pathogen or an epitope- comprising fragment of the pathogen or a variant thereof. In certain embodiments, the donor is an immunized animal or an infected animal. In certain embodiments, the donor is a mammal or a bird. In certain embodiments, the donor is a human, a mouse or a chicken. In certain embodiments, the donor is a human donor who was infected by the target microorganism. In certain embodiments, the donor is a genetically modified non-human animal that produces partially human or fully human antibodies.
[0266] In certain embodiment, the animal infected by or immunized with the target pathogen or epitope-comprising fragment thereof is a genetically modified non-human animal that produces partially human or fully human antibodies. Such animals are known and available in the art and include, but are not limited to e.g., transchromosomic cattle and transgenic rodents, such as the Trianni transgenic mouse, and transgenic chicken, such as the HuMab Chicken from Crystal Biosciences.
[0267] In some embodiments, the antibody-producing cells are cells that have been modified cells, e.g., genetically or physical modified. In some embodiments, the antibody-producing cells are fusion cells, e.g., hybridomas. In some embodiments, the antibody-producing cells have not been modified.
[0268] In some embodiments, the target pathogen or microorganism to which an animal is infected or has been exposed to is a Gram negative bacterium. In some embodiments, the bacterium is a proteobacterium. For example, in some embodiments, the target microorganism is a species of Acinetobacter, Bdellovibrio, Burkholderia, Chlamydia, Enter obacter,
Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella, Neisseria, Pantoea, Pseudomonas, Salmonella, Shigella, Stenotrophomonas, Vibrio, or Yersinia.
[0269] In some embodiments, the target microorganism is Acinetobacter apis, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter beijerinckii, Acinetobacter bereziniae,
Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii, Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter gerneri,
Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii,
Acinetobacter haemolyticus, Acinetobacter harbinensis, Acinetobacter indicus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter kookii, Acinetobacter Iwoffii, Acinetobacter nectaris, Acinetobacter nosocomialis, Acinetobacter pakistanensis, Acinetobacter parvus, Acinetobacter pitii, Acinetobacter pittii, Acinetobacter puyangensis, Acinetobacter
qingfengensis, Acinetobacter radioresistans, Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii, Acinetobacter soli, Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis, Acinetobacter venetianus, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Vibrio cholera, or Yersinia pestis. In some embodiments, the pathogen is Acinetobacter baumannii.
[0270] In some embodiments, the target microorganism is multi-drug resistant
microorganism.
[0271] In some embodiments, the epitope-comprising fragment can be any fragment or portion of a cell that includes an epitope, which include antigenic determinants that are recognized by the immune molecules, e.g., antibodies or immune receptors. For example, in some embodiments, the epitope-comprising fragment is an antigen. In some embodiments, the epitope-comprising fragment is an epitope, or a fragment or a portion of an antigen.
[0272] In some embodiments, the epitope-comprising fragment is a protein or a polypeptide or a fragment thereof. In some embodiments, the epitope-comprising fragment is one or more of a protein, a glycoprotein, a lipid, a phospholipid, a glycolipid, a lipopolysaccharide, a nucleic acid, a polysaccharide and/or a combination thereof.
[0273] In some embodiments, the epitope-comprising fragment is present on the surface of the target microorganism. In some embodiments, the epitope-comprising fragment is accessible by the identified antibody on a live microorganism, e.g., bind to an antigen or epitope on the surface of the microorganism. For example, in some embodiments, the epitope-comprising fragment is a bacterial outer membrane (OM) protein, membrane protein, envelope protein, cell wall protein, surface lipid, glycolipid (e.g. lipopolysaccharide), glycoprotein, surface polysaccharide (e.g. capsule), surface appendage (e.g. flagella or pili), monomolecular surface layer (e.g. S-layer), or any epitope, portion or fragment thereof or a combination thereof. In some embodiments, the epitope-comprising fragment is associated with the outer membrane (OM), cell wall or envelope of the target microorganism. In some embodiments, the target microorganism is a Gram negative bacterium, and the epitope-comprising fragment is an OM protein. In some embodiments, the epitope-comprising fragment is associated with the extracellular side of the OM. In some embodiments, the epitope-comprising fragment is associated with the envelope of a virus, or the cell wall of a bacterium or a fungus. [0274] In some embodiments, the epitope-comprising fragment of the microorganism, e.g., an antigen is an essential component of the target microorganism. In some embodiments, the antigen that contains the epitope-comprising fragment is an essential protein in the target microorganism. In some embodiments, binding of the antibody identified using the methods provided herein to the antigen or the epitope-comprising fragment, can result in blocking, reducing, preventing, modulating and/or inhibiting the function of the epitope-comprising fragment that is an essential component of the microorganism, thereby interfering with an essential function in the target microorganism and rendering the target microorganism susceptible to therapeutic interventions using the antibody.
[0275] In some embodiments, the epitope-comprising fragment comprises a fragment of an OM protein of Gram negative bacteria. OM proteins are fully integrated membrane proteins which serve essential functions for the target microorganism, including nutrient uptake, cell adhesion, cell signaling and waste export. In some target microorganisms, the OM proteins also serve as virulence factors for nutrient scavenging and evasion of host defense mechanisms. In some cases, interfering with the function of an essential OM protein in Gram negative bacteria, e.g., by binding of an antibody, can kill or severely inhibit the growth of the bacteria. In some embodiments, the epitope-comprising fragment comprises a fragment of an OM protein, and the OM protein is BamA, LptD, AdeC, AdeK, BtuB, FadL, FecA, FepA, FhaC, FhuA, LamB, MepC, MexA, NalP, NmpC, NspA, NupA, Ompl l7, Ompl21, Omp200, Omp71, OmpA, OmpC, OmpF, OmpG, OmpT, OmpW, OpcA, OprA, OprB, OprF, OprJ, OprM, OprN, OstA, PagL, PagP, PhoE, PldA, PorA, PorB, PorD, PorP, SmeC, SmeF, SrpC, SucY, TolC, TtgC, or TtgF.
[0276] In some embodiments, epitope-binding fragment of a target microorganism is or comprises a fragment of BamA or LptD. Depletion of either LptD or BamA in Escherichia coli stalls assembly of the outer membrane, an essential organelle in Gram-negative bacteria, thereby causing cell death. LptD and BamA are both integral outer-membrane (OM) β-barrel proteins with critical roles in outer-membrane biogenesis. BamA is a 16-stranded β-barrel with five polypeptide transport-associated (POTRA) domains that sit in the periplasm. LptD catalyzes the terminal step in export of lipopolysaccharide to the cell surface, while BamA is required to fold all outer-membrane proteins, including LptD. LptD forms a complex with the lipoprotein LptE to form a complex in the OM. Antibody inhibition of LptD would decrease LPS levels in the outer-membrane causing dramatic sensitization to traditional antibiotics or cell death. Antibody inhibition of BamA would block folding of outer- membrane proteins thereby dramatically compromising the essential functions of the outer-membrane. LptD and BamA are ubiquitous among Gram-negative bacterial species raising the possibility that antibodies that inhibit these targets could be relevant for a broad range of Gram-negative pathogens, leading to a paradigm shift in the way Gram-negative bacterial infections are treated.
[0277] In some embodiments the epitope-comprising fragments thereof may be generated by expression in cell systems or grown in media that enhance protein production. In some embodiments, all or a portion of the epitope-comprising fragment can be produced using recombinant techniques. In some embodiments, the epitope-comprising fragment can be produced in recombinant bacterial or fungal protein expression systems. In some embodiments, exemplary bacterial cells that can be used for recombinant express include E. coli strains MC4100, B1LK0, RR1, E. coli LE392, E. coli B, E. coli X 1776 (ATCC No. 31537), E. coli BL21-DE3, and E. coli W3110 (F-, λ-, prototrophic, ATCC No. 273325).
[0278] In some embodiments, the epitope-comprising fragments are produced
recombinantly, and are subject to purification. In some embodiments, reporter polynucleotides encoding the epitope-comprising fragments or variants thereof are operably linked to reporter polynucleotides encoding an affinity tag or a purification tag, to facilitate purification.
Exemplary affinity tags include polyhistidine tags, Strep tag, FLAG tag, AviTag™, HA-tag, myc tag and GST tag. In some embodiments, purification columns are used to isolate or purify the epitope-comprising fragment from the rest of the biological material from the recombinant expression system. In some embodiments, the epitope-comprising fragment used in the methods provided herein is optionally linked to a cleavage sequence, such as a protease cleavage site, for subsequent removal of the affinity tag. Exemplary cleavage sequence includes Tobacco Etch Virus (TEV) cleavage site
[0279] In some embodiments, the antibody-producing cells are selected or purified by a positive or negative selection to isolate or enrich for antibody-producing cells, e.g., B cells, plasmablasts and/or plasma cells. In some embodiments, the antibody-producing cells are plasmablasts or plasma cells. In some embodiments, the antibody-producing cells are selected or purified from an organ or a tissue sample from the donor or animal prior to the contacting with the reporter microorganism. In some embodiments, the organ or tissue sample is a spleen or lymph node. In some embodiments, the organ or tissue sample is peripheral blood. In some embodiments, the cells obtained from the donor or immunocompromised animal are peripheral blood mononuclear cells (PBMCs), B cells, plasma cells and/or plasmablasts.
[0280] In some embodiments, cells from the organ or tissue sample, such as the plurality of candidate antibody-producing cells are subject to one or more positive or negative selection based on expression of cell surface markers. In some embodiments, obtaining candidate antibody-producing cells includes a selection of cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, e.g. , surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for selection based on such markers may be used to obtain candidate antibody-producing cells. In some embodiments, the selection is affinity- or immunoaffinity-based selection. For example, the isolation in some aspects includes selection of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding agent that specifically binds to such markers, followed generally by washing steps and selection of cells having bound the antibody or binding agent, from those cells having not bound to the antibody or binding agent.
[0281] Such selection steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding agent are retained.
[0282] The selection need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.
[0283] In some examples, multiple rounds of selection steps are carried out, where the positively or negatively selected fraction from one step is subjected to another selection step, such as a subsequent positive or negative selection. In some examples, a single selection step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding agents, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding agents expressed on the various cell types.
[0284] In some embodiments, the selection is a positive selection and the cell surface marker is one or more of: CD2, CD3, CD4, CD14, CD15, CD16, CD34, CD56, CD61, CD138, CD235a (Glycophorin A) and FceRIa. In some embodiments, one or more selection steps, such as one or more separate selection step is used to obtain candidate antibody-producing cells for
encapsulation and screening. In some embodiments, commercial cell selection kits, such as B cell isolation kits available from Miltenyi Biotech, EasySep™ B Cell Isolation Kit from
Stemcell Technologies, CD138+ cell isolation kit from Stemcell Technologies or Dynabeads B Cells Kit, can be used to obtain candidate antibody-producing cells. Other known markers and/or methods can be used to isolate desired candidate antibody-producing cells, e.g., B cells and/or plasmablasts. In some embodiments, the plurality of candidate antibody-producing cells for encapsulation comprises CD 138+ cells. In some embodiments, at least or at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, or more of the cells are plasma cells or plasmablasts and/or are CD 138+ cells.
B. Methods of Contacting Agents
[0285] In some embodiments, one or more agent is contacted with the reporter
microorganism. In some embodiments, a member of a plurality of agents, such as candidate agents, are each contacted with the reporter microorganism. In certain embodiments, the plurality of agents comprises at least or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, 10000, 100000,1000000 or more agents comprising unique or different agents. In certain embodiments, the plurality of agents is a library of agents.
[0286] In certain embodiments, the contacting is carried out in an array, wherein each of the plurality of agents is individually contacted with the reporter microorganism.
[0287] In some embodiments, the contacting comprises incubating the reporter
microorganism with the agent or each of the plurality of agents. In certain embodiments, the contacting comprises incubating the reporter microorganism with the agent or each of the plurality of agents for greater than or greater than about 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 75, 90, 120 minutes, such as for greater than or greater than about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours or more. In certain embodiments, the contacting can be performed at a temperature of 2 °C to 42 °C, such as generally a temperature from or from about 2 °C to 8 °C, from or from about 15 °C to 30 °C, such as at or about 25 °C + 3° C or from or from about 32 °C to 42°C, such as 37 ° C ± 2 °C.
[0288] In certain embodiments, the contacting of the reporter microorganism with the agent is performed in solution. The contacting can be performed in any container, such as, for example a test tube, flask, or titer plate. In certain embodiments, the contacting is performed without shaking or aeration. In certain embodiments, the contacting is performed with shaking and/or aeration. In certain embodiments, the contacting is performed in the presence of media.
[0289] In some embodiments, the provided methods can detect agents that directly exhibit activity to induce or cause OM-stress, such as directly exhibit or have bactericidal activity. In some embodiments, the methods can be used to detect or identify agents that indirectly exhibit activity to induce or cause OM-stress, such as indirectly exhibit or have bactericidal activity. In some aspects, the agents, e.g. antibody or antigen-binding fragment, activate the complement when reacted or bound to an antigen, which, in some aspects, leads to anti-bactericidal activity. In some embodiments, activity of the agent is only observed in the presence of one or more components of the complement pathway such that interaction of the agent, e.g. antibody or antigen -binding fragment, with a complement component, e.g. CI, triggers activation of the complement cascade resulting in lysis of the microorganism or bacterial cell. In some cases, one or more components of complement is provided by or in the presence of serum. Thus, in some aspects, the methods can be used to detect an agent, e.g. antibody or antigen binding fragment, that exhibits serum-dependent bactericidal activity.
[0290] In certain embodiments, the contacting is performed in the presence of one or more components capable of activating the complement pathway. In certain embodiments, the contacting is performed in the presence of serum. In certain embodiments, the serum is human, bovine, rabbit or mouse serum. In some examples, the agent and reporter microorganism are incubated or contacted with serum at a serum concentration that is at least or about at least 2.5% (vol/vol), 5.0% (vol/vol), 7.5% (vol/vol), 10.0% (vol/vol), 15.0% (vol/vol), 20% (vol/vol), or 25% (vol/vol). In certain embodiments, the concentration of serum is from or from about 2.5% (vol/vol) to 25% (vol/vol), 2.5% (vol/vol) to 15% (vol/vol), 2.5% (vol/vol) to 10% (vol/vol), 2.5% (vol/vol) to 5.0% (vol/vol), 5.0% (vol/vol) to 25% (vol/vol), 5.0% (vol/vol) to 15% (vol/vol), 5.0% (vol/vol) to 10% (vol/vol), 10.0% (vol/vol) to 25% (vol/vol), 10.0% (vol/vol) to 15% (vol/vol), or 15% (vol/vol) to 25% (vol/vol).
[0291] In certain embodiments, the agent does not activate the complement pathway, such that the activity of the agent on the microorganism is direct.
[0292] In certain embodiments, the contacting is carried out inside a microdroplet, wherein each of the plurality of agents is individually contacted with the reporter microorganism in the microdroplet. In some embodiments, the agent, such as an agent-producing cell, is co- encapsulated with the reporter microorganism. In certain embodiments, the agent is produced or secreted by a cell when it is co-encapsulated with the reporter microorganism. Methods for preparing microdroplets by gel encapsulation for screening in accord with the provided methods is further described below.
[0293] In certain embodiments, the agent is bound to a solid support. In certain
embodiments, the solid support is a bead. In certain embodiments, the agent is tethered to a substrate using a suitable linking agent (e.g., a suitable ortho-nitrobenzyl- based linking agent) that possesses one or more of the following features: a tag for linking to a substrate, a spacer moiety, a linker, e.g., a cleavable linker, and a reactive group. In certain embodiments, the tag may be an affinity tag, e.g., a biotin group or the like, or a reactive moiety (e.g. a carboxy group, an amino group, a halo group, a tosylate group, a mesylate group, a reactive hydroxyl groups or metal oxide) that can react with suitable sites (e.g., alcohols, amino nucleophiles, thiol nucleophiles or silane groups on the surface of a substrate to produce a covalent bond between the substrate and the linker or the antigen-containing fragment. In certain embodiments, the spacer may contain an unreactive alkyl chain, e.g., containing 3- 12 carbon atoms (e.g., 5- aminocaproic acid) and the cleavable linker may be chosen as containing appropriate chemistry (see above). The reactive group generally reacts with the effector molecule and forms a covalent bond therewith. Suitable reactive groups include halogens (that are sulhydryl reactive), N- hydroxysuccinimide (NHS)-carbonate (that are amine-reactive) and N,N-diisopropyl-2- cyanoethyl phosphoramidite (that are hydroxyl-reactive), and several other reactive groups are known in the art and may be readily employed in the instant methods.
[0294] In certain non-limiting embodiments, beads can range in size from 20 nm to 200 μιη or larger. In some embodiments, the bead has an average diameter of between about 100 nm and about 100 μιη, about 250 nm and about 75 μιη, about 500 nm and about 50 μιη, about 750 nm and about 25 μπι, about 1 μιη and about 10 μιτι, about 2 μπι and about 8 μπι, about 3 μπι and about 7 μιη or between about 3 μηι and about 5 μιη; or has an average diameter of about 1 μιη, 2 μιη, 3 μηι, 4 μιη, 5 μm, 6 μιη, 7 μm, 8 μιη, 9 μm or 10 μιη.
[0295] In some embodiments, a bead may be made, e.g., of polystyrene, but other materials such as polymethylmethacrylate (PMMA), polyvinyltoluene (PVT), styrene butadiene (S/B) copolymer, styrene/vinyltoluene (S/VT) are also used. Beads useful in the provided methods can be obtained commercially from numerous sources including Molecular Probes (Invitrogen), Bangs Labs, and Polymicrospheres, Inc.
[0296] Beads can be made to display a variety of chemically functional groups on their surface. Reactive groups commonly used include carboxyl, amino, aldehyde, hydroxyl, epoxy, and chloromethyl (See, e.g., U.S. Pat. Nos. 4,217,338, 5,326,692, 5,786,219, 4,717,655, 7,445,844, 5,573,909 and 6,023,540). In certain embodiments, linkers may be attached to these reactive groups, and target antigen-containing fragments may be conjugated directly or indirectly via a linker.
C. Identifying or Detecting a Detectable Signal and Agent
[0297] In some embodiments, the detectable signal is any produced by the reporter molecule. In some embodiments, the detectable signal can be fluorescent, luminescent, chemiluminescent, or colorimetric. The signal can be visual and can be detected by a human or machine. In some embodiments, the detection is carried out using an apparatus, and the apparatus is a light microscope, a fluorescent microscope, a spectrophotometer, a fluorescence- activated cell sorter, a fluorescent sample reader, a 3D tomographer or a camera. In some embodiments, the detection is carried out using a plate reader or a microscope. In some embodiments, detection is by a microscope and the sample containing the reporter
microorganism to which has been contacted with the one or more agents is overlayed or applied to a slide or is imaged or detected using the microscope.
[0298] In some embodiments, the methods further include identifying the agent that is identified as having caused a stress to the outer membrane and/or impacted or modulated the biogenesis or integrity of the outer membrane of the reporter microorganism. In some embodiments, the agent is identified from among a candidate of plurality of agents each contacted with the reporter microorganism. [0299] In some embodiments, the contacting is carried out in an array (e.g. a multi-well plate) and based on the presence of a signal in a particular address or addresses of the array, the identity of the agent can be identified. In some embodiments, identification of the agent is facilitated by a priori knowledge of the agent present at a particular loci or address of the array. For example, members at a locus can be identified by prior recording of their distinct location, a specific code, accession number or tag or other indicator that renders such easily identifiable. Thus, upon identification of a "HIT", i.e. where there is detectable signal, the identity of the agent member or members unique to that address is immediately known from its location in the array without any requirement for further enrichment, amplification or sequencing thereof. Library screening can be high-throughput by screening hundreds to thousands or
more biological samples in the same screening assay. In some embodiments, the contacting is carried out in a microdroplet, such as any microdroplet described herein and the microdroplet is in an array.
[0300] In some embodiments, the reporter microorganism has been contacted with an agent- producing cell. In some embodiment, the agent-producing cell sample, e.g. a cell present in an array or microdroplet, in which a signal was observed or detected can be enriched or separated from other agent-producing cells and/or the agent can be identified or isolated. In some embodiments, the identity (e.g. sequence) of the agent produced or expressed by the cell is determined. In some embodiments, the agent is a protein and determining the sequence of the nucleic acids is carried out using nucleic acid amplification and/or sequencing. Any methods known in the art to determine the sequence of nucleic acids can be used. In particular, techniques that allow determination of nucleic acid sequences from a small amount of starting material, such as single cell PCR, can be used to determine the sequence of the agent produced by the cell. In particular embodiments, the agent (e.g. antibody or other protein) can be identified by reverse transcription (RT)-PCR, proteomics, or any other downstream methods used to obtain the molecular signature of a protein. In some embodiments, determining the sequence of the nucleic acids is carried out using single cell PCR and nucleic acid sequencing. In some embodiments, where the agent is known to be or suspected of being an antibody, the antibody encoding heavy and light chain genes of the antibody-producing cell, e.g., B cell and/or plasmablast, can be PCR amplified, cloned, and sequenced according to established protocols [0301] In particular embodiments, provided methods further comprise isolating reporter polynucleotides encoding the agent (e.g. antibody or other protein) identified by the provided methods, subcloning the reporter polynucleotides into an expression vector, and producing recombinant proteins, such as recombinant antibodies.
[0302] In some embodiments, methods of detecting and/or identifying an agent (e.g. protein or peptides) include isolation or separation of such agent from cells of the agent-producing cell. For example, methods of isolating and/or identifying agents (e.g. protein or peptides) can involve lysis of the cells, affinity purification of the agent and/or elution from cell lysates and analysis of the agent (e.g. protein or peptides). One of skill in the art could readily apply the appropriate analysis tool depending on the type of agent that is or is suspected of being present. For example, using gas chromatography mass spectrometry for small molecules or matrix assisted laser desorption ionization or electrospray ionization for large molecules. In certain embodiments, single cell mass spectrometry is performed. In some cases, affinity purification can involve immunoprecipitation or affinity chromatography. In some cases, one or more of ion exchange chromatography, lectin chromatography, size exclusion high performance liquid chromatography and a combination of any of the above can be used.
[0303] In some embodiments, the identified agents, e.g. antibodies, can then be used to test in vitro and in vivo activity and efficacy on the specific microorganism or a related
microorganism, e.g., bacterial or fungal cell, used for detection. In some embodiments, the in vitro and in vivo activity and efficacy of such antibodies can also be tested on other variants of the same microorganism or other species of microorganisms.
D. Microdroplets and Gel Encapsulation
[0304] Exemplary of methods for carrying out the provided methods involves encapsulating one or more agents, such as one or more agent-producing cells, in microdroplets, e.g. gel microdroplets, with the reporter microorganism. In some embodiments, the microdroplets comprise (i) an agent, such as a candidate agent, which, in some cases, can be an agent- producing cell, e.g. an antibody-producing cell and (ii) a reporter microorganism as provided herein. In some embodiments, the agent is bound to a solid support, such as a bead. In some embodiments, the agent is comprised in a composition comprising an agent-producing cell, such as an antibody-producing cell. In some embodiments, the gel microdroplets may comprise multiple copies of the reporter microorganism and/or agent or agent-producing cells. The microdroplets provide for a rapid and efficient method of screening agents that change or modulate (e.g. increase or decrease), and can substantially reduce the time required to identify antibodies with desired binding specificity to a specific target, compared to any conventional methods.
[0305] In some embodiments, the microdroplets, on average, comprise one or fewer agent or agent-producing cell (e.g. antibody-producing cells). In some embodiments, the average ratio of candidate agents or candidate agent-producing cell (e.g. antibody-producing cell) per microdroplet is less than or less than about 1. In some embodiments, the average ratio of candidate agents or agent-producing cell (e.g. antibody-producing cell) per microdroplet is between about 0.05 and about 1.0, about 0.05 and about 0.5, about 0.05 and about 0.25, about 0.05 and about 0.1, about 0.1 and about 1.0, about 0.1 and about 0.5, about 0.1 and about 0.25, about 0.25 and about 1.0, about 0.25 and about 0.5 or 0.5 and about 1.0, each inclusive. In some embodiments, the average ratio of agent or agent-producing cell (e.g. antibody-producing cells) per microdroplet is or is about 0.1.
[0306] In some embodiments the microdroplets may contain a single agent or agent- producing cell (e.g. antibody-producing cell) and multiple reporter microorganisms.
[0307] The number of agents or agent-producing cells (e.g. antibody-producing cells) and the reporter microorganism may be controlled by Poisson statistics, e.g., as described in Powell (Biotechnology 1990 8: 333-7); Weaver et al (Biotechnology 1991 9: 873-877). During the encapsulation process, the components particles (e.g., agent or agent-producing cells) are randomly distributed into the nascent microdroplets. Since virtually all of the particles become embedded in microdroplets, if the number of particles exceeds the number of microdroplets, each microdroplet may contain, on average, >1 particle. Likewise, if the number of
microdroplets exceeds that of the particles, then each microdroplet may contain, on average, <1 particle.
[0308] In general, for some of the methods described herein, it may be desirable to have one or fewer agent or agent-producing cell (e.g. antibody-producing cell) per microdroplet since this ensures the encapsulation of a single type of agent-producing cell (e.g. antibody-producing cell) that may act upon the reporter microorganism, and thus generate a result that is more clearly interpretable than if multiple types of agents or agent-producing cell (e.g. antibody-producing cell) were present in the microdroplet. In some instances, microdroplets may contain agent- producing cell (e.g. antibody-producing cell) that will be allowed to grow over time, resulting in multiple agent-producing cells (e.g. antibody-producing cell) per microdroplet. In this case, the cells in one microdroplet would be clonal in origin, and hence only produce one type of agent (e.g. antibody).
[0309] In some embodiments, the ratio of agent or agent-producing cell (e.g. antibody- producing cell) to reporter microorganisms and the average number of each in a microdroplet can be optimized based on the desired method of screening, detection and identification and the parameters of gel encapsulation. Exemplary variables for consideration for such optimization include, but are not limited to, e.g., size of the reporter microorganism, size of the microdroplet, number of other particles in the microdroplet, strength of the detection signal, agent output of the agent-producing cells and affinity of the agents. With respect to the reporter microorganism, it may be desirable to have multiple members of each type contained within each microdroplet.
[0310] The number of reporter microorganism can be optimized to ensure visibility of signal during the screening and identification of agent or agent-producing cells (e.g. antibody- producing cells), and in relation to the size of the microdroplet. In some embodiments, the average number of reporter microorganism, per microdroplet can be between about 5 and about 500, such as about 10 and about 250, about 50 and about 200, about 50 and about 150, about 50 and about 100, or about 80 and about 120, such as about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200. The number of reporter microorganism per microdroplet may be lower on average for microorganisms that are larger in cell size, e.g., a fungus or a parasite.
[0311] In some embodiments, the gel microdroplet comprises a polymer matrix and/or a gel matrix. In certain embodiments, gel microdroplets comprise agarose, carrageenan, alginate, alginate -polylysine, collagen, cellulose, methylcellulose, gelatin, chitosan, extracellular matrix, dextran, starch, inulin, heparin, hyaluronan, fibrin, polyvinyl alcohol, poly(N-vinyl-2- pyrrolidone), polyethylene glycol, poly(hydroxyethyl methacrylate), acrylate polymers and sodium polyacrylate, polydimethyl siloxane, cis-polyisoprene, Puramatrix™, poly- divenylbenzene, polyurethane, or polyacrylamide. In particular embodiments, the gel micro- drops comprise a polymer matrix, which may be e.g., agarose, carrageenan, alginate, alginate- polylysine, collagen, a plant-derived gum, cellulose or a derivatives thereof (e.g.,
methylcellulose), gelatin, chitosan or an extracellular matrix (ECM), as described by Kleinman (U.S. Pat. No. 4,829,000), or combinations thereof. Synthetic hydrogels that may be used in the microdroplet include but are not limited to polyvinyl alcohol, block copolymer of ethylene- vinylalcohol, sodium polystyrene sulfonate, vinyl-methyl-tribenzyl ammonium chloride and polypho sphazene .
[0312] Gel microdroplets and screening methodologies that may be used according to the provided methods include any known and available in the art. Examples of gel microdroplets and screening methodologies that may be used include but are not limited to those described in U.S. Patent Nos. 8,415,173, 8,030,095, 7,939,344, 7,413,868, and 8,445,193, U.S. Patent Application Publication Nos. US20080038755 and US20060073095, and PCT Patent
Application Publication No. WO2015/038817.
[0313] In some embodiments, the microdroplets are generated by a microfluidics-based method. Exemplary microfluidics-based devices that can be used to generate the microdroplets include μEncapsulator System (Dolomite Microfluidics) and Cellena ® Microencapsulator (Biomedal Lifescience).
[0314] In some embodiments, gel microdroplets comprise agarose. In some embodiments, the agarose is low gelling temperature agarose, such as an ultra-low gelling temperature agarose. In some embodiments, the low gelling temperature agarose allows for the agarose to stay liquid at lower temperatures, e.g., temperatures that permit viability of the agent-producing cells (e.g. antibody-producing cell) and the reporter microorganism and thereby allow live cell and reporter microorganism encapsulation. In some embodiments, the gelling temperature of the agarose used in encapsulation is such that the temperature of liquid agarose does not adversely affect viability of the agent-producing cells (e.g. antibody-producing cell) and/or the target microorganism, and gel encapsulation can be carried out in a liquid state. In some
embodiments, the agarose has a gelling temperature of lower than about 35°C, such as about 30°C, about 25°C, about 20°C, about 15°C, about 10°C or about 5°C. In some embodiments, the agarose is an ultra-low gelling temperature agarose, such as those with a gelling temperature of lower than about 20°C, about 15°C, about 10°C or about 5°C. In some embodiments, the agarose has a gelling temperature of between about 5°C and about 30°C, about 5°C and about 20°C, about 5°C and about 15°C, about 8°C and about 17°C or about 5°C and about 10°C, such as about 8°C and about 17°C. [0315] In some embodiments, the gel encapsulation is carried out at a temperature that allows viability of the agent-producing cells (e.g. antibody-producing cell) and the reporter microorganism, e.g., pathogen, e.g., about 37°C, about 35°C, about 30°C, about 25°C or about 20°C.
[0316] In some embodiments of the methods provided herein, the methods include a step of incubating the microdroplets at a temperature lower than the gelling temperature of the polymer matrix and/or gel matrix, e.g., at a temperature of between about 0°C and about 5°C, such as about 0°C, about 1°C, about 2°C, about 3°C, about 4°C, or about 5°C. In some embodiments, the incubation is for about 1 min to about 10 min, such as about 1 min, about 2 min, about 3 min, about 4 min, about 5 min, about 6 min, about 7 min, about 8 min, about 9 min, or about 10 min.
[0317] In some embodiments, the provided methods further comprise incubating the gel microdroplets at a temperature of at or about 37 °C prior to detecting a signal. In some aspects, this step facilitates survival and secretion of the agent by the agent-producing cells (e.g.
antibody-producing cell), e.g., B cells or plasmablasts. In some embodiments, the gel microdroplets are incubated in growth media. The time of incubation in media can be determined based on optimal survival and agent secretion by the agent-producing cells (e.g. antibody-producing cell). In some embodiments, the incubation is about 45 minutes to 2 hours, such as about one hour.
[0318] In some embodiments, the gel microdroplets are surrounded by a non-aqueous environment, during or after the encapsulation step. In some embodiments, the gel
microdroplets comprise growth media and are surrounded by a non-aqueous environment. In some embodiments, the composition comprises one or more components capable of activating the complement pathway. In some embodiments, the composition comprises serum, such as human, bovine, rabbit, or mouse serum.
[0319] In some embodiments, the non-aqueous environment comprises an oil. In some embodiments, the oil is gas permeable. The presence of the gas permeable oil allows for physical separation of the microdroplets and can ensure that the secreted agents, e.g. antibodies, do not escape the non-aqueous environment, thereby resulting in a sufficiently high
concentration of the agent, e.g. antibody, in the microdroplets for increased efficiency of screening. Exemplary gas-permeable oils that can be used include fluorinated oils, including but are not limited to, 3M™ Novec™ 7500 and Fluorinert FC40 (Sigma Aldrich). In some embodiments, the gel microdroplets are incubated in a non-aqueous environment after encapsulation. In some embodiments, the gel microdroplets are incubated in a non-aqueous environment at a temperature of at or about 37°C prior to detection of a signal. In some embodiments, the non-aqueous environment comprises a gas-permeable oil, such as 3M™ Novec™ 7500 or Fluorinert FC40.
[0320] In some embodiments, the methods provided herein involve determining whether the agent, which can be an agent produced by an agent-producing cell(s), within the gel
microdroplet modulates or changes a signal from the reporter molecule of the reporter microorganism present in the same gel microdroplet, which includes detecting a signal produced by a reporter molecule as described herein.
[0321] In some embodiments, the provided methods include isolating the microdroplet comprising the identified agent or cell producing the identified agent or isolating reporter polynucleotides encoding the agent (e.g. antibody). In some embodiments, the provided methods also include determining the sequence of the nucleic acids encoding the identified agent (e.g. antibody).
[0322] In some embodiments, the gel microdroplet that contains the identified agent or the cell producing the identified agent (e.g. antibody) is separated away from the plurality of microdroplets. In some embodiments, the isolation is carried out using a micromanipulator or an automated sorter. For example, in some embodiments, the gel microdroplets are visually screened under a microscope, e.g., under a fluorescence microscope, and the microdroplet that contains the identified agent or cell producing the identified agent (e.g. antibody), can be physically separated from other microdroplets as they are identified during the screening process. In some embodiments, the microdroplets are separated using a micromanipulator. In some embodiments, automated sorters can be used to sort particular droplets based on a criterion, e.g., level of detectable signal in the microdroplet.
[0323] In some embodiments, the sequence of the nucleic acid encoding the agent can be determined, such as by using methods as described or known in the art.
VIII. EXEMPLARY EMBODIMENTS
[0324] Among the provided embodiments are: 1. A reporter polynucleotide comprising a sequence comprising a regulatory region of an outer membrane (OM) stress-responsive gene of an Acinetobacter bacterium operably linked to a sequence encoding a reporter molecule, wherein the OM stress-responsive gene is modulated in response to a stress to the outer membrane of the Acinetobacter bacterium.
2. The reporter polynucleotide of embodiment 1, wherein the Acinetobacter bacterium is an Acinetobacter apis, Acinetobacter baumannii, Acinetobacter baylyi,
Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii, Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter gerneri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus, Acinetobacter harbinensis, Acinetobacter indicus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter kookii, Acinetobacter Iwoffii, Acinetobacter nectaris, Acinetobacter nosocomialis, Acinetobacter parvus, Acinetobacter pakistanensis, Acinetobacter pittii, Acinetobacter puyangensis,
Acinetobacter qingfengensis, Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii, Acinetobacter soli, Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis, or Acinetobacter venetianus.
3. The reporter polynucleotide of embodiment 1 or 2, wherein the Acinetobacter bacterium is Acinetobacter baumannii.
4. The reporter polynucleotide of any of embodiments 1-3, wherein the
Acinetobacter bacterium is ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA-1605, ATCC BAA-1709, ATCC BAA- 1710, ATCC BAA-1789, ATCC BAA-1790, ATCC BAA- 1791, ATCC BAA-1792, ATCC BAA- 1793, ATCC BAA-1794, ATCC BAA-1795, ATCC BAA-1796, ATCC BAA-1797, ATCC BAA-1798, ATCC BAA-1799, ATCC BAA-1800, ATCC BAA-1878, ATCC BAA-2093, ATCC BAA-747, SDF, AYE, ACICU, AB0057, AB307-0294, 1656-2, MDR-ZJ06, TCDC- AB0715, MDR-TJ, TYTH-1, D1279779, BJAB07104, BJAB0715, BJAB0868, ZW85-1, AbH120-A2, AB030, AB031, AC29, LAC-4, Ab307-0294, 1656-2, ID 403, AABA041, AABA046, or a modified strain thereof. 5. The reporter polynucleotide of any of embodiments 1-4, wherein the
Acinetobacter bacterium is ATCC 17978, Ab307-0294, AABA041, AABA046.
6. The reporter polynucleotide of any of embodiments 1-5, wherein the stress to the outer membrane of the Acinetobacter bacterium is or is caused by depletion of BamA or treatment with polymyxin B nonapeptide (PMBN).
7. The reporter polynucleotide of any of embodiments 1-6, wherein the stress to the outer membrane of the Acinetobacter bacterium is or is caused by depletion of BamA and treatment with PMBN.
8. The reporter polynucleotide of any of embodiments 1-7, wherein the OM stress- responsive gene is upregulated or downregulated in response to the stress.
9. The reporter polynucleotide of any of embodiments 1-8, wherein the OM stress- responsive gene is upregulated in response to the stress.
10. The reporter polynucleotide of any of embodiments 1-9, wherein the OM stress- responsive gene is upregulated in response to the stress at least or about at least 2-fold, 3 -fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more in response to the stress.
11. The reporter polynucleotide of any of embodiments 1-10, wherein the OM stress- responsive gene is A1S_0012, A1S_0023, A1S_0027, A1S_0028, A1S_0029, A1S_0030, A1S_0031, A1S_0032, A1S_0033, A1S_0037, A1S_0040, A1S_0041, A1S_0044, A1S_0066, A1SJ3092, A1SJ3093, A1S_0109, A1S_0110, A1S_0112, A1S_0113, A1S_0114, A1S_0115, A1S_0116, A1S_0117, A1S_0118, A1S_0126, A1S_0158, A1S_0170, A1S_0175, A1S_0178, A1S_0189, A1S_0224, A1S_0245, A1S_0256, A1S_0276, A1S_0293, A1S_0301, A1S_0309, A1SJ3310, A1SJ3332, A1S_0333, A1S_0363, A1S_0372, A1S_0376, A1S_0391, A1S_0392, A1S_0401, A1S_0441, A1S_0462, A1S_0463, A1S_0464, A1S_0465, A1S_0466, A1S_0494, A1S_0508, A1S_0509, A1S_0510, A1S_0511, A1S_0512, A1S_0514, A1S_0516, A1S_0518, A1S_0519, A1S_0520, A1S_0521, A1S_0522, A1S_0523, A1S_0527, A1S_0535, A1S_0536, A1S_0537, A1S_0538, A1S_0547, A1S_0559, A1S_0561, A1S_0562, A1S_0563, A1S_0564, A1S_0566, A1S_0567, A1S_0568, A1S_0570, A1S_0624, A1S_0630, A1S_0631, A1S_0633, A1S_0634, A1S_0640, A1S_0641, A1S_0642, A1S_0643, A1S_0644, A1S_0645, A1S_0646, A1S_0647, A1S_0650, A1S_0663, A1S_0664, A1S_0665, A1S_0666, A1S_0667, A1S_0669, A1S_0670, A1S_0671, A1S_0673, A1S_0677, A1S_0680, A1S_0683, A1S_0714, A1S_0717, A1SJ3718, A1S_0719, A1S_0736, A1S_0738, A1S_0739, A1S_0749, A1S_0770, A1S_0772, A1S_ _0779, A1S_ _0780 A1S_ _0781, A1S_ _0800 A1S_ _0804, A1S_ _0830 A1S_ _0831, A1S_ _0832,
A1S_ _0834, A1S_ _0835 A1S_ _0861, A1S_ _0884 A1S_ _0889, A1S_ _0929 A1S_ _0930, A1S_ _0931,
A1S_ _0932, A1S_ _0935 A1S_ _0945, A1S_ _0959 A1S_ _0980, A1S_ _1003 A1S_ _1009, A1S_ _1010,
A1S_ _1027, A1S_ _1028 A1S_ _1030, A1S_ _1031 A1S_ _1049, A1S_ _1081 A1S_ _1106, A1S_ _1107,
A1S_ _1120, A1S_ _1121 A1S_ _1123, A1S_ _1132 A1S_ _1133, A1S_ _1134 A1S_ _1139, A1S_ _1143,
A1S_ _1145, A1S_ _1146 A1S_ _1148, A1S_ _1149 A1S_ _1150, A1S_ _1151 A1S_ _1152, A1S_ _1153,
A1S_ _1155, A1S_ _1156 A1S_ _1157, A1S_ _1158 A1S_ _1159, A1S_ _1160 A1S_ _1161, A1S_ _1162,
A1S_ _1163, A1S_ _1164 A1S_ _1165, A1S_ _1167 A1S_ _i m, A1S_ _1172 A1S_ _1173, A1S_ _1180,
A1S_ _1184, A1S_ _1186 A1S_ _H98, A1S_ _1202 A1S_ _1203, A1S_ _1223 A1S_ _1224, A1S_ _1225,
A1S_ _1230, A1S_ _1236 A1S_ _1237, A1S_ _1248 A1S_ _1255, A1S_ _1274 A1S_ _1286, A1S_ _1359,
A1S_ _1360, A1S_ _1361 A1S_ _1362, A1S_ _1363 A1S_ _1383, A1S_ _1384 A1S_ _1385, A1S_ _1386,
A1S_ _1387, A1S_ _1393 A1S_ _1404, A1S_ _1407 A1S_ _1422, A1S_ _1454 A1S_ _1472, A1S_ _1481,
A1S_ _1494, A1S_ _1512 A1S_ _1515, A1S_ _1526 A1S_ _1535, A1S_ _1539 A1S_ _1566, A1S_ _1567,
A1S_ _1569, A1S_ _1583 A1S_ _1584, A1S_ _1585 A1S_ _1589, A1S_ _1590 A1S_ _1593, A1S_ _1595,
A1S_ _1596, A1S_ _1617 A1S_ _1630, A1S_ _1644 A1S_ _1645, A1S_ _1647 A1S_ _1648, A1S_ _1649,
A1S_ _1651, A1S_ _1655 A1S_ _1658, A1S_ _1662 A1S_ _1666, A1S_ _1667 A1S_ _1669, A1S_ _1677,
A1S_ _1680, A1S_ _1681 A1S_ _1687, A1S_ _1735 A1S_ _1741, A1S_ _1743 A1S_ _1744, A1S_ _1750,
A1S_ _1751, A1S_ _1752 A1S_ _1760, A1S_ _1762 A1S_ _1767, A1S_ _1778 A1S_ _1813, A1S_ _1827,
A1S_ _1829, A1S_ _1831 A1S_ _1843, A1S_ _1876 A1S_ _1909, A1S_ _1928 A1S_ _1929, A1S_ _1934,
A1S_ _1952, A1S_ _1955 A1S_ _1956, A1S_ _1957 A1S_ _1959, A1S_ _1960 A1S_ _1961, A1S_ _1962,
A1S_ _1963, A1S_ _1979 A1S_ _1986, A1S_ _1987 A1S_ _1988, A1S_ _2006 A1S_ _2026, A1S_ _2033,
A1S_ _2034, A1S_ _2035 A1S_ _2036, A1S_ _2038 A1S_ _2039, A1S_ _2061 A1S_ _2074, A1S_ _2079,
A1S_ _2082, A1S_ _2092 A1S_ _2093, A1S_ _2106 A1S_ _2139, A1S_ _2140 A1S_ _2141, A1S_ _2142,
A1S_ _2146, A1S_ _2157 A1S_ _2158, A1S_ _2160 A1S_ _2161, A1S_ _2162 A1S_ _2178, A1S_ _2179,
A1S_ _2183, A1S_ _2186 A1S_ _2195, A1S_ _2230 A1S_ _2247, A1S_ _2252 A1S_ _2257, A1S_ _2258,
A1S_ _2259, A1S_ _2262 A1S_ _2271, A1S_ _2272 A1S_ _2273, A1S_ _2283 A1S_ _2285, A1S_ _2298,
A1S_ _2311, A1S_ _2315 A1S_ _2325, A1S_ _2326 A1S_ _2330, A1S_ _2331 A1S_ _2366, A1S_ _2367,
A1S_ _2382, A1S_ _2387 A1S_ _2389, A1S_ _2395 A1S_ _2396, A1S_ _2414 A1S_ _2434, A1S_ _2445,
A1S_ _2446, A1S_ _2447 A1S_ _2448, A1S_ _2454 A1S_ _2455, A1S_ _2456 A1S_ _2458, A1S_ _2459,
A1S_ _2463, A1S_ _2480 A1S_ _2489, A1S_ _2503 A1S_ _2504, A1S_ _2508 A1S_ _2542, A1S_ _2543,
A1S_ _2552, A1S_ _2553 A1S_ _2555, A1S_ _2557 A1S_ _2558, A1S_ _2573 A1S_ _2577, A1S_ _2578, A1S_ _2580, A1S_ _2586 A1S_ _2588, A1S_ _2593, A1S_ _2611, A1S_ _2612, A1S_ _2613, A1S_ _2624,
A1S_ _2650, A1S_ _2651 A1S_ _2654, A1S_ _2656, A1S_ _2660, A1S_ _2664, A1S_ _2668, A1S_ _2675,
A1S_ _2676, A1S_ _2677 A1S_ _2678, A1S_ _2684, A1S_ _2705, A1S_ _2729, A1S_ _2734, A1S_ _2756,
A1S_ _2768, A1S_ _2786 A1S_ _2798, A1S_ _2801, A1S_ _2807, A1S_ _2826, A1S_ _2827, A1S_ _2828,
A1S_ _2839, A1S_ _2863 A1S_ _2882, A1S_ _2883, A1S_ _2884, A1S_ _2885, A1S_ _2889, A1S_ _2892,
A1S_ _2893, A1S_ _2942 A1S_ _2943, A1S_ _2953, A1S_ _2959, A1S_ _2960, A1S_ _2968, A1S_ _2976,
A1S_ _2992, A1S_ _3011 A1S_ _3026, A1S_ _3027, A1S_ _3034, A1S_ _3035, A1S_ _3047, A1S_ _3048,
A1S_ _3099, A1S_ _3100 A1S_ _3101, A1S_ _3104, A1S_ _3105, A1S_ _3114, A1S_ _3115, A1S_ _3116,
A1S_ _3117, A1S_ _3124 A1S_ _3125, A1S_ _3126, A1S_ _3127, A1S_ _3139, A1S_ _3146, A1S_ _3147,
A1S_ _3175, A1S_ _3206 A1S_ _3224, A1S_ _3253, A1S_ _3259, A1S_ _3280, A1S_ _3281, A1S_ _3295,
A1S_ _3317, A1S_ _3326 A1S_ _3339, A1S_ _3360, A1S_ _3361, A1S_ _3367, A1S_ _3368, A1S_ _3371,
A1S_ _3375, A1S_ _3376 A1S_ _3392, A1S_ _3411, A1S_ _3412, A1S_ _3463, A1S_ _3466, A1S_ _3468,
A1S_ _3469, A1S_ _3471 A1S_ _3479, A1S_ _3480, A1S_ _3486, A1S_ _3492, A1S_ _3493, A1S_ _3494,
A1S_ _3499, A1S_ _3510 A1S_ _3512, A1S_ _3518, A1S_ _3522, A1S_ _3523, A1S_ _3533, A1S_ _3534,
A1S_ _3535, A1S_ _3539 A1S_ _3540, A1S_ _3541, A1S_ _3542, A1S_ _3543, A1S_ _3544, A1S_ _3545,
A1S_ _3546, A1S_ _3548 A1S_ _3552, A1S_ _3553, A1S_ _3558, A1S_ _3559, A1S_ _3562, A1S_ _3563,
A1S_ _3567, A1S_ _3570 A1S_ _3577, A1S_ _3580, A1S_ _3580, A1S_ _3585, A1S_ _3586, A1S_ _3594,
A1S_ _3595, A1S_ _3596 A1S_ _3601, A1S_ _3602, A1S_ _3603, A1S_ _3604, A1S_ _3605, A1S_ _3606,
A1S_ _3607, A1S_ _3608 A1S_ _3609, A1S_ _3610, A1S_ _3611, A1S_ _3612, A1S_ _3613, A1S_ _3614,
A1S_ _3617, A1S_ _3618 A1S_ _3621, A1S_ _3630, A1S_ _3632, A1S_ _3634, A1S_ _3635, A1S_ _3636,
A1S_ _3637, A1S_ _3642 A1S_ _3645, A1S_ _3649, A1S_ _3654, A1S_ _3658, A1S_ _3661, A1S_ _3662,
A1S_ _3666, A1S_ _3682 A1S_ _3686, A1S_ _3687, A1S_ _3688, A1S_ _3694, A1S_ _3695, A1S_ _3697,
A1S_ _3704, A1S_ _3707 A1S_ _3708, A1S_ _3712, A1S_ _3716, A1S_ _3725, A1S_ _3726, A1S_ _3727,
A1S_ _3728, A1S_ _3736 A1S_ _3738, A1S_ _3739, A1S_ _3740, A1S_ _3750, A1S_ _3752, A1S_ _3760,
A1S_ _3768, A1S_ _3769 A1S_ _3770, A1S_ _3771, A1S_ _3772, A1S_ _3773, A1S_ _3776, A1S_ _3777,
A1S_ _3778, A1S_ _3782 A1S_ _3783, A1S_ _3786, A1S_ _3789, A1S_ _3790, A1S_ _3791, A1S_ _3792,
A1S_ _3797, A1S_ _3810 A1S_ _3818, A1S_ _3820, A1S_ _3835, A1S_ _3837, A1S_ _3840, A1S_ _3842,
A1S_ _3844, A1S_ _3862 A1S_ _3864, A1S_ _3865, A1S_ _3866, A1S_ _3867, A1S_ _3868, A1S_ _3873,
A1S_ _3875, A1S_ _3879 A1S_ _3889, A1S_ _3900, A1S_ _3901, A1S_ _3902, A1S_ _3908, or
A1S_ _3911. 12. The reporter polynucleotide of any of embodiments 1-11, wherein the OM stress- responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or A1S_3127.
13. The reporter polynucleotide of any of embodiments 1-12, wherein the OM stress- responsive gene is A1S_0032, A1S_2885, or A1S_2889.
14. The reporter polynucleotide of any of embodiments 1-8, wherein the OM stress- responsive gene is downregulated in response to the stress.
15. The reporter polynucleotide of any of embodiments 1-8 and 14,wherein the OM stress-responsive gene is downregulated in response to the stress at least or about at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more in response to the stress.
16. The reporter polynucleotide of any of embodiments 1-8 and 14-15, wherein the OM stress-responsive gene is A1S_0009, A1S_0010, A1S_0025, A1S_0027, A1S_0038,
A1S_ _0067, A1S_ _0070, A1S_ _0071, A1S_ _0073, A1S_ _0076, A1S_ _0077, A1S_0079, A1S_0087,
A1S_ _0090, A1S_ _0091, A1S_ _0095, A1S_ _0096, A1S_ _0097, A1S_ _0098, A1S_0099, A1S_0103,
A1S_ _0104, A1S_ _0105, A1S_ _0106, A1S_ _0107, A1S_ _0108, A1S_ _0109, A1S_0121, A1S_0128,
A1S_ _0129, A1S_ _0141, A1S_ _0148, A1S_ _0150, A1S_ _0151, A1S_ _0152, A1S_0153, A1S_0154,
A1S_ _0155, A1S_ _0156, A1S_ _0157, A1S_ _0177, A1S_ _0184, A1S_ _0200, A1S_0201, A1S_0209,
A1S_ _0218, A1S_ _0238, A1S_ _0239, A1S_ _0253, A1S_ _0257, A1S_ _0258, A1S_0269, A1S_0270,
A1S_ _0279, A1S_ _0286, A1S_ _0292, A1S_ _0302, A1S_ _0303, A1S_ _0304, A1S_0321, A1S_0322,
A1S_ _0323, A1S_ _0347, A1S_ _0365, A1S_ _0369, A1S_ _0370, A1S_ _0388, A1S_0408, A1S_0410,
A1S_ _0427, A1S_ _0429, A1S_ _0447, A1S_ _0448, A1S_ _0474, A1S_ _0480, A1S_0481, A1S_0482,
A1S_ _0486, A1S_ _0490, A1S_ _0491, A1S_ _0498, A1S_ _0526, A1S_ _0533, A1S_0534, A1S_0548,
A1S_ _0549, A1S_ _0566, A1S_ _0567, A1S_ _0568, A1S_ _0591, A1S_ _0594, A1S_0624, A1S_0625,
A1S_ _0626, A1S_ _0627, A1S_ _0629, A1S_ _0630, A1S_ _0631, A1S_ _0632, A1S_0633, A1S_0634,
A1S_ _0635, A1S_ _0639, A1S_ _0640, A1S_ _0641, A1S_ _0642, A1S_ _0643, A1S_0644, A1S_0645,
A1S_ _0646, A1S_ _0647, A1S_ _0649, A1S_ _0650, A1S_ _0651, A1S_ _0690, A1S_0691, A1S_0692,
A1S_ _0695, A1S_ _0698, A1S_ _0721, A1S_ _0731, A1S_ _0732, A1S_ _0771, A1S_0785, A1S_0786,
A1S_ _0787, A1S_ _0788, A1S_ _0818, A1S_ _0822, A1S_ _0846, A1S_ _0849, A1S_0850, A1S_0851,
A1S_ _0852, A1S_ _0853, A1S_ _0854, A1S_ _0855, A1S_ _0869, A1S_ _0877, A1S_0882, A1S_0883,
A1S_ _0890, A1S_ _0891, A1S_ _0901, A1S_ _0910, A1S_ _0911, A1S_ _0912, A1S_0913, A1S_0960,
A1S_ _0965, A1S_ _0973, A1S_ _0984, A1S_ _0996, A1S_ _0997, A1S_ _0999, A1S_1000, A1S_1004, A1S_ _1008, A1S_ .1021 A1S_ _1026, A1S_ _1044 A1S_ _1063, A1S_ _1072 A1S_ _1079, A1S_ _1080,
A1S_ _1088, A1S_ _1089 A1S_ .1091, A1S_ _1092 A1S_ _1093, A1S_ _1094 A1S_ .1109, A1S_ .1113,
A1S_ .1139, A1S_ .1142 A1S_ .1182, A1S_ _1193 A1S_ .1195, A1S_ _1199 A1S_ _1227, A1S_ _1257,
A1S_ _1258, A1S_ .1261 A1S_ _1264, A1S_ _1265 A1S_ _1266, A1S_ _1267 A1S_ _1268, A1S_ _1269,
A1S_ _1270, A1S_ .1281 A1S_ .1317, A1S_ _1318 A1S_ .1319, A1S_ _1327 A1S_ _1334, A1S_ _1335,
A1S_ _1336, A1S_ _1337 A1S_ _1338, A1S_ _1339 A1S_ _1340, A1S_ _1341 A1S_ _1342, A1S_ _1343,
A1S_ _1344, A1S_ _1345 A1S_ _1346, A1S_ _1347 A1S_ _1348, A1S_ _1349 A1S_ _1356, A1S_ _1366,
A1S_ _1367, A1S_ _1368 A1S_ _1369, A1S_ _1370 A1S_ _1372, A1S_ _1373 A1S_ _1374, A1S_ _1375,
A1S_ _1376, A1S_ _1377 A1S_ _1378, A1S_ _1379 A1S_ _1380, A1S_ _1396 A1S_ _1397, A1S_ _1428,
A1S_ _1442, A1S_ _1443 A1S_ _1450, A1S_ _1466 A1S_ _1467, A1S_ _1469 A1S_ _1470, A1S_ _1476,
A1S_ _1490, A1S_ .1491 A1S_ _1492, A1S_ _1493 A1S_ _1498, A1S_ _1499 A1S_ _1505, A1S_ .1510,
A1S_ _1523, A1S_ _1528 A1S_ _1530, A1S_ _1532 A1S_ _1543, A1S_ _1579 A1S_ _1583, A1S_ .1601,
A1S_ _1608, A1S_ _1609 A1S_ .1610, A1S_ _1611 A1S_ .1612, A1S_ .1613 A1S_ _1637, A1S_ _1638,
A1S_ _1639, A1S_ _1655 A1S_ _1692, A1S_ _1698 A1S_ _1699, A1S_ _1700 A1S_ .1701, A1S_ _1703,
A1S_ _1705, A1S_ .1717 A1S_ .1719, A1S_ _1724 A1S_ _1729, A1S_ _1730 A1S_ .1731, A1S_ _1732,
A1S_ _1734, A1S_ _1735 A1S_ _1736, A1S_ _1737 A1S_ _1738, A1S_ _1742 A1S_ _1745, A1S_ _1754,
A1S_ _1756, A1S_ _1758 A1S_ _1775, A1S_ _1776 A1S_ _1790, A1S_ _1791 A1S_ _1792, A1S_ _1794,
A1S_ _1795, A1S_ _1796 A1S_ _1797, A1S_ _1805 A1S_ _1806, A1S_ _1811 A1S_ _1830, A1S_ _1834,
A1S_ _1835, A1S_ _1836 A1S_ _1837, A1S_ _1838 A1S_ _1839, A1S_ _1840 A1S_ .1841, A1S_ _1854,
A1S_ _1855, A1S_ _1856 A1S_ _1857, A1S_ _1858 A1S_ _1859, A1S_ _1860 A1S_ .1861, A1S_ _1862,
A1S_ _1863, A1S_ _1864 A1S_ _1865, A1S_ _1866 A1S_ _1879, A1S_ _1880 A1S_ _1887, A1S_ _1908,
A1S_ _1924, A1S_ _1925 A1S_ _1926, A1S_ _1935 A1S_ _1940, A1S_ _1942 A1S_ _1948, A1S_ .1951,
A1S_ _1984, A1S_ _1996 A1S_ .2041, A1S_ _2042 A1S_ _2052, A1S_ _2053 A1S_ _2068, A1S_ _2072,
A1S_ .2081, A1S_ _2084 A1S_ _2098, A1S_ _2100 A1S_ .2101, A1S_ _2102 A1S_ _2148, A1S_ _2149,
A1S_ _2150, A1S_ _2163 A1S_ _2166, A1S_ _2167 A1S_ _2190, A1S_ _2191 A1S_ _2202, A1S_ _2203,
A1S_ _2207, A1S_ _2209 A1S_ _2218, A1S_ _2221 A1S_ _2225, A1S_ _2232 A1S_ _2234, A1S_ _2248,
A1S_ _2279, A1S_ _2280 A1S_ _2288, A1S_ _2289 A1S_ _2340, A1S_ _2341 A1S_ _2342, A1S_ _2348,
A1S_ _2353, A1S_ _2354 A1S_ _2415, A1S_ _2416 A1S_ _2417, A1S_ _2418 A1S_ _2419, A1S_ _2424,
A1S_ _2425, A1S_ _2431 A1S_ _2435, A1S_ _2443 A1S_ _2449, A1S_ _2450 A1S_ .2451, A1S_ _2452,
A1S_ _2475, A1S_ _2501 A1S_ _2509, A1S_ _2510 A1S_ _2514, A1S_ _2531 A1S_ _2532, A1S_ _2533,
A1S_ _2535, A1S_ _2601 A1S_ _2602, A1S_ _2633 A1S_ _2662, A1S_ _2670 A1S_ .2671, A1S_ _2672, A1S_ _2688, A1S_ _2692, A1S_ _2694, A1S_ _2695, A1S_ _2696, A1S_ _2701, A1S_ _2711, A1S_ _2722,
A1S_ _2724, A1S_ _2738, A1S_ _2740, A1S_ _2741, A1S_ _2748, A1S_ _2753, A1S_ _2755, A1S_ _2758,
A1S_ _2761, A1S_ _2762, A1S_ _2769, A1S_ _2773, A1S_ _2774, A1S_ _2785, A1S_ _2788, A1S_ _2789,
A1S_ _2793, A1S_ _2809, A1S_ _2814, A1S_ _2815, A1S_ _2820, A1S_ _2823, A1S_ _2847, A1S_ _2848,
A1S_ _2849, A1S_ _2852, A1S_ _2860, A1S_ _2904, A1S_ _2905, A1S_ _2906, A1S_ _2911, A1S_ _2913,
A1S_ _2919, A1S_ _2924, A1S_ _2928, A1S_ _2939, A1S_ _2946, A1S_ _2956, A1S_ _3013, A1S_ _3014,
A1S_ _3025, A1S_ _3040, A1S_ _3043, A1S_ _3049, A1S_ _3050, A1S_ _3051, A1S_ _3074, A1S_ _3084,
A1S_ _3110, A1S_ _3120, A1S_ _3121, A1S_ _3122, A1S_ _3128, A1S_ _3129, A1S_ _3130, A1S_ _3131,
A1S_ _3132, A1S_ _3133, A1S_ _3134, A1S_ _3135, A1S_ _3144, A1S_ _3174, A1S_ _3180, A1S_ _3195,
A1S_ _3207, A1S_ _3222, A1S_ _3224, A1S_ _3225, A1S_ _3231, A1S_ _3232, A1S_ _3236, A1S_ _3238,
A1S_ _3248, A1S_ _3250, A1S_ _3268, A1S_ _3269, A1S_ _3273, A1S_ _3278, A1S_ _3290, A1S_ _3297,
A1S_ _3298, A1S_ _3300, A1S_ _3301, A1S_ _3309, A1S_ _3338, A1S_ _3342, A1S_ _3355, A1S_ _3364,
A1S_ _3377, A1S_ _3397, A1S_ _3398, A1S_ _3402, A1S_ _3403, A1S_ _3404, A1S_ _3405, A1S_ _3406,
A1S_ _3407, A1S_ _3410, A1S_ _3413, A1S_ _3414, A1S_ _3415, A1S_ _3416, A1S_ _3418, A1S_ _3431,
A1S_ _3450, A1S_ _3451, A1S_ _3458, A1S_ _3460, A1S_ _3481, A1S_ _3487, A1S_ _3491, A1S_ _3494,
A1S_ _3498, A1S_ _3506, A1S_ _3508, A1S_ _3509, A1S_ _3514, A1S_ _3518, A1S_ _3519, A1S_ _3520,
A1S_ _3521, A1S_ _3522, A1S_ _3523, A1S_ _3524, A1S_ _3526, A1S_ _3528, A1S_ _3530, A1S_ _3531,
A1S_ _3532, A1S_ _3533, A1S_ _3534, A1S_ _3535, A1S_ _3537, A1S_ _3538, A1S_ _3539, A1S_ _3540,
A1S_ _3541, A1S_ _3542, A1S_ _3543, A1S_ _3544, A1S_ _3545, A1S_ _3546, A1S_ _3547, A1S_ _3548,
A1S_ _3549, A1S_ _3550, A1S_ _3552, A1S_ _3553, A1S_ _3554, A1S_ _3568, A1S_ _3569, A1S_ _3578,
A1S_ _3582, A1S_ _3586, A1S_ _3587, A1S_ _3591, A1S_ _3597, A1S_ _3599, A1S_ _3600, A1S_ _3602,
A1S_ _3611, A1S_ _3619, A1S_ _3621, A1S_ _3624, A1S_ _3629, A1S_ _3633, A1S_ _3640, A1S_ _3641,
A1S_ _3644, A1S_ _3647, A1S_ _3651, A1S_ _3652, A1S_ _3659, A1S_ _3663, A1S_ _3667, A1S_ _3673,
A1S_ _3679, A1S_ _3701, A1S_ _3707, A1S_ _3709, A1S_ _3713, A1S_ _3715, A1S_ _3717, A1S_ _3732,
A1S_ _3735, A1S_ _3738, A1S_ _3740, A1S_ _3741, A1S_ _3742, A1S_ _3759, A1S_ _3774, A1S_ _3779,
A1S_ _3787, A1S_ _3788, A1S_ _3794, A1S_ _3801, A1S_ _3802, A1S_ _3806, A1S_ _3809, A1S_ _3811,
A1S_ _3813, A1S_ _3814, A1S_ _3816, A1S_ _3817, A1S_ _3823, A1S_ _3829, A1S_ _3831, A1S_ _3832,
A1S_ _3836, A1S_ _3840, A1S_ _3846, A1S_ _3857, A1S_ _3862, A1S_ _3868, A1S_ _3870, A1S_ _3880,
A1S_ _3884, A1S_ _3886, A1S_ _3887, A1S_ _3891, A1S_ _3894, A1S_ _3898, A1S_ _3907, A1S_ _3908,
A1S_ _3909, A1S_ _3912, A1S_ _3914, or A1S_3915. 17. The reporter polynucleotide of any of embodiments 1-8 and 14-16, wherein the OM stress-responsive gene is A1S_0103, A1S_0645, A1S_1266, A1S_1268, A1S_1335, A1S_1336, A1S_1337, A1S_1338, A1S_1339, A1S_1340, A1S_1341, A1S_1342, A1S_1343, A1S_1344, A1S_1345, A1S_1791, A1S_1792, A1S_1794, A1S_1796, A1S_1835, A1S_1836, A1S_1837, A1S_1838, A1S_1839, A1S_2449, A1S_2450, A1S_2452, A1S_3540, A1S_3541, A1S_3542, A1S_3543, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, A1S_3809, or A1S_3908.
18. The reporter polynucleotide of any of embodiments 1-8 and 14-17, wherein the OM stress-responsive gene is A1S_1336, A1S_1836, A1S_1838, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, or A1S_3809.
19. The reporter polynucleotide of any of embodiments 1-18, wherein the regulatory region comprises a contiguous sequence of nucleotides within 500 base pairs upstream or 5' of the open reading frame (ORF) of the OM stress-responsive gene.
20. The reporter polynucleotide of embodiment 19, wherein the contiguous sequence of nucleotides comprises at least or at least about 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400 or more base pairs.
21. The reporter polynucleotide of any of embodiments 1-20, wherein the regulatory region is or comprises a promoter.
22. The reporter polynucleotide of any of embodiments 1-21, wherein the regulatory region comprises a sequence to further promote translation of the encoded reporter molecule.
23. The reporter polynucleotide of embodiment 22, wherein the sequence further promoting translation is or comprises a bacterial ribosome binding site.
24. The reporter polynucleotide of embodiment 23, wherein the ribosome binding site is a Shine-Dalgarno sequence.
25. The reporter polynucleotide of embodiment 24, wherein the Shine-Dalgarno sequence is native to the regulatory region of the OM stress-responsive gene.
26. The reporter polynucleotide of embodiment 24, wherein the Shine-Dalgarno sequence is synthetic and/or heterologous to the regulatory region of the OM stress-responsive gene. 27. The reporter polynucleotide of embodiment 26, wherein the Shine-Dalgarno sequence comprises the sequence set forth in SEQ ID NO: 14 or a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 14.
28. The reporter polynucleotide of any of embodiments 1-27, wherein the regulatory region comprises the sequence set forth in any of SEQ ID NOS: 1-13 or a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NOs: 1-13.
29. The reporter polynucleotide of any of embodiments 1-28, wherein the reporter molecule is a fluorescent protein, a luminescent protein, a chromoprotein, or an enzyme.
30. The reporter polynucleotide of embodiment 29, wherein the reporter molecule is a fluorescent protein and the fluorescent protein is Sirius, SBFP2, Azurite, mAzurite, EBFP2, moxBFP, mKalamal, mTagBFP2, Aquamarine, ECFP, Cerulean, mCerulean, mCerulean3, moxCerulean3, SCFP3A, mTurquoise2, CyPet, AmCyanl, MiCy (Midoriishi-Cyan), iLOV, AcGFPl, sfGFP, moxGFP, mEmerald, EGFP, mEGFP, AzamiGreen, cfSGFP2, ZsGreen, SGFP2, Clover, mClover2, mClover3, EYFP, Topaz, mTopaz, mVenus, mox Venus, SYFP2, mCitrine, YPet, ZsYellowl, mPapayal, niKusabira-Orange (mKO), mOrange, mOrange2, mK02, TurboRFP, tdTomato, mScarlet-H, mNectarine, mRuby2, eqFP611, DsRed2, mApple, mScarlet, mStrawberry, FusionRed, mRFPl, mCherry, mCherry2, mCrimson3, HcRedl, dKatushka, mKatel.3, mPlum, mRaspberry, TagRFP675, mNeptune, mCardinal, mMaroon, TagRFP657, smURFP, miRFP670, iRFP670, iRFP682, miRFP703, iRFP702, miRFP709, mlFP 683, IFP2.0, iRFP, iSplit, iRFP720, T-sapphire, mT-sapphire, mAmetrine, LSSmOrange, mKeima Red, dKeima Red, LSSmKatel, LSSmKate2, Phamret, PA-sfGFP, mPA-Emerald, PA- GFP, PATagRFP, PAmCherryl, PAmCherry2, PAmCherry3, PAmKate, PAiRFPl, PAiRFP2, Dendra2, mEos3.2, mEos4a, dEos 505, tdEos 505, mKikGR, Kaede 508, PSmOrange,
PSmOrange2, rsTagRFP, rsEmerald, rsGFPl, Dronpa3, mGeos-M, amilGFP, amilCP, or a modified version thereof.
31. The reporter polynucleotide of embodiments 29 or 30, wherein the reporter molecule is a fluorescent protein and wherein the fluorescent protein is sfGFP, mClover3, or mRuby2.
32. The reporter polynucleotide of any of embodiments 1-31, wherein the sequence encoding the reporter molecule comprises the sequence set forth in SEQ ID NO: 15, 33, or 40 or a sequence with at least or at least about 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 15, 33, or 40.
33. The reporter polynucleotide of any of embodiments 1-32, wherein the reporter polynucleotide comprises the sequence set forth in any of SEQ ID Nos: 16-28 or 34-36 or a sequence with at least or at least about 85%, 90%, 95%, 99%, or more sequence identity to any of SEQ ID NOs: 16-28 or 34-36.
34. The reporter polynucleotide of embodiment 29,wherein the reporter molecule is a luminescent protein and the luminescent protein is North American firefly luciferase, Genji- botaru luciferase, Italian firefly luciferase, Heike luciferase, East European firefly luciferase, Pennsylvania firefly luciferase, Click beetle luciferase, Railroad worm luciferase, Renilla luciferase, Rluc8, Green Renilla luciferase, Gaussia luciferase, Gaussia-Dura luciferase, Cypridina luciferase, Vargula luciferase, Metridia luciferase, OLuc, bacterial luciferase (LuxAB), or a modified version thereof.
35. The reporter polynucleotide of embodiment 29, wherein the reporter molecule is an enzyme and the enzyme is chloramphenicol acetyltransferase (CAT), β-galactosidase, alkaline phosphatase, β-glucuronidase, β -lactamase, neomycin phosphotransferase, or a modified version thereof.
36. A reporter vector comprising the reporter polynucleotide of any of embodiments
1-35.
37. The reporter vector of embodiment 36, wherein the reporter polynucleotide is a first reporter polynucleotide and the reporter vector further comprises a second reporter polynucleotide of any of embodiments 1-35.
38. The reporter vector of embodiment 37, wherein the first reporter polynucleotide and the second reporter polynucleotide are different.
39. The reporter vector of embodiment 37 or embodiment 38, wherein the regulatory region of the first reporter polynucleotide and the regulatory region of the second reporter polynucleotide are from different OM stress-responsive genes.
40. The reporter vector of embodiment 38 or embodiment 39, wherein the reporter molecule encoded by the first reporter polynucleotide is different from the reporter molecule encoded by the second reporter polynucleotide. 41. The reporter vector of any of embodiments 38-40, wherein the reporter molecule encoded by the first reporter polynucleotide and the reporter molecule encoded by the second reporter polynucleotide do not exhibit an overlapping emission and absorption spectra and/or are distinguishably detectable.
42. The reporter vector of any of embodiments 36-42, wherein the reporter vector is capable of being expressed in a host microorganism.
43. The reporter vector of embodiment 42, wherein the host microorganism is a Gram-negative bacterium.
44. The reporter vector of embodiment 42 or embodiment 43, wherein the host microorganism is Acinetobacter, Bdellovibrio, Burkholderia, Chlamydia, Enterobacter, Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella, Neisseria, Pantoea, Pseudomonas, Salmonella, Shigella, Stenotrophomonas, Vibrio, or Yersinia.
45. The reporter vector of any of embodiments 42-44, wherein the host
microorganism is Acinetobacter.
46. The reporter vector of any of embodiments 42-45, wherein the host
microorganism is Acinetobacter apis, Acinetobacter baumannii, Acinetobacter baylyi,
Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii, Acinetobacter calcoaceticus,
Acinetobacter gandensis, Acinetobacter gerneri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus, Acinetobacter harbinensis, Acinetobacter indicus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter kookii, Acinetobacter Iwoffii, Acinetobacter nectaris, Acinetobacter nosocomialis, Acinetobacter parvus, Acinetobacter pakistanensis, Acinetobacter pittii, Acinetobacter puyangensis,
Acinetobacter qingfengensis, Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii, Acinetobacter soli, Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis, or Acinetobacter venetianus.
47. The reporter vector of any of embodiments 42-46, wherein the host
microorganism is Acinetobacter baumannii.
48. The reporter vector of any of embodiments 42-47, wherein the host
microorganism is ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA- 1605, ATCC BAA- 1709, ATCC BAA- 1710, ATCC BAA-1789, ATCC BAA-1790, ATCC BAA- 1791, ATCC BAA-1792, ATCC BAA-1793, ATCC BAA-1794, ATCC BAA-1795, ATCC BAA-1796, ATCC BAA-1797, ATCC BAA- 1798, ATCC BAA-1799, ATCC BAA-1800, ATCC BAA-1878, ATCC BAA-2093, ATCC BAA-747, SDF, AYE, ACICU, AB0057, AB307-0294, 1656-2, MDR-ZJ06, TCDC-AB0715, MDR-TJ, TYTH-1, D1279779, BJAB07104, BJAB0715, BJAB0868, ZW85-1, AbH120-A2, AB030, AB031, AC29, LAC-4, Ab307-0294, 1656-2, ID 403, AABA041, AABA046, or a modified strain thereof.
49. The reporter vector of any of embodiments 42-48, wherein the host
microorganism is ATCC 17978, Ab307-0294, AABA041, AABA046.
50. The reporter vector of any of embodiments 36-49, wherein the reporter polynucleotide, optionally the first reporter polynucleotide and second reporter polynucleotide, is comprised in a backbone vector, and the backbone vector is pACH106, pWH1266, or pET- RA.
51. The reporter vector of embodiment 50, wherein a nucleotide sequence comprising the reporter polynucleotide, optionally the first reporter polynucleotide and the second reporter polynucleotide, is inserted into or replaces a portion of the nucleotide sequence of the backbone vector.
52. The reporter vector of embodiment 50 or embodiment 51 , wherein the backbone vector comprises the sequence of nucleotides set forth in SEQ ID NO: 29 and a nucleotide sequence comprising the reporter polynucleotide replaces nucleotides 5,715-7,395 of the backbone vector.
53. The reporter vector of any of embodiments 36 and 42-52, wherein the reporter vector comprises the sequence set forth in any of SEQ ID NOs: 30-32 or a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NOs: 30-32.
54. A reporter microorganism comprising one or more reporter polynucleotide of any of embodiments 1-35 or one or more reporter vector of any of embodiments 36-53. 55. The reporter microorganism of embodiment 54, wherein the one or more reporter polynucleotides comprise a first reporter polynucleotide and a second reporter polynucleotide that are different.
56. The reporter microorganism of embodiment 55, wherein the regulatory region of the first reporter polynucleotide and the regulatory region of the second reporter polynucleotide are from different OM stress-responsive genes.
57. The reporter microorganism of embodiment 55 or embodiment 56, wherein the reporter molecule encoded by the first reporter polynucleotide is different from the reporter molecule encoded by the second reporter polynucleotide.
58. The reporter microorganism of any of embodiment 55-57, wherein the reporter molecule encoded by the first reporter polynucleotide and the reporter molecule encoded by the second reporter polynucleotide do not exhibit an overlapping emission and absorption spectra and/or are distinguishably detectable.
59. The reporter microorganism of any of embodiments 55-58, wherein the first reporter polynucleotide and second reporter polynucleotide are comprised in the same reporter vector.
60. The reporter microorganism of any of embodiments 55-59, wherein the first reporter polynucleotide and second reporter polynucleotide are comprised in different reporter vectors.
61. The reporter microorganism of any of embodiments 54-60, wherein the reporter microorganism is a Gram-negative bacterium.
62. The reporter microorganism of any of embodiments 54-61 , wherein the reporter microorganism is Acinetobacter, Bdellovibrio, Burkholderia, Chlamydia, Enterobacter, Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella, Neisseria, Pantoea, Pseudomonas, Salmonella, Shigella, Stenotrophomonas, Vibrio, or Yersinia.
63. The reporter microorganism of any of embodiments 54-62, wherein the reporter microorganism is Acinetobacter.
64. The reporter microorganism of any of embodiments 54-63, wherein the reporter microorganism is Acinetobacter apis, Acinetobacter baumannii, Acinetobacter baylyi,
Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii, Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter gerneri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus, Acinetobacter harbinensis, Acinetobacter indicus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter kookii, Acinetobacter Iwoffii, Acinetobacter nectaris, Acinetobacter nosocomialis, Acinetobacter parvus, Acinetobacter pakistanensis, Acinetobacter pittii, Acinetobacter puyangensis,
Acinetobacter qingfengensis, Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii, Acinetobacter soli, Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis, or Acinetobacter venetianus.
65. The reporter microorganism of any of embodiments 54-64, wherein the reporter microorganism is Acinetobacter baumannii.
66. The reporter microorganism of any of embodiments 54-65, wherein the reporter microorganism is derived from ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA-1605, ATCC BAA- 1709, ATCC BAA- 1710, ATCC BAA-1789, ATCC BAA-1790, ATCC BAA-1791, ATCC BAA- 1792, ATCC BAA-1793, ATCC BAA-1794, ATCC BAA- 1795, ATCC BAA-1796, ATCC BAA-1797, ATCC BAA-1798, ATCC BAA-1799, ATCC BAA-1800, ATCC BAA- 1878, ATCC BAA- 2093, ATCC BAA-747, SDF, AYE, ACICU, AB0057, AB307-0294, 1656-2, MDR-ZJ06, TCDC-AB0715, MDR-TJ, TYTH-1, D1279779, BJAB07104, BJAB0715, BJAB0868, ZW85-1, AbH120-A2, AB030, AB031, AC29, LAC-4, Ab307-0294, 1656-2, ID 403, AABA041, AABA046, or a modified strain thereof.
67. The reporter microorganism of any of embodiments 54-66, wherein the reporter microorganism is ATCC 17978, Ab307-0294, AABA041, or AABA046.
68. A plurality of reporter microorganisms comprising two or more reporter microorganisms of any of embodiments 54-67.
69. The plurality of reporter microorganisms of embodiment 68, wherein each of at least two reporter microorganisms in the plurality comprise a different reporter polynucleotide in which comprises the same regulatory region of an OM-responsive gene but that is operatively linked to a different reporter molecule. 70. The plurality of reporter microorganisms of embodiment 69, wherein the different reporter molecules do not exhibit overlapping emission and absorption spectra and/or are distinguishably detectable.
71. The plurality of reporter microorganisms of any of embodiments 68-70, comprising 2, 3, 4, 5, or more different reporter microorganisms.
72. The plurality of reporter microorganisms of any of embodiments 69-71, wherein the at least two reporter microorganisms are derived from the same host strain.
73. The plurality of microorganisms of any of embodiments 69-71, wherein each of the at least two reporter microorganisms is derived from a different host strain, optionally wherein each of the at least two reporter microorganisms is derived from a different isolate or subtype of the strain.
74. A composition comprising the microorganism of any of embodiments 54-67 or the plurality of microorganisms of any of embodiments 68-73.
75. The composition of embodiment 74, further comprising one or more components capable of activating the complement pathway.
76. The composition of embodiment 74 or embodiment 75, further comprising serum.
77. The composition of embodiment 76, wherein the serum is human serum, rabbit serum, bovine serum, or mouse serum.
78. The composition of embodiment 76 or embodiment 77, wherein the concentration of serum is at least or at least about 2.5% (vol/vol), 5.0% (vol/vol), 7.5% (vol/vol), 10.0% (vol/vol), 15.0% (vol/vol), 20% (vol/vol), or 25% (vol/vol).
79. The composition of any of embodiments 76-78, wherein the concentration of serum is from or from about 2.5% (vol/vol) to 25% (vol/vol), 2.5% (vol/vol) to 15% (vol/vol), 2.5% (vol/vol) to 10% (vol/vol), 2.5% (vol/vol) to 5.0% (vol/vol), 5.0% (vol/vol) to 25% (vol/vol), 5.0% (vol/vol) to 15% (vol/vol), 5.0% (vol/vol) to 10% (vol/vol), 10.0% (vol/vol) to 25% (vol/vol), 10.0% (vol/vol) to 15% (vol/vol), or 15% (vol/vol) to 25% (vol/vol).
80. The composition of any of embodiments 74-79, further comprising an agent, optionally a candidate antibacterial agent.
81. The composition of embodiment 80, wherein the agent is a small molecule compound, a peptide or a protein. 82. The composition of embodiment 80 or embodiment 81, wherein the agent is an antibody or antigen-binding fragment thereof.
83. A microdroplet comprising the microorganism of any of embodiments 54-67, the plurality of microorganisms of any of embodiments 68-73, or the composition of any of embodiments 69-82.
84. The microdroplet of embodiment 83, wherein the microdroplet comprises an agent.
85. The microdroplet of embodiment 84, wherein the agent is a candidate antibacterial agent.
86. The microdroplet of embodiment 84 or 85, wherein the agent is a small molecule compound, a peptide, or a protein.
87. The microdroplet of any of embodiments 84-86, wherein the agent is a small molecule antibiotic or peptide antibiotic.
88. The microdroplet of any of embodiments 84-87, wherein the agent is an antibody or antigen-binding fragment thereof.
89. The microdroplet of any of embodiments 84-88, wherein the microdroplet further comprises a cell that produces or secretes the agent.
90. The microdroplet of embodiment 89, wherein the cell is an antibody-producing cell.
91. The microdroplet of embodiment 89 or embodiment 90, wherein the cell is a B cell.
92. The microdroplet of any of embodiments 89-91 , wherein the cell is a plasma cell or a plasmablast.
93. The microdroplet of any of embodiments 89-92, wherein the cell is a mammalian cell.
94. The microdroplet of any of embodiments 89-93, wherein the cell is a
microorganism.
95. The microdroplet of any of embodiments 89-94, wherein the cell is a fungal or bacterial cell.
96. The microdroplet of any of embodiments 83-95, wherein the microdroplet comprises agarose, carrageenan, alginate, alginate-polylysine, collagen, cellulose, methylcellulose, gelatin, chitosan, extracellular matrix, dextran, starch, inulin, heparin, hyaluronan, fibrin, polyvinyl alcohol, poly(N-vinyl-2-pyrrolidone), polyethylene glycol, poly(hydroxyethyl methacrylate), acrylate polymers and sodium polyacrylate, polydimethyl siloxane, cis- polyisoprene, Puramatrix™, poly-divenylbenzene, polyurethane, polyacrylamide or combinations thereof.
97. The microdroplet of any of embodiments 83-96, wherein the microdroplet comprises agarose.
98. The microdroplet of any of embodiments 83-97, wherein the microdroplet comprises growth media.
99. The microdroplet of any of embodiments 83-98, wherein the microdroplet comprises serum.
100. The microdroplet of embodiment 99, wherein the serum is human serum, rabbit serum, bovine serum, or mouse serum.
101. The microdroplet of embodiment 99 or embodiment 100, wherein the
concentration of serum is at least or at least about 2.5% (vol/vol), 5.0% (vol/vol), 7.5% (vol/vol), 10.0% (vol/vol), 15.0% (vol/vol), 20% (vol/vol), or 25% (vol/vol).
102. The microdroplet of any of embodiments 99-101, wherein the concentration of serum is from or from about 2.5% (vol/vol) to 25% (vol/vol), 2.5% (vol/vol) to 15% (vol/vol), 2.5% (vol/vol) to 10% (vol/vol), 2.5% (vol/vol) to 5.0% (vol/vol), 5.0% (vol/vol) to 25%
(vol/vol), 5.0% (vol/vol) to 15% (vol/vol), 5.0% (vol/vol) to 10% (vol/vol), 10.0% (vol/vol) to 25% (vol/vol), 10.0% (vol/vol) to 15% (vol/vol), or 15% (vol/vol) to 25% (vol/vol).
103. A composition comprising a microdroplet of any of embodiments 83-102 or a plurality of the microdroplets of any of embodiments 83-102.
104. A kit comprising:
the reporter polynucleotide of any of embodiments 1-35, the reporter vector of any of embodiments 36-53, the reporter microorganism of any of embodiments 54-67, the plurality of reporter microorganisms of any of embodiments 68-73, the composition of any of embodiments 74-82 and 103, or the microdroplet of any of embodiments 83-102; and
instructions for use.
105. A method of assessing outer membrane (OM) stress of a microorganism, comprising: (a) exposing the reporter microorganism of any of embodiments 54-67, the plurality of reporter microorganisms of any of embodiments 68-73, or the composition of any of embodiments 74-82 to a condition that is known to cause or suspected of causing stress to the outer membrane; and
(b) detecting the presence, absence, or level of a detectable signal from the reporter molecule(s).
106. The method of embodiment 105, wherein the method further comprises (c) determining if there a change in the level of the detectable signal from the reporter molecule(s) compared to in the absence of exposing the reporter microorganism to the condition, wherein a change in the level of the detectable signal indicates the condition causes OM stress to the microorganism.
107. The method of embodiment 105 or embodiment 106, wherein the exposing is carried out in suspension, in an array, or in a microdroplet.
108. The method of any of embodiments 105-107, wherein the condition is treatment with an agent.
109. A method of screening an agent, comprising:
(a) contacting the reporter microorganism of any of embodiments 54-67, the plurality of reporter microorganisms of any of embodiments 68-73, or the composition of any of embodiments 74-82 with an agent; and
(b) detecting the presence, absence, or level of a detectable signal from the reporter molecule(s).
110. The method of embodiment 109, wherein the method further comprises (c) identifying the agent that causes a change in the level of the detectable signal from the reporter molecule compared to in the absence of the contacting of the agent.
111. A method of screening an agent, comprising:
(a) contacting an agent with a first reporter microorganism of any of embodiments 54-
67;
(b) contacting the agent with at least one additional reporter microorganism of any of embodiments 54-67 with an agent, wherein the at least one additional reporter microorganism is not the same as the first reporter microorganism; and (c) detecting the presence, absence, or level of a detectable signal from the reporter molecule from the first and/or at least one additional reporter microorganism.
112. The method of embodiment 111, wherein the contacting in (a) and (b) is carried out separately.
113. The method of embodiment 111, wherein the contacting in (a) and (b) is carried out together.
114. The method of embodiments 111 or 113, wherein the first microorganism, the at least one additional reporter microorganism, and the agent are encapsulated together in a microdroplet.
115. The method of any of embodiments 111-114, wherein the first and the at least one additional reporter microorganism comprise a different reporter polynucleotide in which comprises the same regulatory region of an OM-responsive gene but that is operatively linked to a different reporter molecule.
116. The method of any of embodiments 111-114, wherein the first and the at least one additional microorganism comprise a different reporter polynucleotide in which comprises a different regulatory region of an OM-responsive gene and is operatively linked to a different reporter molecule.
117. The method of embodiment 115 or 116, wherein the different reporter molecules do not exhibit overlapping emission and absorption spectra and/or are distinguishably detectable.
118. The method of any of embodiments 111-117, wherein the first and the at least one additional microorganism are derived from the same host strain.
119. The method of any of embodiments 111-118, wherein the first and the at least one additional microorganism are derived from a different host strain, optionally wherein each of the first and the at least one additional microorganism is derived from a different isolate or subtype of the strain.
120. The method of any of embodiments 109-111, wherein the contacting is carried out in suspension, in an array, or in a microdroplet.
121. The method of any of embodiments 109-120, wherein the contacting is carried out for at least or about at least 5 minutes, 10 minutes, 30 minutes, 60 minutes, 2 hours or 3 hours. 122. A method of screening an agent comprising:
(a) encapsulating in a microdroplet: (i) the microorganism of any of embodiments 54-67, a plurality of reporter microorganisms of any of embodiments 68-73, or the composition of any of embodiments 74-82; and (ii) a cell, wherein the cell produces an agent; and
(b) detecting, in the microdroplet, the presence, absence, or level of a detectable signal from the reporter molecule(s).
123. The method of embodiment 122, wherein the method further comprises (c) isolating the cell from the microdroplet in which there is a change in the level of the detectable signal from the reporter molecule compared to in the absence of exposing the reporter microorganism to the agent.
124. The method of embodiment 123, wherein the isolating is carried out using a micromanipulator or an automated sorter.
125. The method of any of embodiments 122 or embodiment 124, further comprising identifying the agent produced by the cell.
126. The method of embodiment 125, wherein identifying comprises determining the sequence of the agent, optionally using single cell PCR and nucleic acid sequencing.
127. The method of any of embodiments 108-126, wherein step (a) is repeated with a plurality of agents.
128. The method of any of embodiments 108-127, wherein:
if there is a change in the presence, absence, or level of the detectable signal, the microorganism is identified as potentially not being resistant to the agent; and
if there is not a change in the presence, absence, or level of the detectable signal, the microorganism is identified as potentially being resistant to the agent.
129. The method of any of embodiments 105-128 or 169-173, wherein the OM-stress responsive gene that is operably linked to the reporter molecule in the reporter vector comprised in the reporter microorganism is downregulated in response to an outer membrane stress and the level of the detectable signal decreases.
130. The method of any of embodiments 105-129 or 169-173, wherein the OM stress- responsive gene is A1S_0009, A1S_0010, A1S_0025, A1S_0027, A1S_0038, A1S_0067, A1S_0070, A1S_0071, A1S_0073, A1S_0076, A1S_0077, A1S_0079, A1S_0087, A1S_0090, A1S_0091, A1SJ3095, A1S_0096, A1S_0097, A1S_0098, A1S_0099, A1S_0103, A1S_0104, A1S_ _0105, A1S_ _0106 A1S_ _0107, A1S_ _0108 A1S_ _0109, A1S_ _0121 A1S_ _0128, A1S_ _0129,
A1S_ _0141, A1S_ _0148 A1S_ _0150, A1S_ _0151 A1S_ _0152, A1S_ _0153 A1S_ _0154, A1S_ _0155,
A1S_ _0156, A1S_ _0157 A1S_ _0177, A1S_ _0184 A1S_ _0200, A1S_ _0201 A1S_ _0209, A1S_ _0218,
A1S_ _0238, A1S_ _0239 A1S_ _0253, A1S_ _0257 A1S_ _0258, A1S_ _0269 A1S_ _0270, A1S_ _0279,
A1S_ _0286, A1S_ _0292 A1S_ _0302, A1S_ _0303 A1S_ _0304, A1S_ _0321 A1S_ _0322, A1S_ _0323,
A1S_ _0347, A1S_ _0365 A1S_ _0369, A1S_ _0370 A1S_ _0388, A1S_ _0408 A1S_ _0410, A1S_ _0427,
A1S_ _0429, A1S_ _0447 A1S_ _0448, A1S_ _0474 A1S_ _0480, A1S_ _0481 A1S_ _0482, A1S_ _0486,
A1S_ _0490, A1S_ _0491 A1S_ _0498, A1S_ _0526 A1S_ _0533, A1S_ _0534 A1S_ _0548, A1S_ _0549,
A1S_ _0566, A1S_ _0567 A1S_ _0568, A1S_ _0591 A1S_ _0594, A1S_ _0624 A1S_ _0625, A1S_ _0626,
A1S_ _0627, A1S_ _0629 A1S_ _0630, A1S_ _0631 A1S_ _0632, A1S_ _0633 A1S_ _0634, A1S_ _0635,
A1S_ _0639, A1S_ _0640 A1S_ _0641, A1S_ _0642 A1S_ _0643, A1S_ _0644 A1S_ _0645, A1S_ _0646,
A1S_ _0647, A1S_ _0649 A1S_ _0650, A1S_ _0651 A1S_ _0690, A1S_ _0691 A1S_ _0692, A1S_ _0695,
A1S_ _0698, A1S_ _0721 A1S_ _0731, A1S_ _0732 A1S_ _0771, A1S_ _0785 A1S_ _0786, A1S_ _0787,
A1S_ _0788, A1S_ _0818 A1S_ _0822, A1S_ _0846 A1S_ _0849, A1S_ _0850 A1S_ _0851, A1S_ _0852,
A1S_ _0853, A1S_ _0854 A1S_ _0855, A1S_ _0869 A1S_ _0877, A1S_ _0882 A1S_ _0883, A1S_ _0890,
A1S_ _0891, A1S_ _0901 A1S_ _0910, A1S_ _0911 A1S_ _0912, A1S_ _0913 A1S_ _0960, A1S_ _0965,
A1S_ _0973, A1S_ _0984 A1S_ _0996, A1S_ _0997 A1S_ _0999, A1S_ _1000 A1S_ _1004, A1S_ _1008,
A1S_ _1021, A1S_ _1026 A1S_ _1044, A1S_ _1063 A1S_ _1072, A1S_ _1079 A1S_ _1080, A1S_ _1088,
A1S_ _1089, A1S_ _1091 A1S_ _1092, A1S_ _1093 A1S_ _1094, A1S_ _1109 A1S_ _1113, A1S_ _1139,
A1S_ _1142, A1S_ _1182 A1S_ _1193, A1S_ _1195 A1S_ _1199, A1S_ _1227 A1S_ _1257, A1S_ _1258,
A1S_ _1261, A1S_ _1264 A1S_ _1265, A1S_ _1266 A1S_ _1267, A1S_ _1268 A1S_ _1269, A1S_ _1270,
A1S_ _1281, A1S_ _1317 A1S_ _1318, A1S_ _1319 A1S_ _1327, A1S_ _1334 A1S_ _1335, A1S_ _1336,
A1S_ _1337, A1S_ _1338 A1S_ _1339, A1S_ _1340 A1S_ _1341, A1S_ _1342 A1S_ _1343, A1S_ _1344,
A1S_ _1345, A1S_ _1346 A1S_ _1347, A1S_ _1348 A1S_ _1349, A1S_ _1356 A1S_ _1366, A1S_ _1367,
A1S_ _1368, A1S_ _1369 A1S_ _1370, A1S_ _1372 A1S_ _1373, A1S_ _1374 A1S_ _1375, A1S_ _1376,
A1S_ _1377, A1S_ _1378 A1S_ _1379, A1S_ _1380 A1S_ _1396, A1S_ _1397 A1S_ _1428, A1S_ _1442,
A1S_ _1443, A1S_ _1450 A1S_ _1466, A1S_ _1467 A1S_ _1469, A1S_ _1470 A1S_ _1476, A1S_ _1490,
A1S_ _1491, A1S_ _1492 A1S_ _1493, A1S_ _1498 A1S_ _1499, A1S_ _1505 A1S_ _1510, A1S_ _1523,
A1S_ _1528, A1S_ _1530 A1S_ _1532, A1S_ _1543 A1S_ _1579, A1S_ _1583 A1S_ _1601, A1S_ _1608,
A1S_ _1609, A1S_ _1610 A1S_ _1611, A1S_ _1612 A1S_ _1613, A1S_ _1637 A1S_ _1638, A1S_ _1639,
A1S_ _1655, A1S_ _1692 A1S_ _1698, A1S_ _1699 A1S_ _1700, A1S_ _1701 A1S_ _1703, A1S_ _1705, A1S_ _1717, A1S_ _1719 A1S_ _1724, A1S_ _1729 A1S_ _1730, A1S_ _1731 A1S_ _1732, A1S_ _1734,
A1S_ _1735, A1S_ _1736 A1S_ _1737, A1S_ _1738 A1S_ _1742, A1S_ _1745 A1S_ _1754, A1S_ _1756,
A1S_ _1758, A1S_ _1775 A1S_ _1776, A1S_ _1790 A1S_ _1791, A1S_ _1792 A1S_ _1794, A1S_ _1795,
A1S_ _1796, A1S_ _1797 A1S_ _1805, A1S_ _1806 A1S_ _1811, A1S_ _1830 A1S_ _1834, A1S_ _1835,
A1S_ _1836, A1S_ _1837 A1S_ _1838, A1S_ _1839 A1S_ _1840, A1S_ _1841 A1S_ _1854, A1S_ _1855,
A1S_ _1856, A1S_ _1857 A1S_ _1858, A1S_ _1859 A1S_ _1860, A1S_ _1861 A1S_ _1862, A1S_ _1863,
A1S_ _1864, A1S_ _1865 A1S_ _1866, A1S_ _1879 A1S_ _1880, A1S_ _1887 A1S_ _1908, A1S_ _1924,
A1S_ _1925, A1S_ _1926 A1S_ _1935, A1S_ _1940 A1S_ _1942, A1S_ _1948 A1S_ _1951, A1S_ _1984,
A1S_ _1996, A1S_ _2041 A1S_ _2042, A1S_ _2052 A1S_ _2053, A1S_ _2068 A1S_ _2072, A1S_ _2081,
A1S_ _2084, A1S_ _2098 A1S_ _2100, A1S_ _2101 A1S_ _2102, A1S_ _2148 A1S_ _2149, A1S_ _2150,
A1S_ _2163, A1S_ _2166 A1S_ _2167, A1S_ _2190 A1S_ _2191, A1S_ _2202 A1S_ _2203, A1S_ _2207,
A1S_ _2209, A1S_ _2218 A1S_ _2221, A1S_ _2225 A1S_ _2232, A1S_ _2234 A1S_ _2248, A1S_ _2279,
A1S_ _2280, A1S_ _2288 A1S_ _2289, A1S_ _2340 A1S_ _2341, A1S_ _2342 A1S_ _2348, A1S_ _2353,
A1S_ _2354, A1S_ _2415 A1S_ _2416, A1S_ _2417 A1S_ _2418, A1S_ _2419 A1S_ _2424, A1S_ _2425,
A1S_ _2431, A1S_ _2435 A1S_ _2443, A1S_ _2449 A1S_ _2450, A1S_ _2451 A1S_ _2452, A1S_ _2475,
A1S_ _2501, A1S_ _2509 A1S_ _2510, A1S_ _2514 A1S_ _2531, A1S_ _2532 A1S_ _2533, A1S_ _2535,
A1S_ _2601, A1S_ _2602 A1S_ _2633, A1S_ _2662 A1S_ _2670, A1S_ _2671 A1S_ _2672, A1S_ _2688,
A1S_ _2692, A1S_ _2694 A1S_ _2695, A1S_ _2696 A1S_ _2701, A1S_ _2711 A1S_ _2722, A1S_ _2724,
A1S_ _2738, A1S_ _2740 A1S_ _2741, A1S_ _2748 A1S_ _2753, A1S_ _2755 A1S_ _2758, A1S_ _2761,
A1S_ _2762, A1S_ _2769 A1S_ _2773, A1S_ _2774 A1S_ _2785, A1S_ _2788 A1S_ _2789, A1S_ _2793,
A1S_ _2809, A1S_ _2814 A1S_ _2815, A1S_ _2820 A1S_ _2823, A1S_ _2847 A1S_ _2848, A1S_ _2849,
A1S_ _2852, A1S_ _2860 A1S_ _2904, A1S_ _2905 A1S_ _2906, A1S_ _2911 A1S_ _2913, A1S_ _2919,
A1S_ _2924, A1S_ _2928 A1S_ _2939, A1S_ _2946 A1S_ _2956, A1S_ _3013 A1S_ _3014, A1S_ _3025,
A1S_ _3040, A1S_ _3043 A1S_ _3049, A1S_ _3050 A1S_ _3051, A1S_ _3074 A1S_ _3084, A1S_ _3110,
A1S_ _3120, A1S_ _3121 A1S_ _3122, A1S_ _3128 A1S_ _3129, A1S_ _3130 A1S_ _3131, A1S_ _3132,
A1S_ _3133, A1S_ _3134 A1S_ _3135, A1S_ _3144 A1S_ _3174, A1S_ _3180 A1S_ _3195, A1S_ _3207,
A1S_ _3222, A1S_ _3224 A1S_ _3225, A1S_ _3231 A1S_ _3232, A1S_ _3236 A1S_ _3238, A1S_ _3248,
A1S_ _3250, A1S_ _3268 A1S_ _3269, A1S_ _3273 A1S_ _3278, A1S_ _3290 A1S_ _3297, A1S_ _3298,
A1S_ _3300, A1S_ _3301 A1S_ _3309, A1S_ _3338 A1S_ _3342, A1S_ _3355 A1S_ _3364, A1S_ _3377,
A1S_ _3397, A1S_ _3398 A1S_ _3402, A1S_ _3403 A1S_ _3404, A1S_ _3405 A1S_ _3406, A1S_ _3407,
A1S_ _3410, A1S_ _3413 A1S_ _3414, A1S_ _3415 A1S_ _3416, A1S_ _3418 A1S_ _3431, A1S_ _3450, A1S_ _3451, A1S_ _3458, A1S_ _3460, A1S_ _3481, A1S_ _3487, A1S_ _3491, A1S_ _3494, A1S_ _3498,
A1S_ _3506, A1S_ _3508, A1S_ _3509, A1S_ _3514, A1S_ _3518, A1S_ _3519, A1S_ _3520, A1S_ _3521,
A1S_ _3522, A1S_ _3523, A1S_ _3524, A1S_ _3526, A1S_ _3528, A1S_ _3530, A1S_ _3531, A1S_ _3532,
A1S_ _3533, A1S_ _3534, A1S_ _3535, A1S_ _3537, A1S_ _3538, A1S_ _3539, A1S_ _3540, A1S_ J3541,
A1S_ _3542, A1S_ _3543, A1S_ _3544, A1S_ _3545, A1S_ _3546, A1S_ _3547, A1S_ _3548, A1S_ _3549,
A1S_ _3550, A1S_ _3552, A1S_ _3553, A1S_ _3554, A1S_ _3568, A1S_ _3569, A1S_ _3578, A1S_ _3582,
A1S_ _3586, A1S_ _3587, A1S_ _3591, A1S_ _3597, A1S_ _3599, A1S_ _3600, A1S_ _3602, A1S_ _3611,
A1S_ _3619, A1S_ _3621, A1S_ _3624, A1S_ _3629, A1S_ _3633, A1S_ _3640, A1S_ _3641, A1S_ _3644,
A1S_ _3647, A1S_ _3651, A1S_ _3652, A1S_ _3659, A1S_ _3663, A1S_ _3667, A1S_ _3673, A1S_ _3679,
A1S_ _3701, A1S_ _3707, A1S_ _3709, A1S_ _3713, A1S_ _3715, A1S_ _3717, A1S_ _3732, A1S_ _3735,
A1S_ _3738, A1S_ _3740, A1S_ _3741, A1S_ _3742, A1S_ _3759, A1S_ _3774, A1S_ _3779, A1S_ _3787,
A1S_ _3788, A1S_ _3794, A1S_ _3801, A1S_ _3802, A1S_ _3806, A1S_ _3809, A1S_ _3811, A1S_ _3813,
A1S_ _3814, A1S_ _3816, A1S_ _3817, A1S_ _3823, A1S_ _3829, A1S_ _3831, A1S_ _3832, A1S_ _3836,
A1S_ _3840, A1S_ _3846, A1S_ _3857, A1S_ _3862, A1S_ _3868, A1S_ _3870, A1S_ _3880, A1S_ _3884,
A1S_ _3886, A1S_ _3887, A1S_ _3891, A1S_ _3894, A1S_ _3898, A1S_ _3907, A1S_ _3908, A1S_ _3909,
A1S_3912, A1S_3914, or A1S_3915.
131. The method of any of embodiments 105-130 or 169-173, wherein the OM stress- responsive gene is A1S_0103, A1S_0645, A1S_1266, A1S_1268, A1S_1335, A1S_1336, A1S_1337, A1S_1338, A1S_1339, A1S_1340, A1S_1341, A1S_1342, A1S_1343, A1S_1344, A1S_1345, A1S_1791, A1S_1792, A1S_1794, A1S_1796, A1S_1835, A1S_1836, A1S_1837, A1S_1838, A1S_1839, A1S_2449, A1S_2450, A1S_2452, A1S_3540, A1S_3541, A1S_3542, A1S_3543, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, A1S_3809, or
A1S_3908.
132. The method of any of embodiments 105-131 or 169-173, wherein the OM stress- responsive gene is A1S_1336, A1S_1836, A1S_1838, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, or A1S_3809.
133. The method of any of embodiments 105-128 or 169-173, wherein the OM-stress responsive gene that is operably linked to the reporter molecule in the reporter vector comprised in the reporter microorganism is upregulated in response to an outer membrane stress and the level of the detectable signal increases. 134. The method of any of embodiments 105-128, 133, or 169-173, wherein the OM stress-responsive gene is A1SJ3012, A1SJ3023, A1S_0027, A1S_0028, A1S_0029, A1S_0030,
A1S_ _0031, A1S_ _0032, A1S_ _0033, A1S_ _0037, A1S_ _0040, A1S_ _0041, A1S_ _0044, A1S_ _0066,
A1S_ _0092, A1S_ _0093, A1S_ _0109, A1S_ _0110, A1S_ _0112, A1S_ _0113, A1S_ _0114, A1S_ _0115,
A1S_ _0116, A1S_ _0117, A1S_ _0118, A1S_ _0126, A1S_ _0158, A1S_ _0170, A1S_ _0175, A1S_ _0178,
A1S_ _0189, A1S_ _0224, A1S_ _0245, A1S_ _0256, A1S_ _0276, A1S_ _0293, A1S_ _0301, A1S_ _0309,
A1S_ _0310, A1S_ _0332, A1S_ _0333, A1S_ _0363, A1S_ _0372, A1S_ _0376, A1S_ _0391, A1S_ _0392,
A1S_ _0401, A1S_ _0441, A1S_ _0462, A1S_ _0463, A1S_ _0464, A1S_ _0465, A1S_ _0466, A1S_ _0494,
A1S_ _0508, A1S_ _0509, A1S_ _0510, A1S_ _0511, A1S_ _0512, A1S_ _0514, A1S_ _0516, A1S_ _0518,
A1S_ _0519, A1S_ _0520, A1S_ _0521, A1S_ _0522, A1S_ _0523, A1S_ _0527, A1S_ _0535, A1S_ _0536,
A1S_ _0537, A1S_ _0538, A1S_ _0547, A1S_ _0559, A1S_ _0561, A1S_ _0562, A1S_ _0563, A1S_ _0564,
A1S_ _0566, A1S_ _0567, A1S_ _0568, A1S_ _0570, A1S_ _0624, A1S_ _0630, A1S_ _0631, A1S_ _0633,
A1S_ _0634, A1S_ _0640, A1S_ _0641, A1S_ _0642, A1S_ _0643, A1S_ _0644, A1S_ _0645, A1S_ _0646,
A1S_ _0647, A1S_ _0650, A1S_ _0663, A1S_ _0664, A1S_ _0665, A1S_ _0666, A1S_ _0667, A1S_ _0669,
A1S_ _0670, A1S_ _0671, A1S_ _0673, A1S_ _0677, A1S_ _0680, A1S_ _0683, A1S_ _0714, A1S_ _0717,
A1S_ _0718, A1S_ _0719, A1S_ _0736, A1S_ _0738, A1S_ _0739, A1S_ _0749, A1S_ _0770, A1S_ _0772,
A1S_ _0779, A1S_ _0780, A1S_ _0781, A1S_ _0800, A1S_ _0804, A1S_ _0830, A1S_ _0831, A1S_ _0832,
A1S_ _0834, A1S_ _0835, A1S_ _0861, A1S_ _0884, A1S_ _0889, A1S_ _0929, A1S_ _0930, A1S_ _0931,
A1S_ _0932, A1S_ _0935, A1S_ _0945, A1S_ _0959, A1S_ _0980, A1S_ _1003, A1S_ _1009, A1S_ _1010,
A1S_ _1027, A1S_ _1028, A1S_ _1030, A1S_ _1031, A1S_ _1049, A1S_ _1081, A1S_ _1106, A1S_ _1107,
A1S_ _1120, A1S_ _1121, A1S_ _1123, A1S_ _H32, A1S_ _H33, A1S_ _H34, A1S_ _H39, A1S_ _1143,
A1S_ _1145, A1S_ _1146, A1S_ _1148, A1S_ _1149, A1S_ _1150, A1S_ _1151, A1S_ _1152, A1S_ _1153,
A1S_ _1155, A1S_ _1156, A1S_ _1157, A1S_ _1158, A1S_ _1159, A1S_ _1160, A1S_ _1161, A1S_ _1162,
A1S_ _1163, A1S_ _1164, A1S_ _1165, A1S_ _1167, A1S_ _1171, A1S_ _1172, A1S_ _1173, A1S_ _1180,
A1S_ _1184, A1S_ _1186, A1S_ _1198, A1S_ _1202, A1S_ _1203, A1S_ _1223, A1S_ _1224, A1S_ _1225,
A1S_ _1230, A1S_ _1236, A1S_ _1237, A1S_ _1248, A1S_ _1255, A1S_ _1274, A1S_ _1286, A1S_ _1359,
A1S_ _1360, A1S_ _1361, A1S_ _1362, A1S_ _1363, A1S_ _1383, A1S_ _1384, A1S_ _1385, A1S_ _1386,
A1S_ _1387, A1S_ _1393, A1S_ _1404, A1S_ _1407, A1S_ _1422, A1S_ _1454, A1S_ _1472, A1S_ _1481,
A1S_ _1494, A1S_ _1512, A1S_ _1515, A1S_ _1526, A1S_ _1535, A1S_ _1539, A1S_ _1566, A1S_ _1567,
A1S_ _1569, A1S_ _1583, A1S_ _1584, A1S_ _1585, A1S_ _1589, A1S_ _1590, A1S_ _1593, A1S_ _1595,
A1S_ _1596, A1S_ _1617, A1S_ _1630, A1S_ _1644, A1S_ _1645, A1S_ _1647, A1S_ _1648, A1S_ _1649, A1S_ _1651, A1S_ _1655 A1S_ _1658, A1S_ _1662 A1S_ _1666, A1S_ _1667 A1S_ _1669, A1S_ _1677,
A1S_ _1680, A1S_ _1681 A1S_ _1687, A1S_ _1735 A1S_ _1741, A1S_ _1743 A1S_ _1744, A1S_ _1750,
A1S_ _1751, A1S_ _1752 A1S_ _1760, A1S_ _1762 A1S_ _1767, A1S_ _1778 A1S_ _1813, A1S_ _1827,
A1S_ _1829, A1S_ _1831 A1S_ _1843, A1S_ _1876 A1S_ _1909, A1S_ _1928 A1S_ _1929, A1S_ _1934,
A1S_ _1952, A1S_ _1955 A1S_ _1956, A1S_ _1957 A1S_ _1959, A1S_ _1960 A1S_ _1961, A1S_ _1962,
A1S_ _1963, A1S_ _1979 A1S_ _1986, A1S_ _1987 A1S_ _1988, A1S_ _2006 A1S_ _2026, A1S_ _2033,
A1S_ _2034, A1S_ _2035 A1S_ _2036, A1S_ _2038 A1S_ _2039, A1S_ _2061 A1S_ _2074, A1S_ _2079,
A1S_ _2082, A1S_ _2092 A1S_ _2093, A1S_ _2106 A1S_ _2139, A1S_ _2140 A1S_ _2141, A1S_ _2142,
A1S_ _2146, A1S_ _2157 A1S_ _2158, A1S_ _2160 A1S_ _2161, A1S_ _2162 A1S_ _2178, A1S_ _2179,
A1S_ _2183, A1S_ _2186 A1S_ _2195, A1S_ _2230 A1S_ _2247, A1S_ _2252 A1S_ _2257, A1S_ _2258,
A1S_ _2259, A1S_ _2262 A1S_ _2271, A1S_ _2272 A1S_ _2273, A1S_ _2283 A1S_ _2285, A1S_ _2298,
A1S_ _2311, A1S_ _2315 A1S_ _2325, A1S_ _2326 A1S_ _2330, A1S_ _2331 A1S_ _2366, A1S_ _2367,
A1S_ _2382, A1S_ _2387 A1S_ _2389, A1S_ _2395 A1S_ _2396, A1S_ _2414 A1S_ _2434, A1S_ _2445,
A1S_ _2446, A1S_ _2447 A1S_ _2448, A1S_ _2454 A1S_ _2455, A1S_ _2456 A1S_ _2458, A1S_ _2459,
A1S_ _2463, A1S_ _2480 A1S_ _2489, A1S_ _2503 A1S_ _2504, A1S_ _2508 A1S_ _2542, A1S_ _2543,
A1S_ _2552, A1S_ _2553 A1S_ _2555, A1S_ _2557 A1S_ _2558, A1S_ _2573 A1S_ _2577, A1S_ _2578,
A1S_ _2580, A1S_ _2586 A1S_ _2588, A1S_ _2593 A1S_ _2611, A1S_ _2612 A1S_ _2613, A1S_ _2624,
A1S_ _2650, A1S_ _2651 A1S_ _2654, A1S_ _2656 A1S_ _2660, A1S_ _2664 A1S_ _2668, A1S_ _2675,
A1S_ _2676, A1S_ _2677 A1S_ _2678, A1S_ _2684 A1S_ _2705, A1S_ _2729 A1S_ _2734, A1S_ _2756,
A1S_ _2768, A1S_ _2786 A1S_ _2798, A1S_ _2801 A1S_ _2807, A1S_ _2826 A1S_ _2827, A1S_ _2828,
A1S_ _2839, A1S_ _2863 A1S_ _2882, A1S_ _2883 A1S_ _2884, A1S_ _2885 A1S_ _2889, A1S_ _2892,
A1S_ _2893, A1S_ _2942 A1S_ _2943, A1S_ _2953 A1S_ _2959, A1S_ _2960 A1S_ _2968, A1S_ _2976,
A1S_ _2992, A1S_ _3011 A1S_ _3026, A1S_ _3027 A1S_ _3034, A1S_ _3035 A1S_ _3047, A1S_ _3048,
A1S_ _3099, A1S_ _3100 A1S_ _3101, A1S_ _3104 A1S_ _3105, A1S_ _3114 A1S_ _3115, A1S_ _3116,
A1S_ _3117, A1S_ _3124 A1S_ _3125, A1S_ _3126 A1S_ _3127, A1S_ _3139 A1S_ _3146, A1S_ _3147,
A1S_ _3175, A1S_ _3206 A1S_ _3224, A1S_ _3253 A1S_ _3259, A1S_ _3280 A1S_ _3281, A1S_ _3295,
A1S_ _3317, A1S_ _3326 A1S_ _3339, A1S_ _3360 A1S_ _3361, A1S_ _3367 A1S_ _3368, A1S_ _3371,
A1S_ _3375, A1S_ _3376 A1S_ _3392, A1S_ _3411 A1S_ _3412, A1S_ _3463 A1S_ _3466, A1S_ _3468,
A1S_ _3469, A1S_ _3471 A1S_ _3479, A1S_ _3480 A1S_ _3486, A1S_ _3492 A1S_ _3493, A1S_ _3494,
A1S_ _3499, A1S_ _3510 A1S_ _3512, A1S_ _3518 A1S_ _3522, A1S_ _3523 A1S_ _3533, A1S_ _3534,
A1S_ _3535, A1S_ _3539 A1S_ _3540, A1S_ _3541 A1S_ _3542, A1S_ _3543 A1S_ _3544, A1S_ _3545, A1S_3546, A1S_3548, A1S_3552, A1S_3553, A1S_3558, A1S_3559, A1S_3562, A1S_3563, A1S_3567, A1S_3570, A1S_3577, A1S_3580, A1S_3580, A1S_3585, A1S_3586, A1S_3594, A1S_3595, A1S_3596, A1S_3601, A1S_3602, A1S_3603, A1S_3604, A1S_3605, A1S_3606, A1S_3607, A1S_3608, A1S_3609, A1S_3610, A1S_3611, A1S_3612, A1S_3613, A1S_3614, A1S_3617, A1S_3618, A1S_3621, A1S_3630, A1S_3632, A1S_3634, A1S_3635, A1S_3636, A1S_3637, A1S_3642, A1S_3645, A1S_3649, A1S_3654, A1S_3658, A1S_3661, A1S_3662, A1S_3666, A1S_3682, A1S_3686, A1S_3687, A1S_3688, A1S_3694, A1S_3695, A1S_3697, A1S_3704, A1S_3707, A1S_3708, A1S_3712, A1S_3716, A1S_3725, A1S_3726, A1S_3727, A1S_3728, A1S_3736, A1S_3738, A1S_3739, A1S_3740, A1S_3750, A1S_3752, A1S_3760, A1S_3768, A1S_3769, A1S_3770, A1S_3771, A1S_3772, A1S_3773, A1S_3776, A1S_3777, A1S_3778, A1S_3782, A1S_3783, A1S_3786, A1S_3789, A1S_3790, A1S_3791, A1S_3792, A1S_3797, A1S_3810, A1S_3818, A1S_3820, A1S_3835, A1S_3837, A1S_3840, A1S_3842, A1S_3844, A1S_3862, A1S_3864, A1S_3865, A1S_3866, A1S_3867, A1S_3868, A1S_3873, A1S_3875, A1S_3879, A1S_3889, A1S_3900, A1S_3901, A1S_3902, A1S_3908, or
A1S_3911.
135. The method of any of embodiments 105-128, 133-134, or 169-173, wherein the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or A1S_3127.
136. The method of any of embodiments 105-128, 133-135, or 169-173, wherein the OM stress-responsive gene is A1S_0032, A1S_2885, or A1S_2889.
137. The method of any of embodiments 105-121, 127-136, or 169-173, wherein the reporter microorganism of (a) is encapsulated in a microdroplet with a cell.
138. The method of any of embodiments 108-137, wherein the agent is a small molecule compound, a peptide, or a protein.
139. The method of any of embodiments 108-138, wherein the agent is an antibiotic.
140. The method of any of embodiments 108-139, wherein the agent is an antibody or antigen-binding fragment thereof.
141. The method of any of embodiments 122-140, wherein the agent is an antibody and the cell is an antibody-producing cell.
142. The method of any of embodiments 122-141, wherein the cell is a
microorganism. 143. The method of embodiment 142, wherein the cell is a fungal or bacterial cell.
144. The method of any of embodiments 122-141, wherein the cell is a mammalian cell.
145. The method of any of embodiments 122-141, 144, wherein the cell is a B cell.
146. The method of any of embodiments 122-141 or 144-145, wherein the cell is a plasma cell or a plasmablast.
147. The method of any of embodiments 122-141 or 144-146, wherein the cell is obtained from a donor that has been exposed to the target microorganism or an epitope- comprising fragment of a target microorganism or a variant thereof.
148. The method of embodiment 147, wherein the donor has been immunized or infected with the target microorganism or an epitope-comprising fragment of the target microorganism or a variant thereof.
149. The method of embodiment 147 or 148, wherein the donor is an immunized animal or an infected animal.
150. The method of any of embodiments 147-149, wherein the donor is a mammal or a bird.
151. The method of any of embodiments 147-150, wherein the donor is a human, a mouse or a chicken.
152. The method of any of embodiments 147-151, wherein the donor is a human donor who was infected by the target microorganism.
153. The method of any of embodiments 147-152, wherein the donor is a genetically modified non-human animal that produces partially human or fully human antibodies.
154. The method of any of embodiments 120-153, wherein the microdroplet is generated by a microfluidics-based method.
155. The method of any of embodiments 120-154, wherein the microdroplet comprises agarose, carrageenan, alginate, alginate-polylysine, collagen, cellulose, methylcellulose, gelatin, chitosan, extracellular matrix, dextran, starch, inulin, heparin, hyaluronan, fibrin, polyvinyl alcohol, poly(N-vinyl-2-pyrrolidone), polyethylene glycol, poly(hydroxyethyl methacrylate), acrylate polymers and sodium polyacrylate, polydimethyl siloxane, cis- polyisoprene,
Puramatrix™, poly-divenylbenzene, polyurethane, polyacrylamide, or combinations thereof. 156. The method of any of embodiments 120-155, wherein the microdroplet comprises agarose.
157. The method of any of embodiments 137-156, wherein the method further comprises (c) isolating the cell from the microdroplet in which there is a change in the level of the detectable signal from the reporter molecule compared to in the absence of exposing the reporter microorganism to the agent.
158. The method of embodiment 157, wherein the isolating is carried out using a micromanipulator or an automated sorter.
159. The method of any of embodiments 157 or embodiment 158, further comprising identifying the agent produced by the cell.
160. The method of embodiment 159, wherein identifying comprises determining the sequence of the agent, optionally using single cell PCR and nucleic acid sequencing.
161. The method of any of embodiments 105-160 or 169-173, comprising prior to (a) introducing the reporter polynucleotide of any of embodiments 1-35 or reporter vector of any of embodiments 36-47 into a host microorganism.
162. The method of embodiment 161, wherein the host microorganism is a bacterium.
163. The method of embodiment 161 or 162, wherein the host microorganism is a Gram negative bacterium.
164. The method of any of embodiments 161-163, wherein the host microorganism is a proteobacterium.
165. The method of any of embodiments 161- 164, wherein the host microorganism is a species of Acinetobacter, Bdellovibrio, Burkholderia, Chlamydia, Enterobacter, Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella, Neisseria, Pantoea, Pseudomonas, Salmonella, Shigella, Stenotrophomonas, Vibrio, or Yersinia.
166. The method of any of embodiments 161-165, wherein the host microorganism is Acinetobacter apis, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii, Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter gerneri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus, Acinetobacter harbinensis, Acinetobacter indicus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter kookii, Acinetobacter Iwoffii, Acinetobacter nectaris, Acinetobacter nosocomialis, Acinetobacter pakistanensis, Acinetobacter parvus, Acinetobacter pitii, Acinetobacter pittii, Acinetobacter puyangensis, Acinetobacter qingfengensis, Acinetobacter radioresistans, Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii, Acinetobacter soli, Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis, Acinetobacter venetianus, Escherichia coli, Haemophilus influenzae, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella typhimurium, Shigella boydii, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Vibrio cholera, or Yersinia pestis.
167. The method of embodiment 161-166, wherein the host microorganism is Acinetobacter baumannii.
168. An agent identified by the method of any of embodiments 108- 167 or 169-173.
169. A method of determining the drug resistance of a microorganism, the method comprising:
(a) contacting a microorganism comprising the reporter polynucleotide of any of embodiments 1-35 or the reporter vector of any of embodiments 36-47 with a drug; and
(b) identifying a microorganism in which there is a change in a detectable signal from the reporter molecule compared to in the absence of the contacting with the drug, wherein if there is a change in the detectable signal, the microorganism is not resistant to the drug and if there is not a change in the detectable signal, the microorganism is identified as potentially being resistant to the drug.
170. The method of embodiment 169, wherein the drug is a small molecule compound, a peptide or a protein.
171. The method of embodiments 169 or 170, wherein the drug is an antibiotic.
172. The method of any of embodiments 169-171, wherein the drug is an antibody or antigen-binding fragment thereof.
173. The method of any of embodiments 169-172, wherein the drug is an antibody and the cell is an antibody-producing cell.
IX. EXAMPLES
[0325] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. Example 1 : Identifying Outer Membrane-Stress-Reactive Genes in Acinetol>acter
[0326] Assays were performed to identify genes in Acinetobacter baumannii (A. baumannii) that are modulated (increased or decreased) in response to outer membrane stress. An outer membrane stress response was induced in A. baumannii by BamA inhibition (e.g. BamA depletion) or by using polymyxin B nonapeptide (PMBN), which is an agent known to disrupt or permeabilize the outer membrane of Gram-negative bacteria. Changes in expression of gene expression were assessed using RNA-Seq.
A. BamA depletion
1. Methods
[0327] Outer membrane (OM) stress response genes that are modulated in response to BamA depletion were identified using one of two modified A. baumannii strains, designated AABA041 and AABA046, that allow for exogenous control of the expression of BamA, an essential OM biogenesis factor, through the presence of an arabinose inducer. The AABA041 and AABA046 strains carry: (1) a deletion of the chromosomal bamA gene by replacement of the gene with a selective marker endowing resistance to kanamycin; and (2) a replicating extrachromosomal plasmid that carries the open reading frame of the bamA gene from A.
baumannii (ATCC No. 19606) fused to the araBAD promoter from E. coli which allows regulation of transcription by addition of the inducing sugar arabinose. Because BamA is an essential protein, this strain requires arabinose to grow by inducing expression of BamA.
[0328] Strain AABA046 was grown in duplicate with arabinose (Ara+) or without arabinose (Ara-) and cultures were backdiluted every 3 hours to maintain the cells in exponential growth phase. The growth curve for the Ara-i- and Ara- cultures are shown in FIG. 1, which depicts in circles the time points where back-dilution was carried out. As shown, after six hours the cultures lacking arabinose failed to grow, indicating that BamA was essential for
growth/viability. At the time points indicated by squares in FIG. 1, i.e. 2, 4 and 6 hours, 1 mL samples were harvested, pelleted, and snap-frozen for gene expression analysis using RNA-Seq. For analysis, the samples were lysed in the presence of TRIzol™ reagent to extract RNA and total RNA was purified using a column with enzymatic removal of DNA.
[0329] For each sample, after depleting ribosomal RNA by negative selection, RNA was reverse transcribed to cDNA and a next generation sequencing (NGS) library was prepared by random fragmentation of cDNA with simultaneous 5' and 3' adapter ligation using Nextera transposase-mediated "tagmentation." Multiplexing was carried out and each sample was sequenced using an Illumina HiSeq for analysis of gene expression. Raw reads output from HiSeq were analyzed using FastQC and Trim (Trimmomatic, Bolger et al, Bioinformatics 2014). High quality reads were mapped to the genome sequence of A. baumannii (ATCC No. 17978; GenBank No. CP000521.1) genome using Bowtie (Langmead & Salzberg, Nature Methods, 2012), and transcript levels were normalized and compared using Cufflinks (Trapnell et al, Nature Biotechnology, 2010). Genes that were differentially expressed by culture with or without arabinose were identified.
2. Kesu/ts
[0330] For each sample dataset, gene expression was quantified and normalized to fragments per kilobase of transcript per million mapped reads (FPKM). A strong correlation was observed between replicate datasets, since data points representing an individual transcript in the A. baumannii 17978 genome aligned together in duplicate samples indicating similar gene expression. FIGS. 2A and 2B provide exemplary datasets from the 6-hour time point and demonstrate that data points on plots comparing duplicate samples cultured with arabinose (FIG. 2A) and duplicate samples cultured without arabinose (FIG. 2B) generally had the same gene expression level in both conditions.
[0331] In contrast, differential gene expression was observed when comparing BamA- depleted (+Ara) and non-BamA-depleted (-Ara) cells (Fig. 2C). As shown in FIG. 2C, the differential gene expression was evident by the number of transcript data points that lie off the diagonal, with the distance from the diagonal correlating with the magnitude of difference in gene expression. As shown in FIGS. 3A-3C, these differences in gene expression in cultures grown with inducer (+Ara) as compared to those grown without inducer (-Ara) increased with time, indicating that the global gene expression profile changes following BamA depletion in a time-dependent manner (Fig. 3A-3C).
[0332] Exemplary gene transcripts that were increased or decreased in expression after BamA depletion are summarized in subsection "C" below in Tables 1 and 2, respectively. The fold change in the expression level of exemplary gene transcripts after BamA-depletion (-Ara) compared to expression in BamA-expressing cells (+Ara) is shown at each of the 2, 4, and 6 hour time points in Fig. 4. As shown, the largest increase in expression was for A1S_0032 which had 1210-fold change in expression after 6 hours of BamA depletion. FIG. 5A-5B sets forth exemplary gene transcripts that were increased in expression after BamA-depletion, including increased >10-fold after 4 or 6 hours of BamA depletion. FIG. 6A-D sets forth exemplary gene transcripts that were decreased in expression after BamA-depletion, including decreased >10-fold after 4 or 6 hours of BamA depletion.
B. PMBN
I. Methods
[0333] Wild type A. baumannii (ATCC 17978) were grown in the presence of 25 μg/mL or 250 μg/mL PMBN for either 10 minutes or 30 minutes. Samples were harvested, RNA extracted and used to prepare an NGS library for analysis using RNA-Seq as described above. Genes that were differentially expressed after treatment with PMBN were identified.
2. iesults
[0334] Exemplary gene transcripts that were increased or decreased in expression in the presence of PMBN are summarized in subsection "C" below in Tables 1 and 2, respectively. As shown in FIG. 8A-8D, several exemplary gene transcripts were observed to be variously increased after treatment with PMBN at the indicated times and concentrations. As shown in FIG. 9, treatment with 250 μg/mL PMBN increased expression of gene transcripts to greater levels than treatment with 25 μg/mL, although each of the exemplary transcripts were upregulated to some degree under both conditions compared to expression without PMBN treatment. After 10 minutes in the presence of 250 μg/mL PMBN in this exemplary experiment, 9 gene transcripts showed a >10-fold increase in expression compared to expression without treatment, while only 2 of the gene transcripts showed a >10-fold increase in expression after 10 minutes of in the presence of 25 μg/mL PMBN. After 30 minutes of treatment in the presence of 250 μg/mL PMBN, 4 genes showed a >10-fold increase in expression, while only 1 gene showed a >10-fold increase in expression after 30 minutes of treatment in the presence of 25 μg/mL PMBN. C. Summary and Comparison of Gene Expression Following BamA depletion and PMBN treatment
[0335] The findings showed that BamA depletion and PMBN treatment induced expression of an overlapping set of genes, indicating a concerted regulatory response to defects in OM biogenesis or integrity. BamA depletion, which is a more severe physiological event, caused a more robust response. While there are genes that were induced after BamA depletion and not after PMBN treatment, or vice versa, the majority of genes induced by one event were also induced by the other, indicating that a common stress response or signal transduction cascade is being activated by each of these cues.
[0336] Table 1 sets forth exemplary gene transcripts that were increased in expression after 6 hours of BamA depletion, as well as exemplary gene transcripts that were increased in expression after PMBN treatment. Table 2 sets forth exemplary gene transcripts that were decreased in expression after 6 hours of BamA depletion, as well as exemplary gene transcripts that were decreased in expression after 250 μg/mL PMBN treatment.
Table 1. Exemplary transcripts that were increased in expression after outer membrane stress
Gene ID Locus (relative to BamA 10 min. 30 min. Gene ID Locus (relative to BamA 10 min. 30 min.
ATCC 17978 depletion PMBN PMBN ATCC 17978 depletion PMBN PMBN genome) (fold (fold (fold genome) (fold (fold (fold change) change) change) change) change) change)
A1S_0012 13997-14843 2.4 A1S_0256 283070-283775 2.5
A1S_0023 30806-31940 2.3 A1S_0276 300005-300089 2.0
A1S_0027 34316-36286 3.3 A1S_0293 319228-319582 3.1
A1S_0028 34316-36286 2.3 A1S_0301 326394-326952 4.7
A1S_0029 36312-37296 2.1 A1S_030 336584-337115 3.0
A1S_0030 37367-38336 2.4 A1SJ1310 337239-339048 3.3
A1S_0031 38669-40025 8.0 A1SJB32 359439-359524 6.0
A1S_0032 40145-40466 1210.7 106.9 5.0 A1SJB33 359566-359643 2.8
A1S_0033 40688-41099 45.3 3.3 A1SJB63 398936-399266 2.6
A1S_0037 43718-44336 5.1 2.2 A1SJB72 409035-409374 2.3
A1S_0040 46009-47035 2.8 A1S_0376 411954-412212 2.6
A1S_0041 47059-48235 2.2 A1SJB91 423435-423690 6.3
A1S_0044 51946-52956 7.0 A1S_0392 423791-425090 3.3
A1S_0066 78157-79528 2.3 A1S_0401 433002-433078 2.3
A1S_0092 110458-112432 2.4 A1S_0441 480807-481380 5.2
A1S_0093 112633-113779 2.4 A1S_0462 498659-500993 2.2
A1S_0109 130375-130927 2.1 8.1 A1S_0463 501200-503381 2.6
AISJMIO 131005-131371 3.3 A1S_0464 503554-504759 3.6
A1S_0112 133514-135404 4.3 17.8 A1S_0465 503554-504759 7.0
A1S_0113 135436-141443 6.2 29.1 A1S_0466 504770-504989 11.8
A1S_0114 135436-141443 3.9 17.6 A1S_0494 540486-541095 2.4
A1S_0115 135436-141443 3.6 17.6 A1S_0508 552086-553489 4.1
A1S_0116 141467-146389 2.8 13.8 A1S_0509 553490-553619 2.2
A1S_0117 141467-146389 4.2 A1S_0510 553763-554012 3.6
A1S_0118 146442-149065 3.5 A1S_0511 554032-554563 5.5
A1S_0126 151830-152421 2.2 2.1 A1S_0512 554572-556231 2.3
A1S_0158 178789-179389 18.1 2.6 A1S_0514 556976-557903 2.1
A1SJM70 188522-190598 2.4 A1S_0516 559449-562797 13.0
A1S_0175 195047-195123 3.5 A1S_0518 559449-562797 12.4
A1S_0178 200607-201189 2.1 A1S_0519 559449-562797 8.9
A1S_0189 212191-212815 10.5 A1S_0520 562826-564122 3.4
A1S_0224 250435-250654 2.9 A1S_0521 564124-566294 2.7
A1SJG45 270841-272188 2.1 A1S_0522 564124-566294 2.4
A1S_0523 564124-566294 2.2 A1S_0667 792218-792508 3.4 Table 1. Exemplary transcripts that were increased in expression after outer membrane stress
Gene ID Locus (relative to BamA 10 min. 30 min. Gene ID Locus (relative to BamA 10 min. 30 min.
ATCC 17978 depletion PMBN PMBN ATCC 17978 depletion PMBN PMBN genome) (fold (fold (fold genome) (fold (fold (fold change) change) change) change) change) change)1S_0527 568738-569395 2.2 A1S_0669 793092-793614 3.5
1S_0535 574378-575785 26.4 8.0 A1S_0670 793983-794532 2.0
1S_0536 575795-577790 32.1 7.0 A1S_0671 793983-794532 4.6
1S_0537 577792-578041 37.7 5.7 A1S_0673 796968-797619 2.3
1S_0538 578043-578910 40.5 6.5 A1S_0677 800463-800631 2.2
1S_0547 590795-591257 2.7 A1S_0680 810374-810450 2.1
1S_0559 607842-608604 2.0 A1S_0683 814282-814621 2.2
1S_0561 609793-610303 2.2 A1S_0714 845402-845480 4.6
1S_0562 610637-611642 2.4 A1S_0717 845725-845803 2.8
1S_0563 611790-612975 2.6 A1S_0718 845957-846953 2.3
1S_0564 612993-613401 5.1 2.0 2.0 A1S_0719 847354-848530 2.6
1S_0566 614632-615760 2.8 A1S_0736 868665-869652 2.31S_0567 615771-616086 2.6 A1S_0738 870902-871394 2.1
1S_0568 616098-617553 2.3 A1S_0739 872487-874331 2.7
1S_0570 618677-619079 2.9 A1S_0749 889025-890618 2.3
1S_0624 677112-677907 2.3 A1S_0770 911539-911962 3.0
1S_0630 703842-704970 2.3 A1S_0772 914694-915894 2.2
1S_0631 704972-706106 2.6 A1S_0779 923963-924464 2.0 2.1
1S_0633 715174-717829 2.0 A1S_0780 924609-925839 2.5
1S_0634 718 14-720347 2.0 A1S_0781 925984-926857 2.9
1S_0640 731422-732367 2.7 A1S_0800 950957-951422 2.4
1S_0641 732447-733818 2.9 A1S_0804 952978-955248 2.9
1S_0642 734599-735658 4.0 A1S_0830 975666-975741 3.7
1S_0643 735942-737232 3.7 A1S_0831 975833-975908 2.7
1S_0644 737244-738063 6.2 A1S_0832 975929-976004 4.3
1S_0645 744710-745841 5.0 A1S_0834 976033-976937 5.1
1S_0646 751374-754647 4.1 A1S_0835 976955-977534 16.9 2.7
1S_0647 755128-757866 3.5 A1S_0861 1002253-1003114 2.1
1S_0650 765517-767063 2.8 A1S_0884 1025530-1026184 3.8 2.7
1S_0663 786446-788142 3.4 A1S_0889 1031554-1032337 5.4
1S_0664 786446-788142 4.5 A1S_0929 1078189-1079812 2.6
1S_0665 789503-790283 4.9 A1S_0930 1080151-1081495 2.9
1S_0666 790521-791967 5.3 A1S_0931 1081557-1081995 2.8
1S_0932 1082006-1082621 2.8 A1S_1158 1353562-1353946 5.2
1S_0935 1085243-1086506 2.3 A1S_1159 1353955-1354339 3.1 Table 1. Exemplary transcripts that were increased in expression after outer membrane stress
Gene ID Locus (relative to BamA 10 min. 30 min. Gene ID Locus (relative to BamA 10 min. 30 min.
ATCC 17978 depletion PMBN PMBN ATCC 17978 depletion PMBN PMBN genome) (fold (fold (fold genome) (fold (fold (fold change) change) change) change) change) change)1S_0945 1094826-1095132 2.1 A1S_1160 1354547-1355466 3.0
1S_0959 1107083-1109640 8.2 A1S_1161 1354547-1355466 4.0
1S_0980 1136705-1138292 2.3 A1S_1162 1355528-1356296 4.0
1S_1003 1162333-1162816 2.1 A1S_1163 1356643-1356880 2.8
1S_1009 1169599-1169956 2.3 A1S_1164 1357545-1360585 2.5
1S_1010 1170159-1170483 2.0 3.0 A1S_1165 1357545-1360585 3.4
1S_1027 1185805-1186915 3.0 2.5 A1S_1167 1360595-1362103 2.5
1S_1028 1187084-1187861 3.1 A1S_1171 1368144-1368687 4.9
1S_1030 1188754-1190305 4.4 A1S_1172 1369011-1369725 2.9
1S_1031 1190429-1191086 4.4 A1S_1173 1370416-1371715 2.1
1S_1049 1210798-1211320 2.5 A1S_1180 1383016-1383802 2.7 2.2
1S_1081 1262184-1262736 2.4 A1S_1184 1386736-1387965 2.1
1S_1106 1293743-1294280 2.1 A1S_1186 1388265-1390845 2.4
1S_1107 1294336-1295587 2.2 A1S_1198 1402980-1403877 2.4
1S_1120 1310973-1311294 2.2 A1S_1202 1407106-1407325 2.0
1S_1121 1311317-1311866 2.0 A1S_1203 1407361-1407949 2.5
1S_1123 1312837-1314325 2.7 2.5 2.8 A1S_1223 1432694-1432772 7.5
1S_1132 1323351-1324182 2.5 A1S_1224 M32944- 143 178 13.1
1S_1133 1324205-1324697 2.5 A1S_1225 1433329-1434001 2.1
1S_1134 1324804-1325755 2.3 A1S_1230 1440550-1441381 2.1
1S_1139 1329944-1330334 2.2 A1S_1236 1448204-1449182 3.8
1S_1143 1336525-1336966 4.4 A1S_1237 1449185-1449725 2.5
1S_1145 1337987-1338236 5.4 A1S_1248 1459875-1460547 6.1 3.8
1S_1146 1338841-1343140 3.9 A1S_1255 1468345-1469278 3.8
1S_1148 1338841-1343140 4.2 A1S_1274 1495013-1496177 4.7
1S_1149 1344550-1345589 2.6 A1S_1286 1508990-1510297 2.4
1S_1150 1344550-1345589 3.1 A1S_1359 1592070-1593185 2.2
1S_1151 1345647- 1347628 2.8 3.3 2.3 A1S_1360 1593207-1593588 3.2
1S_1152 1345647-1347628 2.9 A1S_1361 1593882-1596113 2.7
1S_1153 1347636-1350251 2.4 A1S_1362 1593882-1596113 2.1
1S_1155 1350881-1352084 3.5 A1S_1363 1596214-1597369 2.8
1S_1156 1352097-1352568 3.1 A1S_1383 1622739-1623057 31.4 2.4
1S_1157 1352573-1353548 2.7 A1S_1384 1623757-1624147 7.8
1S_1385 1624462-1625872 4.0 A1S_1655 1922911-1925230 2.01S_1386 1625932-1628071 2.6 A1S_1658 1928553-1930701 4.5 Table 1. Exemplary transcripts that were increased in expression after outer membrane stress
Gene ID Locus (relative to BamA 10 min. 30 min. Gene ID Locus (relative to BamA 10 min. 30 min.
ATCC 17978 depletion PMBN PMBN ATCC 17978 depletion PMBN PMBN genome) (fold (fold (fold genome) (fold (fold (fold change) change) change) change) change) change)1S_1387 1628126-1629005 6.6 A1S_1662 1933711-1935280 2.4
1S_1393 1635045-1639466 2.1 A1S_1666 1938432-1940288 2.5
1S_1404 1647134-1647284 2.6 A1S_1667 1940642-1942808 2.8
1S_1407 1649695-1650619 2.0 A1S_1669 1942999-1944819 2.5
1S_1422 1662988-1665552 2.1 A1S_1677 1952187-1953294 2.3
1S_1454 1695534-1696569 2.2 A1S_1680 1955070-1955271 4.2
1S_1472 1716132-1716765 2.1 A1S_1681 1956719-1957487 2.2
1S_1481 1726152-1727244 2.0 A1S_1687 1964330-1964495 10.0 2.3
1S_1494 1739600-1740917 2.9 A1SJ735 2018428-2018829 2.5
1S_1512 1760904-1761993 2.3 A1S_1741 2025836-2027087 2.5
1S_1515 1764261-1764855 2.5 A1S_1743 2027928-2029167 2.6
1S_1526 1774680-1774 11 2.4 A1S_1744 2029195-2029726 2.3
1S_1535 1785566-1787854 2.0 A1S_1750 2036446-2039985 7.8
1S_1539 1791079-1791388 4.6 A1S_1751 2036446-2039985 15.9 3.01S_1566 1816128-1816725 5.9 A1S_1752 2040250-2040754 17.8 2.81S_1567 1816775-1818610 2.1 A1S_1760 2049637-2050384 2.2
1S_1569 1818643-1819120 2.6 A1S_1762 2052476-2053559 2.3
1S_1583 ^34442- 1836954 2.7 A1S_1767 2058752-2059721 2.3
1S_1584 1834442-1836954 2.7 A1S_1778 2074016-2074271 2.0
1S_1585 1834442-1836954 2.1 A1S_1813 2113585-2114164 2.2
1S_1589 1844073-1848256 2.2 A1S_1827 2129135-2129828 2.4
1S_1590 1844073-1848256 3.5 2.1 A1S_1829 2130475-2131282 2.2
1S_1593 1849125-1849985 2.0 A1S_1831 2132134-2133202 2.7
1S_1595 1850054-1850950 2.3 A1S_1843 2145312-2146254 2.5
1S_1596 18 1681-1852779 2.2 A1S_1876 2177931-2178798 2.0
1S_1617 1885791-1886058 2.3 A1S_1909 2211081-2211405 2.0
1S_1630 1898439-1898760 2.1 A1S_1928 2235930-2236602 4.0 2.3
1S_1644 1910053-1911427 2.6 A1S_1929 2236827-2237424 2.4
1S_1645 1911663-1911942 8.2 A1S_1934 2240345-2240771 2.3
1S_1647 1912994-1916205 2.9 A1S_1952 2262196-2262826 3.9 2.3
1S_1648 1912994-1916205 2.7 A1S_1955 2266982-2267729 3.2
1S_1649 1916225-1917665 2.8 A1S_1956 2267736-2269143 4.6
1S_1651 1918559-1919519 2.0 A1S_1957 2269183-2270434 10.4
1S_1959 2271076-2271613 2.1 A1S_2178 2539164-2539944 2.2
1S_1960 2272963-2273626 2.2 A1S_2179 2539946-2540543 2.1 Table 1. Exemplary transcripts that were increased in expression after outer membrane stress
Gene ID Locus (relative to BamA 10 min. 30 min. Gene ID Locus (relative to BamA 10 min. 30 min.
ATCC 17978 depletion PMBN PMBN ATCC 17978 depletion PMBN PMBN genome) (fold (fold (fold genome) (fold (fold (fold change) change) change) change) change) change)1S_1961 2273649-2274051 3.5 A1S_2183 2545849-2546080 2.0
1S_1962 2274196-2275246 4.9 A1S_2186 2548374-2548644 2.5 2.3
1S_1963 2275299-2275782 3.4 A1S_2195 2555872-2556076 3.8
1S_1979 2292696-2293350 2.5 A1S_2230 2594847-2595081 14.8 5.1
1S_1986 2299906-2301301 2.8 2.0 A1S_2247 2607936-2609373 3.9
1S_1987 2301415-2302423 4.3 A1S_2252 2614774-2615362 2.2
1S_1988 2302488-2302857 3.3 A1S_2257 2618615-2619689 2.0
1S_2006 2321933-2322524 2.1 A1S_2258 2619724-2620300 2.4
1S_2026 2353926-2354154 2.1 A1S_2259 2620503-2620884 15.5 5.8
1S_2033 2360772-2361811 2.1 A1S_2262 2623140-2624007 4.7
1S_2034 2364152-2364953 2.6 A1S_2271 2630762-2632031 37.4
1S_2035 2366468-2366942 2.9 A1S_2272 2632429-2632888 10.4
1S_2036 2367266-2370789 2.9 A1S_2273 2633147-2634158 6.8
1S_2038 2374390-2374831 2.2 A1S_2283 2645388-2646566 2.1
1S_2039 2375167-2377494 2.2 A1S_2285 2649168-2649720 2.3 2.1
1S_2061 2402774-2404166 3.1 A1S_2298 2663063-2663393 2.3
1S_2074 2419235-2419661 3.7 A1S_2311 2678573-2681088 2.0
1S_2079 2427161-2428397 2.6 A1S_2315 2681890-2683033 2.8
1S_2082 2431180-2431735 2.3 A1S_2325 2691620-2692739 10.5 2.2
1S_2092 2442483-2445090 2.2 A1S_2326 2693151-2694498 2.6
1S_2093 2445408-2445666 45.8 A1S_2330 2699287-2699770 2.9
1S_2106 2458921-2459368 2.4 A1S_2331 2700057-2701500 2.8
1S_2139 2498044-2498650 2.2 A1S_2366 2740080-2740635 2.1
1S_2140 2498663-2500697 3.1 A1S_2367 2740796-2741948 2.1
1S_2141 2500711-2501296 3.3 A1S_2382 2758469-2759441 2.2
1S_2142 2501348-2502345 3.7 A1S_2387 2766152-2768808 2.2
1S_2146 2504825-2505602 2.0 A1S_2389 2766152-2768808 2.0
1S_2157 2515546-2515969 41.5 8.6 A1S_2395 2776723-2777410 2.8
1S_2158 2516840-2517845 6.2 2.0 A1S_2396 2777845-2778409 2.1
1S_2160 2518595-2524571 2.5 2.4 A1S_2414 2798185-2800132 2.2
1S_2161 2518595-2524571 2.7 2.4 A1S_2434 2819120-2819543 4.3 2.8
1S_2162 251 595-2524571 2.7 2.4 A1S_2445 2829746-2830649 2.4
1S_2446 2830662-2833436 3.5 A1S_2654 3076343-3077369 58.7 3.2
1S_2447 2830662-2833436 3.6 A1S_2656 3078050-3078956 2.4
1S_2448 2833536-2834568 5.0 A1S_2660 3082184-3085280 11.5 4.0 Table 1. Exemplary transcripts that were increased in expression after outer membrane stress
Gene ID Locus (relative to BamA 10 min. 30 min. Gene ID Locus (relative to BamA 10 min. 30 min.
ATCC 17978 depletion PMBN PMBN ATCC 17978 depletion PMBN PMBN genome) (fold (fold (fold genome) (fold (fold (fold change) change) change) change) change) change)1S_2454 2842197-2843571 2.0 A1S_2664 3088347-3089982 2.2
1S_2455 2844038-2844740 14.0 4.8 A1S_2668 3092585-3094421 2.2
1S_2456 2844858-2845755 5.2 A1S_2675 3101817-3101892 2.6
1S_2458 2846516-2847611 3.0 A1S_2676 3101902-3101989 3.1
1S_2459 2847623-2848694 2.3 A1S_2677 3102088-3103852 2.6
1S_2463 2853750-2854389 3.4 A1S_2678 3104048-3104135 2.3
1S_2480 2877825-2879112 2.5 A1S_2684 3109204-3109741 2.6
1S_2489 2887077-2887878 2.1 A1S_2705 3136166-3136454 2.1
1S_2503 2901447-2902035 2.0 A1S_2729 3166014-3166710 42.9 5.9
1S_2504 2902128-2904150 2.4 A1S_2734 3171216-3171843 3.8
1S_2508 2907148-2908378 2.4 A1S_2756 3197704-3198622 2.2
1S_2542 2939051-2939723 2.1 A1S_2768 3207903-3208722 2.0
1S_2543 2939736-2940153 3.0 2.6 A1S_2786 3227314-3227833 2.4
1S_2552 2944671-2949201 2.0 A1S_2798 3243913-3246460 2.7
1S_2553 2949216-2950113 2.5 A1S_2801 3247722-3247799 2.0
1S_2555 2951085-2953363 2.5 A1S_2807 3249095-3249464 2.1
1S_2557 2953599-2953995 3.4 A1S_2826 3271589-3272414 2.1
1S_2558 2954038-2954203 2.2 2.3 A1S_2827 3272447-3273371 2.4
1S_2573 2972592-2974211 2.7 A1S_2828 3273436-3274546 2.3
1S_2577 2978719-2980603 2.5 A1S_2839 3286067-3287273 8.5
1S_2578 2980687-2983192 2.1 A1S_2863 3312957-3313599 3.8 2.4
1S_2580 2984033-2984663 2.1 A1S_2882 3330831-3332274 3.0
1S_2586 2994946-2996287 2.2 A1S_2883 3332337-3333024 6.1 2.3
1S_2588 2996986-2997991 2.1 A1S_2884 3333043-3334507 11.9 2.7
1S_2593 3001325-3002672 2.4 A1S_2885 3334834-3335215 299.1 49.7 2.51S_2611 3022291-3023333 3.8 2.6 A1S_2889 3339895-3340279 999.4 29.8
1S_2612 3022291-3023333 4.2 2.8 A1S_2892 3342623-3344699 3.2
1S_2613 3023352-3024117 4.6 2.6 A1S_2893 3344834-3345599 10.6
1S_2624 3034971-3035631 2.2 A1S_2942 3414466-3415516 2.6
1S_2650 3069419-3069926 2.3 A1S_2943 3420548-3421754 2.2
1S_2651 3069995-3073994 2.4 A1S_2953 3439474-3439550 2.1
1S_2959 3446804-3447359 2.3
1S_2960 3447512-3449453 2.3 A1S_3295 3784099-3786934 2.6
1S_2968 3459568-3460165 2.7 2.3 A1S_3317 3813815-3814583 3.6 3.0
1S_2976 3467119-3467818 3.1 2.0 A1S_3326 3823780-3824356 3.4 Table 1. Exemplary transcripts that were increased in expression after outer membrane stress
Gene ID Locus (relative to BamA 10 min. 30 min. Gene ID Locus (relative to BamA 10 min. 30 min.
ATCC 17978 depletion PMBN PMBN ATCC 17978 depletion PMBN PMBN genome) (fold (fold (fold genome) (fold (fold (fold change) change) change) change) change) change)1S_2992 3503856-3503932 2.0 A1S_3339 3841191-3843351 2.9
1S_3011 3497325-3497925 2.1 A1S_3360 3862246-3862828 2.4
1S_3026 3511909-3512572 2.5 A1S_3361 3863039-3864383 2.2
1S_3027 3512629-3 14573 3.6 2.1 A1S_3367 3876268-3876808 2.1
1S_3034 3519957-3520671 14.5 2.7 A1S_3368 3876821-3877431 2.3
1S_3035 3520743-3521319 5.0 A1S_3371 3880043-3881411 4.4
1S_3047 3535309-3536581 2.9 A1S_3375 3883669-3884134 3.1
1S_3048 3536621-3536831 2.3 A1S_3376 3884143-3885502 2.0
1S_3099 3578914-3579202 10.4 2.6 A1S_3392 3899393-3900804 2.1
1S_3100 3579278-3579854 11.8 2.7 A1S_3411 3921303-3922569 6.1
1S_3101 3579880-3582164 5.8 2.0 A1S_3412 3922711-3923395 3.4 3.0
1S_3104 3582555-3584403 2.7 2.7 A1S_3463 pABl:3537-4788 3.6
1S_3105 3584812-3585643 2.0 A1S_3466 pAB 1:7073-7685 2.4
1S_3114 3596342-3597443 2.2 A1S_3468 pABl:10071- 2.4
10723
1S_3115 3597575-3598373 4.5 A1S_3469 pABl:10071- 2.8
10723
1S_3116 3598375-3599638 3.4 A1S_3471 pAB 1:0-1504 2.1
1S_3117 3599811-3600996 2.2 A1S_3479 49195-49333 4.6
1S_3124 3609067-3609487 4.2 2.4 A1S_3480 51946-52956 64.8 5.6
1S_3125 3609581-3610013 2.2 A1S_3486 97895-98132 3.0
1S_3126 3610109-3611465 3.0 A1S_3492 314151-314517 38.9 17.1
1S_3127 3612402-3613164 19.2 15.3 A1S_3493 314779-315088 12.4 3.0
1S_3139 3626404-3626968 17.4 3.5 A1S_3494 340739-340835 2.3
1S_3146 3632692-3633922 3.2 A1S_3499 553641-553752 3.2
1S_3147 3634291-3634657 2.6 A1S_3510 682605-683410 2.2
1S_3175 3661564-3663606 2.1 A1S_3512 685572-685686 2.61S_3206 3692145-3693401 2.0 A1S_3518 691526-692903 2.01S_3224 3716297-3717188 2.4 A1S_3522 702026-703842 2.61S_3253 3741782-3742175 10.6 4.8 A1S_3523 702026-703842 2.41S_3259 3748754-3749090 2.1 A1S_3533 730543-731401 2.31S_3280 3769315-3772053 5.3 A1S_3534 733836-734586 3.21S_3281 3769 15-3772053 2.0 A1S_3535 738104-738575 5.01S_3539 743976-744693 5.2 A1S_3611 1338841-1343140 4.4
1S_3540 745850-747482 5.4 A1S_3612 1338841-1343140 3.2
1S_3541 747506-748361 5.2 A1S_3613 1338841-1343140 7.4
1S_3542 748399-748978 6.1 A1S_3614 1343150-1343885 3.6 Table 1. Exemplary transcripts that were increased in expression after outer membrane stress
Gene ID Locus (relative to BamA 10 min. 30 min. Gene ID Locus (relative to BamA 10 min. 30 min.
ATCC 17978 depletion PMBN PMBN ATCC 17978 depletion PMBN PMBN genome) (fold (fold (fold genome) (fold (fold (fold change) change) change) change) change) change)1S_3543 749082-749502 5.1 A1S_3617 1354348-1354540 2.5
1S_3544 749580-751358 3.1 A1S_3618 1356302-1356617 3.2
1S_3545 749580-751358 2.3 A1S_3621 1367712-1368102 2.0
1S_3546 754708-75 119 3.3 A1S_3630 1434564-1434954 2.2
1S_3548 755128-757866 3.2 A1S_3632 1439238-1439697 3.5
1S_3552 765517-767063 4.4 A1S_3634 1443391-1443721 2.8 2.0
1S_3553 767079-767373 2.8 A1S_3635 1445438-1446069 5.8 3.9
1S_3558 784604-784781 4.5 3.2 A1S_3636 1445438-1446069 4.5 2.6
1S_3559 788860-788968 2.6 A1S_3637 1445438-1446069 4.3 2.6
1S_3562 882514-882616 2.1 A1S_3642 M81741-1482059 2.5
1S_3563 884662-884797 2.5 A1S_3645 1505531-1505657 3.1 2.8 2.41S_3567 920512-921217 3.0 A1S_3649 1549014-1549113 6.4
1S_3570 950745-950847 2.6 A1S_3654 1582221-1582848 3.5
1S_3577 1060492-1061839 2.5 A1S_3658 1621505-1621649 3.7
1S_3580 1083508-1083709 3.6 A1S_3661 1623162-1623384 33.5 2.4 2,11S_3580 1083508-1083709 3.5 A1S_3662 1630044-1630749 8.9 2.4
1S_3585 1131946-1132111 6.4 A1S_3666 1655879-1656311 2.3
1S_3586 1134942-1135071 2.8 A1S_3682 1796386-1796629 3.5
1S_3594 1234458-1234767 2.3 A1S_3686 1831813-1832446 2.9
1S_3595 1237338-1237950 2.4 A1S_3687 1832713-1833151 3.9
1S_3596 1238719-1238836 2.1 A1S_3688 1837225-1837828 2.6
1S_3601 1316391-1317976 2.6 A1S_3694 1841125-1842084 2.6
1S_3602 1321090-1321904 3.4 A1S_3695 1841125-1842084 3.3 3.9
1S_3603 1333785-1334193 3.0 A1S_3697 1842702-1843185 2.4
1S_3604 1334193-1334445 3.0 A1S_3704 1861348-1863311 2.5
1S_3605 1334446-1335406 4.1 A1S_3707 1868781-1869498 3.5
1S_3606 1335420-1336188 3.7 A1S_3708 1868781-1869498 2.1
1S_3607 1338290-1338791 4.9 A1S_3712 1938432-1940288 2.2
1S_3608 1338841-1343140 3.7 A1S_3716 1963924-1964260 4.5
1S_3609 1338841-1343140 4.3 A1S_3725 2050390-2051065 2.0
1S_3610 1338841-1343140 3.3 A1S_3726 2051919-2052435 2.6
1S_3727 2053576-2054197 2.7 A1S_3862 3433087-3434281 6.3 2.81S_3728 2054981-2055080 3.4 A1S_3864 3443652-3443979 38.2 10.5
1S_3736 2114184-2114301 2.3 A1S_3865 3444152-3444383 7.4 4.1
1S_3738 2135777-2136674 2.1 A1S_3866 3449783-3450701 2.1 Table 1. Exemplary transcripts that were increased in expression after outer membrane stress
Gene ID Locus (relative to BamA 10 min. 30 min. Gene ID Locus (relative to BamA 10 min. 30 min.
ATCC 17978 depletion PMBN PMBN ATCC 17978 depletion PMBN PMBN genome) (fold (fold (fold genome) (fold (fold (fold change) change) change) change) change) change)1S_3739 2151550-2154013 2.6 A1S_3867 3451000-3451372 4.8 2.4
1S_3740 2156967-2159355 2.1 A1S_3868 3451602-3451791 2.9 2.1
1S_3750 2299476-2299773 7.2 2.4 A1S_3873 3467913-3468900 5.0 2.3
1S_3752 2321244-2321352 6.1 A1S_3875 3521371-3522029 2.2
1S_3760 2346538-2347054 2.0 A1S_3879 3578725-3578845 2.3
1S_3768 2365213-2365651 3.6 A1S_3889 3731833-3732464 2.6
1S_3769 2366057-2366468 3.5 A1S_3900 3866477-3867203 3.9
1S_3770 2367266-2370789 2.5 A1S_3901 3867318-3868125 4.1
1S_3771 2367266-2370789 3.0 A1S_3902 3868129-3869872 2.3
1S_3772 2367266-2370789 2.5 A1S_3908 3905565-3906780 2.4 2.41S_3773 2371364-2371541 2.7 A1S_3911 3941919-3942756 2.01S_3776 2375167-2377494 2.1
1S_3777 2377495-2377747 4.1
1S_3778 2377747-2378155 2.3
1S_3782 2416009-2416183 2.7 2.2
1S_3783 2416354-2416588 6.7
1S_3786 2472597-2472816 2.6
1S_3789 2501348-2502345 6.0 4.2
1S_3790 2508121-2509395 2.3
1S_3791 2516202-2516706 182.2 21.3
1S_3792 2518595-2524571 2.5 2.8
1S_3797 2590929-2591067 2.1
1S_3810 2854521-2855226 2.4
1S_3818 2986306-2987233 8.0
1S_3820 2988952-2989528 3.4
1S_3835 3185608-3186561 2.1
1S_3837 3248326-3248425 2.7
1S_3840 3321647-3322643 2.5
1S_3842 3361647-3361863 2.1
1S_3844 3367024-3367801 2.6 Table 2. Exemplary transcripts that were decreased in expression after outer membrane stress
Gene ID Locus (relative to BamA 10 min. 30 min. Gene ID Locus (relative to BamA 10 min. 30 min.
ATCC 17978 depletion PMBN PMBN ATCC 17978 depletion PMBN PMBN genome) (fold (fold (fold genome) (fold (fold (fold change) change) change) change) change) change)1S_0009 11127-12135 -15.9 A1S_0150 172414-172660 -4.9
1S_0010 12477-13482 -2.4 A1S_0151 172698-173169 -4.7
1S_0025 32621-33242 -3.6 A1S_0152 173181-173718 -4.5
1S_0027 34316-36286 -2.1 A1SJU53 173764-175309 -4.3
1S_0038 44413-45061 -2.3 A1S_0154 175386-176256 -4.3
1S_0067 79908-81570 -2.3 A1SJU55 176286-177681 -4.0
1S_0070 83781-85488 -2.2 A1S_0156 177698-178118 -4.0
1S_0071 85536-86751 -4.9 A1S_0157 178266-178749 -16.5
1S_0073 87266-88854 -2.5 A1S_0177 199553-200552 -3.1
1S_0076 90282-92889 -2.6 -2.0 A1SJU84 207389-207890 -2.1
1S_0077 92998-95467 -4.4 -2.1 A1S_0200 222773-223301 -2.2
1S_0079 97026-97536 -2.9 A1S_0201 223407-224682 -16.5
1S_0087 106538-107240 -10.1 A1S_0209 232389-233127 -2.1
1S_0090 109630-110005 -3.0 A1S_0218 244875-245214 -2.8
1S_0091 110033-110414 -2.7 A1S_0238 263528-264668 -2.1
1S_0095 114423-115689 -6.1 A1S_0239 264723-266025 -2.5
1S_0096 115709-116813 -6.6 -2.0 A1S_0253 280037-281517 -2.3
1S_0097 116823-117183 -13.2 -2.2 A1S_0257 284090-284360 -2.0
1S_0098 117344-118784 -12.4 -2.6 A1S_0258 284369-284483 -2.1
1S_0099 119154-120585 -2.6 A1S_0269 292871-294451 -2.3
1S_0103 123217-124108 -45.5 A1S_0270 292871-294451 -2.5
1S_0104 124192-125839 -24.7 A1S_0279 300452-301643 -2.1
1S_0105 125850-126978 -23.5 A1S_0286 304940-305312 -2.2
1SJM06 126999-127773 -29.9 A1S_0292 318463-319045 -12.9
1S_0107 127785-128811 -17.4 A1S_0302 327020-327737 -6.3
1S_0108 128930-130283 -6.7 A1S_0303 328012-328504 -2.5
1S_0109 130375-130927 -2.3 A1SJB04 328557-331536 -3.3
1S_0121 149280-149356 -3.1 A1S_0321 348233-349901 -2.4
1SJM28 153263-153701 -2.1 A1S_0322 350015-351326 -2.4
1S_0129 153759-154989 -5.3 A1S_0323 351402-351729 -3.2
1S_0141 166036-166957 -2.1 A1S_0347 375475-376789 -3.3
1S_0148 171454-172389 -4.0 A1S_0365 400384-400996 -2.1
1S_0369 406469-407675 -2.2 A1S_0633 715174-717829 -4.4 Table 2. Exemplary transcripts that were decreased in expression after outer membrane stress
Gene ID Locus (relative to BamA 10 min. 30 min. Gene ID Locus (relative to BamA 10 min. 30 min.
ATCC 17978 depletion PMBN PMBN ATCC 17978 depletion PMBN PMBN genome) (fold (fold (fold genome) (fold (fold (fold change) change) change) change) change) change)1S_0370 407698-408280 -3.3 A1S_0634 718514-720347 -6.5
1S_0388 421321-421732 -2.8 A1S_0635 721727-723065 -2.1
1S_0408 441820-442426 -3.6 A1S_0639 727284-728169 -2.5
1S_0410 443426-444326 -2.4 A1S_0640 731422-732367 -12.7
1S_0427 465164-466283 -2.4 A1S_0641 732447-733818 -8.1
1S_0429 468283-469570 -3.9 A1S_0642 734599-735658 -14.7
1S_0447 486868-487024 -2.1 A1S_0643 735942-737232 -11.6
1S_0448 487036-487273 -2.2 A1S_0644 737244-738063 -26.5
1S_0474 512466-514677 -2.2 A1S_0645 744710-745841 -43.4
1S_0480 520303-521830 -2.3 A1S_0646 751374-754647 -14.7
1S_0481 522058-524203 -3.0 A1S_0647 755128-757866 -10.0
1S_0482 524246-525449 -3.5 A1S_0649 762203-764054 -2.7
1S_0486 530275-530788 -2.3 A1S_0650 765517-767063 -11.2
1S_0490 534256-535570 -4.7 A1S_0651 770542-772804 -2.3
1S_0491 535623-536466 -2.1 A1S_0690 821217-822057 -4.9
1S_0498 542610-543042 -2.3 A1S_0691 822353-823187 -4.2
1S_0526 568280-568715 -2.5 A1S_0692 823655-824423 -2.0
1S_0533 572636-573368 -2.2 A1S_0695 827339-829271 -2.2
1S_0534 573441-574308 -2.2 A1S_0698 830635-831166 -2.2
1S_0548 591473-592046 -16.4 -2.1 A1S_0721 849825-851037 -2.8
1S_0549 592376-593240 -7.0 A1S_0731 862786-864154 -5.2 -2.1
1S_0566 614632-615760 -18.4 -2.2 A1S_0732 864212-865220 -2.8
1S_0567 615771-616086 -15.8 A1S_0771 912706-914383 -3.8
1S_0568 616098-617553 -13.5 A1S_0785 929384-930725 -2.4
1S_0591 637832-639461 -3.5 A1S_0786 930943-932974 -2.3
1S_0594 642557-643178 -2.7 A1S_0787 932989-933715 -2.6
1S_0624 677112-677907 -12.1 A1S_0788 933747-936159 -2.2
1S_0625 678350-678923 -3.7 A1S_0818 965341-967059 -2.1
1S_0626 679421-680321 -2.7 A1S_0822 968954-969629 -2.3
1S_0627 683474-684857 -4.3 A1S_0846 988299-988377 -2.1
1S_0629 692917-698877 -3.3 A1S_0849 990843-991956 -2.2
1S_0630 703842-704970 -10.1 A1S_0850 991982-993629 -2.9
1S_0631 704972-706106 -11.1 A1S_0851 993640-994615 -2.7
1S_0632 706229-709964 -4.1 A1S_0852 994657-995773 -5.5
1S_0853 995972-997291 -4.6 A1S_1093 1274183-1275793 -14.2 Table 2. Exemplary transcripts that were decreased in expression after outer membrane stress
Gene ID Locus (relative to BamA 10 min. 30 min. Gene ID Locus (relative to BamA 10 min. 30 min.
ATCC 17978 depletion PMBN PMBN ATCC 17978 depletion PMBN PMBN genome) (fold (fold (fold genome) (fold (fold (fold change) change) change) change) change) change)1S_0854 995972-997291 -4.2 A1S_1094 1276192-1277656 -7.1
1S_0855 997338-998295 -4.4 A1S_1109 1296884-1298762 -2.4
1S_0869 1010222-1011371 -2.1 A1S_1113 1302730-1303189 -2.1
1S_0877 1020261-1020882 -2.4 A1S_1139 1329944-1330334 -2.2
1S_0882 1023296-1024343 -2.2 A1S_1142 1331536-1332817 -2.0
1S_0883 1024359-1025286 -2.6 A1S_1182 1385133-1385706 -2.1
1S_0890 1032445-1033294 -2.3 A1S_1193 1397486-1398902 -5.0
1S_0891 1033546-1034002 -5.1 A1S_1195 1399921-1400404 -2.5
1S_0901 1044841-1045333 -2.2 A1S_1199 1404084-1404696 -2.9
1S_0910 1053666-1055631 -6.5 A1S_1227 1435603-1436221 -3.9
1S_0911 1056401-1057004 -4.9 A1S_1257 1472276-1473581 -2.4
1S_0912 1057016-1057 17 -5.7 A1S_1258 1473675-1474113 -8.5
1S_0913 1057525-1058632 -6.4 A1S_1261 1476636-1477587 -6.4
1S_0960 1107083-1109640 -4.2 A1S_1264 1480139-1481387 -3.8
1S_0965 1115597-1116743 -2.5 A1S_1265 1482342-1483254 -2.2
1S_0973 1128483-1129818 -2.1 A1S_1266 1483620-1484853 -89.2 -2.5
1S_0984 1141283-1141898 -3.2 A1S_1267 1484887-1485652 -38.1 -2.3
1S_0996 1154160-1154598 -2.8 A1S_1268 1485663-1486470 -46.3 -2.6
1S_0997 1154667-1155156 -2.1 A1S_1269 1486502-1489804 -17.1 -2.2
1S_0999 1157132-1158575 -2.8 A1S_1270 1486502-1489804 -22.2
1S_1000 1158957-1159866 -2.2 A1S_1281 1502656-1505369 -5.7
1S_1004 1162891-1164229 -6.2 A1S_1317 1544198-1545356 -2.3
1S_1008 1167810-1169415 -2.2 A1S_1318 1545435-1545894 -3.2
1S_1021 1179719-1180238 -2.0 -2.9 A1S_1319 1546206-1546776 -2.0
1S_1026 1184406-1185750 -2.2 A1S_1327 1554878-1555109 -2.4
1S_1044 1207071-1207747 A1S_1334 1562431-1563811 -2.3
1S_1063 1227345-1229460 -2.2 A1S_1335 1563854-1565957 -162.7
1S_1072 1239532-1240884 -2.7 A1S_1336 1566222-1567161 -318.7
1S_1079 1257771-1259520 -2.1 A1S_1337 1567193-1567484 -222.6 -2.1
1S_1080 1259981-1260860 -7.0 A1S_1338 1567496-1568252 -176.0
1S_1088 1270360-1271131 -3.0 A1S_1339 1568268-1568769 -116.6 -2.1
1S_1089 1271159-1272590 -4.1 A1S_1340 1568792-1569854 -140.2
1S_1091 1273507-1273981 -5.9 A1S_1341 1569862-1571427 -131.4 -2.2
1S_1092 1274183-1275793 -10.4 A1S_1342 1569862-1571427 -155.8
1S_1343 1571429-1572968 -98.1 A1S_1492 1738115-1738793 -13.3 -3.0 Table 2. Exemplary transcripts that were decreased in expression after outer membrane stress
Gene ID Locus (relative to BamA 10 min. 30 min. Gene ID Locus (relative to BamA 10 min. 30 min.
ATCC 17978 depletion PMBN PMBN ATCC 17978 depletion PMBN PMBN genome) (fold (fold (fold genome) (fold (fold (fold change) change) change) change) change) change)1S_1344 1572980-1574189 -40.0 A1S_1493 1738812-1739550 -7.0 -2.5
1S_1345 1574370-1574898 -65.0 A1S_1498 1743404-1743965 -5.1
1S_1346 1575142-1575520 -25.1 A1S_1499 1744240-1745154 -4.0
1S_1347 1575579-1577131 -6.8 A1S_1505 1751075-1752005 -3.8 -3.8 -3.31S_1348 1575579-1577131 -7.5 -2.1 A1S_1510 1758162-1758531 -5.6
1S_1349 1577206-1577590 -11.1 -2.7 A1S_1523 1772486-1773077 -2.1
1S_1356 1587432-1588647 -2.0 A1S_1528 1776927-1780680 -2.2
1S_1366 1599663-1600290 -2.5 A1S_1530 1781373-1782816 -7.0
1S_1367 1600847-1601648 -4.0 A1S_1532 1784019-1784394 -3.0
1S_1368 1601716-1602178 -13.8 -2.4 A1S_1543 1795074-1796262 -2.1
1S_1369 1602210-1605107 -12.0 -2.7 A1S_1579 1827636-1828689 -2.1
1S_1370 1602210-1605107 -9.5 -2.3 A1S_1583 1834442-1836954 -2.1
1S_1372 1606595-1609486 -7.2 -3.9 A1S_1601 1864204-1866370 -3.5
1S_1373 1606595-1609486 -9.6 -4.0 A1S_1608 1873080-1874871 -2.8
1S_1374 1609497-1610301 -9.8 -4.5 A1S_1609 1874889-1876710 -3.3
1S_1375 1610310-1611912 -12.3 -3.9 A1S_1610 1876710-1878744 -3.9
1S_1376 1611936-1613109 -8.9 -3.3 A1S_1611 1878756-1879695 -3.0
1S_1377 1613253-1613871 -2.9 A1S_1612 1879702-1880845 -3.0
1S_1378 1613981-1615676 -7.0 -4.0 A1S_1613 1880857-1882588 -2.6
1S_1379 1615746-1616418 -8.0 -4.6 A1S_1637 1902528-1902801 -2.7
1S_1380 1616658-1617945 -10.9 -2.5 A1S_1638 1903200-1904061 -2.2
1S_1396 1640178-1641494 -3.0 A1S_1639 1904256-1904799 -2.2
1S_1397 1640178-1641494 -2.1 A1S_1655 1922911-1925230 -2.1
1S_1428 1669120-1670294 -2.3 A1SJ692 1967742-1968948 -2.8
1S_1442 1683286-1684309 -4.6 A1S_1698 1975034-1976021 -2.1
1S_1443 1684318-1685979 -2.8 A1S_1699 1976385-1977348 -12.0
1S_1450 1691408-1692095 -2.1 A1S_1700 1977361-1978381 -11.2
1S_1466 1709124-1710192 -13.0 A1S_1701 1978427-1979918 -4.0
1S_1467 1710876-1712025 -10.5 A1S_1703 1981169-1981334 -3.5
1S_1469 1714322-1715224 -2.1 A1S_1705 1982175-1983234 -2.7
1S_1470 1714322-1715224 -2.5 A1SJ717 1994907-1995651 -3.1
1S_1476 1722031-1722421 -5.5 A1S_1719 1997433-1997679 -2.5 -2.31S_1490 1736282-1737176 -10.3 -2.0 A1S_1724 2002158-2003445 -5.0
1S_1491 1737270-1738113 -16.5 -2.8 A1S_1729 2011426-2012599 -3.2 -2.0
1S_1730 2012664-2014074 -6.7 A1S_1855 2159491-2160406 -2.3 Table 2. Exemplary transcripts that were decreased in expression after outer membrane stress
Gene ID Locus (relative to BamA 10 min. 30 min. Gene ID Locus (relative to BamA 10 min. 30 min.
ATCC 17978 depletion PMBN PMBN ATCC 17978 depletion PMBN PMBN genome) (fold (fold (fold genome) (fold (fold (fold change) change) change) change) change) change)1S_1731 2014190-2014832 -8.7 A1S_1856 2160604-2161558 -3.5
1S_1732 2014833-2015538 -8.9 A1S_1857 2161606-2162560 -6.2
1S_1734 2017398-2018226 -4.1 A1S_1858 2162594-2163332 -5.9
1S_1735 2018428-2018829 -2.4 A1S_1859 2163342-2163827 -9.2
1S_1736 2020537-2021959 -8.6 A1S_1860 2163837-2164662 -10.9
1S_1737 2021992-2022778 -4.1 -2.2 A1S_1861 2164712-2165114 -7.1
1S_1738 2023129-2024095 -2.9 A1S_1862 2165143-2165671 -11.4
1S_1742 2027099-2027885 A1S_1863 2165681-2166041 -16.2
1S_1745 2029779-2030727 -2.1 A1S_1864 2166069-2167236 -12.6
1S_1754 2041665-2042751 -3.0 A1S_1865 2167254-2168364 -9.2
1S_1756 2044492-2045518 -2.5 A1S_1866 2168456-2169080 -2.8
1S_1758 2046654-2047509 -2.0 A1S_1879 2179769-2180237 -6.4
1S_1775 2069767-2070031 -3.3 A1S_1880 2180866-2183296 -2.2
1S_1776 2070099-2070441 -2.2 A1S_1887 2188933-2189209 -2.3
1S_1790 2085457-2087706 -17.5 A1S_1908 2209598-2210654 -2.8
1S_1791 2085457-2087706 -51.3 A1S_1924 2232177-2234906 -4.3 -2.2
1S_1792 2087739-2088711 -79.4 A1S_1925 2232177-2234906 -2.2 -2.3
1S_1794 2089969-2091145 -66.6 A1S_1926 2235024-2235321 -2.1
1S_1795 2091232-2093020 -20.6 A1S_1935 2240911-2241343 -2.4
1S_1796 2093129-2093627 -124.5 A1S_1940 2244737-2245532 -2.2
1S_1797 2093789-2094620 -23.0 A1S_1942 2247377-2247797 -3.8
1S_1805 2100281-2101625 -13.3 A1S_1948 2256206-2256708 -2.3
1S_1806 2101921-2102800 -16.8 A1S_1951 2260399-2261842 -2.1
1S_1811 2107310-2108057 -3.1 A1S_1984 2296375-2297620 -9.0 -2.3
1S_1830 2131349-2131925 -3.3 A1S_1996 2306966-2309125 -2.2
1S_1834 2135777-2136674 -2.5 A1S_2041 2380733-2381603 -16.1
1S_1835 2136725-2138300 -280.1 A1S_2042 2381920-2382493 -5.9
1S_1836 2138311-2139310 -459.6 A1S_2052 2390341-2391586 -2.0
1S_1837 2139320-2140136 -288.9 A1S_2053 2391811-2393403 -2.4
1S_1838 2140246-2140507 -432.1 A1S_2068 2409995-2411168 -2.5
1S_1839 2140793-2142527 -191.8 A1S_2072 2416875-2417319 -7.0
1S_1840 2142610-2143327 -4.5 A1S_2081 2430686-2431118 -2.9
1S_1841 2143394-2144189 -2.1 A1S_2084 2434135-2434693 -4.7
1S_1854 2156967-2159355 -2.2 A1S_2098 2450303-2451476 -4.1
1S_2100 2452413-2453828 -2.0 A1S_2419 2803209-2803779 -2.2 Table 2. Exemplary transcripts that were decreased in expression after outer membrane stress
Gene ID Locus (relative to BamA 10 min. 30 min. Gene ID Locus (relative to BamA 10 min. 30 min.
ATCC 17978 depletion PMBN PMBN ATCC 17978 depletion PMBN PMBN genome) (fold (fold (fold genome) (fold (fold (fold change) change) change) change) change) change)1S_2101 2452413-2453828 -2.0 A1S_2424 2809228-2809561 -4.7
1S_2102 2453971-2455483 -3.5 A1S_2425 2809587-2810133 -4.9
1S_2148 2506394-2508038 -5.0 A1S_2431 2815755-2816685 -3.6
1S_2149 2508121-2509395 -5.6 A1S_2435 2819669-2820818 -3.1
1S_2150 2509413-2510184 -11.4 -2.2 A1S_2443 2826172-2828362 -5.4
1S_2163 2524956-2525793 -2.1 A1S_2449 2835023-2836142 -62.6
1S_2166 2529462-2530 15 -2.6 A1S_2450 2836542-2838264 -304.8
1S_2167 2530518-2532510 -2.2 A1S_2451 2838398-2838884 -6.8
1S_2190 2551999-2552518 -2.1 A1S_2452 2838955-2840386 -56.1
1S_2191 2552534-2552834 -2.0 A1S_2475 2869528-2870785 -2.4 -2.0
1S_2202 2563129-2563858 -2.0 A1S_2501 2898717-2900175 -2.5
1S_2203 2564571-2566933 -2.2 A1S_2509 2908600-2910562 -2.8
1S_2207 2568590-2569481 -6.7 A1S_2510 2910613-2912314 -3.5
1S_2209 2570999-2571509 -2.6 A1S_2514 2914648-2915407 -4.5
1S_2218 2581216-2581753 -2.6 A1S_2531 2930745-2931498 -6.0
1S_2221 2585012-2586248 -3.5 A1S_2532 2931573-2931785 -2.8
1S_2225 2590295-2590766 -2.6 A1S_2533 2931815-2932424 -2.3
1S_2232 2596729-2598298 -2.3 A1S_2535 2932504-2934219 -2.21S_2234 2599369-2599717 -2.2 A1S_2601 3008419-3010867 -4.0
1S_2248 2609511-2610477 -2.9 A1S_2602 3010881-3012735 -4.4
1S_2279 2641008-2642154 -5.4 A1S_2633 3047180-3048659 -5.0
1S_2280 2642317-2643676 -2.2 A1S_2662 3086729-3087692 -10.8
1S_2288 2651013-2653031 -2.2 A1S_2670 3096131-3097640 -2.4
1S_2289 2653138-2653498 -6.4 A1S_2671 3098026-3099346 -2.5
1S_2340 2712683-2713385 -2.2 A1S_2672 3099462-3099885 -3.1
1S_2341 2713597-2713828 -2.9 A1S_2688 3115171-31 15648 -2.0
1S_2342 2714052-2714811 -3,3 A1S_2692 3117713-31 18151 -2.5
1S_2348 2716963-2718421 -18.1 -2.0 A1S_2694 3118617-3122707 -2.4
1S_2353 2722192-2725533 -5.3 A1S_2695 3118617-3122707 -2.3
1S_2354 2722192-2725533 -2.6 A1S_2696 3122855-3129161 -2.6 -2.4
1S_2415 2800397-2800578 -5.1 A1S_2701 3131878-3133264 -3.0
1S_2416 2800641-2800944 -2.7 -2.1 A1S_2711 3144820-3145587 -2.1
1S_2417 2800946-2801372 -2.9 -2.4 A1S_2722 3159070-3159970 -2.1
1S_2418 2801588-2802695 -4.7 -2.2 A1S_2724 3162259-3163587 -3.6
1S_2738 3177870-3178221 -3.1 A1S_2946 3429906-3430401 -2.2 Table 2. Exemplary transcripts that were decreased in expression after outer membrane stress
Gene ID Locus (relative to BamA 10 min. 30 min. Gene ID Locus (relative to BamA 10 min. 30 min.
ATCC 17978 depletion PMBN PMBN ATCC 17978 depletion PMBN PMBN genome) (fold (fold (fold genome) (fold (fold (fold change) change) change) change) change) change)1S_2740 3179147-3179954 -2.3 A1S_2956 3442371-3443442 -2.2
1S_2741 3179979-3180186 -2.9 A1S_3013 3499256-3499820 -2.4
1S_2748 3187834-3189052 -2.2 A1S_3014 3499839-3500190 -2.7
1S_2753 3193785-3195090 -16.6 -2.2 A1S_3025 3510711-3511698 -2.2
1S_2755 3197010-3197637 -2.9 A1S_3040 3525411-3526053 -3.0
1S_2758 3199217-3200072 -3.2 A1S_3043 3528294-3530661 -5.1
1S_2761 3201639-3202827 -3.6 A1S_3049 3537422-3539618 -4.9
1S_2762 3203269-3203830 -2.1 A1S_3050 3539805-3540372 -3.2
1S_2769 3209077-3209878 -2.7 A1S_3051 3540450-3541536 -5.5
1S_2773 3213513-3215025 -17.3 -2.0 A1S_3074 3554574-3555439 -2.1
1S_2774 3215092-3215710 -8.3 A1S_3084 3561982-3563530 -2.7
1S_2785 3226657-3227236 -4.0 A1S_3110 3590661-3592965 -3.9
1S_2788 3229108-3229858 -2.0 A1S_3120 3605537-3605717 -2.5
1S_2789 3229899-3231948 -2.7 A1S_3121 3605932-3607048 -2.0
1S_2793 3236219-3237695 -3.2 A1S_3122 3607050-3607944 -14.9
1S_2809 3250578-3250719 -2.2 A1S_3128 3613226-3614201 -5.2
1S_2814 3259866-3260229 -2.1 A1S_3129 3614213-3615557 -5.6
1S_2815 3260252-3260636 -2.5 A1S_3130 3615574-3618075 -5.1
1S_2820 3267676-3268018 -2.1 A1S_3131 3615574-3618075 -4.4
1S_2823 3269162-3269612 -2.7 A1S_3132 3618093-3618930 -4.2
1S_2847 3294149-3295376 -4.0 A1S_3133 3619077-3619311 -4.1
1S_2848 3295582-3296347 -4.2 A1S_3134 3619455-3620727 -2.8
1S_2849 3296727-3297981 -7.7 A1S_3135 3621761-3623195 -14.5
1S_2852 3300626-3301832 -2.3 A1S_3144 3630637-3631585 -2.3
1S_2860 3307860-3309171 -5.0 A1S_3174 3661125-3661320 -2.01S_2904 3356336-3357263 -3.2 A1S_3180 3666315-3666906 -2.6
1S_2905 3357287-3360038 -2.0 A1S_3195 3679879-3682280 -2.7
1S_2906 3360106-3361375 -3.9 A1S_3207 3693623-3694625 -2.4
1S_2911 3373995-3374565 -3.0 A1S_3222 3712619-3713897 -2.4
1S_2913 3375972-3376302 -2.0 A1S_3224 3716297-3717188 -5.0
1S_2919 3381431-3382343 -2.2 A1S_3225 3717255-3719376 -4.0
1S_2924 3386628-3386943 -2.3 A1S_3231 3725183-3726698 -2.0
1S_2928 3390666-3393354 -3.1 A1S_3232 3726828-3727260 -2.3
1S_2939 3410235-3412593 -2.3 -2.0 A1S_3236 3729663-3730212 -10.5
1S_3238 3731034-3731766 -2.5 A1S_3458 3973198-3974677 -9.3 Table 2. Exemplary transcripts that were decreased in expression after outer membrane stress
Gene ID Locus (relative to BamA 10 min. 30 min. Gene ID Locus (relative to BamA 10 min. 30 min.
ATCC 17978 depletion PMBN PMBN ATCC 17978 depletion PMBN PMBN genome) (fold (fold (fold genome) (fold (fold (fold change) change) change) change) change) change)1S_3248 3738545-3739235 -9.5 A1S_3460 3976055-3976655 -2.3
1S_3250 3739412-3739655 -2.1 A1S_3481 67185-68349 -2.8
1S_3268 3757736-3758129 -3.1 A1S_3487 171454-172389 -4.3
1S_3269 3758173-3758843 -2.1 A1S_3491 241566-243363 -2.0
1S_3273 3762823-3763207 -19.7 A1S_3494 340739-340835 -2.21S_3278 3767589-3768216 -2.3 A1S_3498 512466-514677 -2.0
1S_3290 3780683-3781358 -2.0 A1S_3506 651992-652244 -4.2
1S_3297 3789666-3790566 -4.6 A1S_3508 680322-680808 -2.4
1S_3298 3790632-3792600 -5.1 A1S_3509 681319-681889 -2.5
1S_3300 3793627-3795343 -18.6 -2.8 A1S_3514 687878-688160 -5.3
1S_3301 3795344-3795677 -18.2 -2.5 A1S_3518 691526-692903 -4.1
1S_3309 3804682-3806632 -5.4 A1S_3519 692917-698877 -3.2
1S_3338 3840719-3840932 -2.1 A1S_3520 698890-700942 -4.2
1S_3342 3845998-3846823 -2.3 A1S_3521 701358-702009 -2.6
1S_3355 3856559-3857304 -4.8 A1S_3522 702026-703842 -8.1
1S_3364 3870877-3873679 -4.2 A1S_3523 702026-703842 -7.8
1S_3377 3885571-3886768 -2.7 A1S_3524 710417-711071 -2.7
1S_3397 3907011-3908422 -2.7 A1S_3526 713732-714230 -3.9
1S_3398 3907011-3908422 -3.7 A1S_3528 721098-721383 -7.2
1S_3402 3911261-3912182 -4.7 A1S_3530 728965-729157 -3.8
1S_3403 3912192-3913398 -4.8 A1S_3531 729255-730534 -3.3
1S_3404 3913449-3914862 -5.0 A1S_3532 729255-730534 -4.3
1S_3405 3914926-3916465 -3.5 A1S_3533 730543-731401 -6.6
1S_3406 3916552-3916849 -2.8 A1S_3534 733836-734586 -6.7
1S_3407 3916898-3918131 -2.3 A1S_3535 738104-738575 -27.4
1S_3410 3920203-3921277 -3.9 A1S_3537 741575-741908 -6.4
1S_3413 3923558-3924947 -16.2 A1S_3538 742372-742675 -5.0
1S_3414 3925305-3927232 -7.3 A1S_3539 743976-744693 -32.8
1S_3415 3925305-3927232 -7.8 A1S_3540 745850-747482 -48.4
1S_3416 3927249-3927798 -12.4 A1S_3541 747506-748361 -49.2
1S_3418 3928941-3929997 -37.1 A1S_3542 748399-748978 -70.0
1S_3431 3942842-3943205 -3.8 A1S_3543 749082-749502 -83.9
1S_3450 3963994-3964621 -3.6 A1S_3544 749580-751358 -10.4
1S_3451 3964779-3965166 -2.9 A1S_3545 749580-751358 -6.3
1S_3546 754708-755119 -12.9 A1S_3679 1767313-1767499 -2.1 Table 2. Exemplary transcripts that were decreased in expression after outer membrane stress
Gene ID Locus (relative to BamA 10 min. 30 min. Gene ID Locus (relative to BamA 10 min. 30 min.
ATCC 17978 depletion PMBN PMBN ATCC 17978 depletion PMBN PMBN genome) (fold (fold (fold genome) (fold (fold (fold change) change) change) change) change) change)1S_3547 755128-757866 -7.1 A1S_3701 1851220-1851622 -3.1
1S_3548 755128-757866 -9.2 A1S_3707 1868781-1869498 -4.31S_3549 759189-760109 -3.4 A1S_3709 1883408-1883777 -2.6
1S_3550 759189-760109 -2.5 A1S_3713 1955778-1955994 -2.2
1S_3552 765517-767063 -18.6 A1S_3715 1963521-1963788 -8.4
1S_3553 767079-767373 -7.5 A1S_3717 1973738-1973843 -3.0
1S_3554 768038-768803 -3.0 A1S_3732 2108264-2108510 -4.0
1S_3568 921309-921486 -2.7 A1S_3735 2112990-2113506 -2.8
1S_3569 936320-937274 -2.1 A1S_3738 2135777-2136674 -5.1 -4.7
1S_3578 1065513-1065609 -4.0 A1S_3740 2156967-2159355 -4.6
1S_3582 1114210-1115446 -5.1 A1S_3741 2163342-2163827 -10.1
1S_3586 1134942-1135071 -4.9 A1S_3742 2168456-2169080 -2.1
1S_3587 1139973-1141021 -2.1 A1S_3759 2344529-2344892 -6.8
1S_3591 1199995-1200313 -2.1 A1S_3774 2372430-2373321 -3.3
1S_3597 1239532-1240884 -4.4 A1S_3779 2378476-2378563 -3.2
1S_3599 1262867-1263338 -3.1 A1S_3787 2475670-2476099 -6.6
1S_3600 1278999-1279095 -2.8 A1S_3788 2478674-2479109 -3.3
1S_3602 1321090-1321904 -2.7 A1S_3794 2563997-2564492 -2.4
1S_3611 1338841-1343140 -3.4 A1S_3801 2743969-2744170 -9.2
1S_3619 1356950-1357451 -6.9 A1S_3802 2744347-2744440 -11.6 -2.2
1S_3621 1367712-1368102 -2.5 A1S_3806 2800397-2800578 -5.0
1S_3624 1391658-1392075 -2.8 A1S_3809 2836225-2836402 -121.2 -5.3
1S_3629 1430539-1430764 -2.1 A1S_3811 2868525-2868936 -4.9
1S_3633 1441532-1442747 -2.7 A1S_3813 2931573-2931785 -3.5
1S_3640 1475687-1476502 -2.5 A1S_3814 2949216-2950113 -2.41S_3641 1478191-1478650 -7.0 A1S_3816 2955076-2955415 -2.2
1S_3644 1502656-1505369 -4.7 A1S_3817 2967457-2968409 -2.3
1S_3647 1519019-1519712 -14.0 A1S_3823 3064273-3064879 -2.1
1S_3651 1578073-1578265 -5.4 A1S_3829 3118617-3122707 -6.2
1S_3652 1578727-1579087 -5.5 A1S_3831 3139828-3140950 -4.4
1S_3659 1621918-1622347 -2.6 A1S_3832 3144638-3144734 -3.0
1S_3663 1645263-1645979 -4.0 A1S_3836 3238117-3238399 -2.3
1S_3667 1689472-1690284 -2.2 A1S_3840 3321647-3322643 -19.1
1S_3673 1744240-1745154 -2.2 A1S_3846 3372881-3373910 -2.1
1S_3857 3419556-3419760 -3.2 A1S_3894 3758173-3758843 -2.8 Table 2. Exemplary transcripts that were decreased in expression after outer membrane stress
Gene ID Locus (relative to BamA 10 min. 30 min. Gene ID Locus (relative to BamA 10 min. 30 min.
ATCC 17978 depletion PMBN PMBN ATCC 17978 depletion PMBN PMBN genome) (fold (fold (fold genome) (fold (fold (fold change) change) change) change) change) change)
A1S_3862 3433087-3434281 -2.0 A1S_3898 3856559-3857304 -4.3
A1S_3868 3451602-3451791 -2.0 A1S_3907 3904441-3904615 -2.3
A1S_3870 3458183-3458507 -2.2 A1S_3908 3905565-3906780 -44.7
A1S_3880 3604635-3605466 -12.3 A1S_3909 3936958-3937162 -3.8
A1S_3884 3700150-3701098 -2.0 A1S_3912 3948391-3949652 -2.7
A1S_3886 3709202-3709895 -2.1 A1S_3914 3973042-3973186 -3.2
A1S_3887 3710237-3710618 -3.5 -2.2 A1S_3915 3974687-3974831 -8.3
A1S_3891 3749400-3749877 -2.5
Example 2 : Generation of Outer Membrane Stress Transcriptional Reporter Constructs
A. Reporter Constructs
[0338] Reporter constructs were generated containing transcriptional regulatory regions upstream of exemplary genes identified in Example 1 as being responsive to outer membrane- induced stress. A reporter construct containing a transcriptional regulatory region upstream of A1S_2890, which was not identified in the screen described above, was generated as a negative control.
[0339] A roughly 250 base pair (bp) DNA sequence upstream of the open reading frame (ORF) of each gene was operatively linked to a fast-folding green fluorescent protein gene
(sfGFP; set forth in SEQ ID NO: 15) using standard molecular biology techniques. Also included immediately upstream of the sfGFP ORF was either the native Shine-Dalgarno sequence of the gene or a synthetic Shine-Dalgarno sequence (set forth in SEQ ID NO: 14).
[0340] The transcription fusion constructs were incorporated into A. baumannii pACH106 (also called pMMB67EH, ATCC No. 37622; SEQ ID NO: 29). As shown in FIG. 9, pACH106 encodes replication factors and selective markers necessary for autonomous replication in A.
baumannii and maintenance by antibiotic selection. The final reporter vector constructs were constructed by Gibson assembly and were generated by replacing the 5,715-7,395 bp region of the A. baumannii plasmid pACH106 (Fig. 9) with the upstream sequence: sfGFP fusion
constructs. Exemplary generated transcriptional fusion constructs and final reporter vectors are set forth in Table 3, with sequence identifiers (SEQ ID NO) for individual components indicated. All fusion constructs contained a native Shine-Dalgarno (SD) sequence, except for A1S_0032 and A1S_2889 that were generated to contain a synthetic SD sequence in place of the native sequence.
Table 3: Exemplary transcriptional fusion constructs and reporter vectors
Figure imgf000161_0001
Example 3 : Assessment of Response of Modified A. Itaumarinii R porter Bacterial Strains to Outer Membrane Stress
[0341] Various exemplary modified A. baumannii bacterial strains were generated by incorporating one of the exemplary reporter vectors generated as described in Example 2 into A. baumannii strains using standard techniques. As described below, vectors were introduced into wildtype A. baumannii strain ATCC 17978, an A. baumannii strain depleted for BamA, or a multi-drug resistant A. baumannii (strain Ab307-0294). The generated A. baumannii reporter bacterial strains were monitored for induction of fluorescence in response to various known outer membrane stress inducers. A. PMBN
[0342] Exemplary reporter vectors were introduced into wildtype A. baumannii strain ATCC 17978 and were grown for 30 minutes in the presence or absence of 250 μg/mL PMBN, which is a concentration of PMBN that is well below the minimal inhibitory concentration and therefore has no antimicrobial activity. The cells were then subjected to flow cytometry to assess fluorescence in response to treatment with PMBN.
[0343] The three most responsive modified A. baumannii reporter bacterial strains were those containing reporter vectors PAis_oo32-sfGFP (SEQ ID NO: 30), PAis_2889-sfGFP (SEQ ID NO: 31), and PAis_2885-sfGFP (SEQ ID NO: 32). These three modified bacterial strains also showed little to no background fluorescence. Exemplary results for bacterial reporter cells containing the PAis_2S85-sfGFP reporter vector are shown in Fig. 10A. As shown, the histogram of fluorescence intensities showed a 10-to-20-fold average increase in fluorescence following treatment with PMBN, demonstrating that this OM permeabilizing agent was an inducer of this fluorescent reporter. Fig. 10B shows fluorescent micrographs of reporter bacterial cells containing the PAis_2889-sfGFP reporter vector with and without treatment in the presence of PMBN. As shown, GFP fluorescence was readily detectable only in the PMBN treatment condition.
[0344] To assess effect of culture conditions to induction of fluorescence by the reporter strains, wildtype A. baumannii strain ATCC 17878 containing PAis_2885-sfGFP that was treated with 250 μg/mL PMBN was grown either in culture flasks with aeration or in microtiter dishes without shaking or aeration. As shown in Fig. 11A and 1 IB, strong fluorescence was induced in both growth conditions showing that the reporter strains can be used in a variety of assay formats, including high-throughput 96-well-plate -based assays.
B. BamA Depletion
[0345] Reporter vectors PAis_oo32-sfGFP or PAis_2889-sfGFP were introduced into the A. baumannii strain described in Example 1 that was depleted for BamA unless exogenous arabinose was added. The reporter strains were grown in the presence or absence of arabinose and fluorescence intensity was measured by flow cytometry. As shown in FIG. 12A, mean fluorescence intensity was >100-fold higher when the reporter strains were grown in the absence of arabinose, indicating that these reporter constructs were responsive to defects in OM biogenesis caused by depletion of BamA. The extent of responsiveness of the reporter bacterial cells is shown in FIG. 12B, which shows that a majority of cells exhibited a level of fluorescence at or near the upper limit of detection when cells were grown without arabinose for only 3 hrs.
C. Vancomycin
[0346] Reporter vectors PAis_2885-sfGFP or PAis_2889-sfGFP were introduced into A.
baumannii Ab307-0294, which is known to be a multi-drug resistant strain. The resulting strains were treated either with 250 μg/mL PMBN or Vancomycin, a cell- wall-targeting antibiotic which prevents biogenesis of the peptidoglycan cell wall and consequently influences the integrity of the cell envelope.
[0347] Whereas PMBN was a potent inducer of fluorescence in these reporters, the antibiotic vancomycin was a mild inducer (Fig. 13A-13B). An approximate 5-fold increase in median fluorescence intensity was observed when the A. baumannii strain Ab307-0294 was exposed to a sub-lethal concentration of vancomycin (Fig. 13A), whereas the response was substantially greater when treated with PMBN.
D. LpxC inhibitor
[0348] Reporter vectors PAis_oo32-sfGFP or PAis_2885-sfGFP were introduced into wildtype A. baumannii strain ATCC 17978 and were treated with 10 g/mL ACHN-975 (Cas No: 1410809- 36-7), an antibiotic that inhibits synthesis of lipopolysaccharide (LPS) by inhibiting the activity of the LpxC enzyme. As shown in Fig. 14A and 14B, ACHN-975 induced fluorescence in both reporter cells, which further indicated these reporters were responsive to defects in OM biogenesis/integrity, since LPS, whose synthesis is prevented by ACHN-975, is the primary component of the outer leaflet of the OM. PMBN, which was shown above also to be a potent inducer of the reporters, also acts by specifically disrupting the LPS layer.
E. Other agents that induce OM stress
[0349] Reporter vectors PAis_oo32-sfGFP or PAis_2885-sfGFP were introduced into wildtype A. baumannii strain ATCC 17978 and their responsiveness, by induction of fluorescence, to outer membrane stress by various agents known to disrupt/permeabilize the outer membrane was assessed. Specifically, the reporter cells were variously treated with the following agents: (1) 10 μg/mL ACHN-975 as described above; (2) 2 μg/mL sub-lethal concentration of colistin, a broad- spectram antibiotic that binds and perturbs the LPS leaflet of the OM; (3) 5 mM EDTA, a chelating agent that disrupts the LPS layer by stripping away divalent cations that stabilize the OM by neutralizing the negative charges in LPS; (4) 5 mM EDTA with 1.0% SDS, a detergent that acts synergistically with EDTA to permeabilize the destabilized OM; and (5) 250 μg/mL PMBN as described above. As shown in Fig. 15A and 15B, the agents caused induction of fluorescence in the A. ba mannii reporter strains to varying degrees.
Example 4 : Use of A. b um nnii^LvaoxX&x cells in microparticle-based screen for
antibodies that perturb the Gram-negative cell envelope
[0350] The outer membrane (OM) stress transcriptional reporter cells described above were assessed in a microparticle-based assay. In the assay, the reporter cells were encapsulated within agarose particles with a known or suspected outer membrane stress agent and fluorescence induced in individual cells in response to OM stress was monitored by fluorescent microscopy. The microparticle-based assay also was used in a screening assay to screen a plurality of candidate molecules that perturbed or disrupted the Gram-negative cell envelope. Fig. 16 provides a general outline of the screen.
A. Outer Membrane Stress Agent
[0351] Reporter A. baumannii cells, carrying the reporter construct PAis_2889-sfGFP in wildtype A. baumannii strain ATCC 17978, were incubated for 10 minutes in the presence or absence of a sub-lethal concentration of colistin (0.5 ug/mL), which is an agent known to induce OM stress. The reporter cells were then encapsulated within 65 pL agarose microparticles using a microfluidic device. Encapsulated microparticles were spread onto a microscope slide and cells were assessed for fluorescence using a fluorescent microscope.
[0352] As shown in Fig. 17A, reporter cells that were not treated with colistin exhibited no detectable fluorescence within microparticles. In contrast, FIG. 17B, showed that reporter cells that were treated with colistin were brightly fluorescent and readily detected by conventional epifluorescence microscopy. These results confirmed that induction of the fluorescent reporter in the reporter bacterial cells can be readily detected in agarose microparticles. B. Screening for molecules that perturb or disrupt the Gram-negative cell envelope and/or are induced by outer membrane stress.
[0353] An antibody-secreting B cell pool was generated by immunizing Balb/C mice with A. baumannii Ab307-0294. Antibody-producing B cells were isolated from spleen and lymph node cells of the immunized Balb/C animals in a two-step cell purification process. First, spleen and lymph node cells were harvested and purified using the Pan B Cell Isolation Kit (Miltenyi Biotec, Cat. No. 130-095-813) to remove tissue debris and other material. Then, the cell preparation was further purified using EasySep™ CD138+ cell isolation kit (Stemcell
Technologies) to obtain a B cell preparation.
[0354] Single B cells were co -encapsulated with reporter bacterial cells. A preparation of 4% ultra-low gelling temperature agarose (Sigma- Aldrich, Cat. No. A5030) in phosphate buffered saline (PBS) was melted at 70 °C for 15 minutes, then cooled to 37 °C for
encapsulation of live B cells and the reporter bacterial cells. The B cell preparation was centrifuged and resuspended in a media comprising a combination of 54% Iscove's Modified Dulbecco's Medium (IMDM) and 46% OptiPrep™ Density Gradient Medium (Sigma-Aldrich, Cat. No. D1556). Reporter bacterial cells were centrifuged and resuspended in 125 pL of the B cell suspension generated above. Then, 125 μΐ^ of 4% ultra-low gelling temperature agarose was added to the B cell and bacterial reporter cell suspension making the final ultra-low gelling temperature agarose concentration 2%. This 250 pL mixture was loaded on pEncapsulator (Dolomite Microfluidics) microfluidic device, following the Manufacturer's instructions to generate encapsulated particles. The encapsulated particles were collected and incubated on ice for 5 minutes to gel the agarose. The gelled encapsulated particles were transferred onto a 6- well dish and incubated for 1 hour at 37 °C, with agitation, to allow for antibody secretion by the B cells. The samples were then transferred into tubes, emulsion was broken using Pico Surf surfactant (Dolomite Microfluidics), and washed with 2% fetal bovine serum (FBS) in PBS.
[0355] The samples were then washed in 2% FBS/PBS, and screened under a fluorescent microscope. As shown in FIG. 18C, merging of the brightfield (FIG. 18 A) with the fluorescent field (FITC; FIG. 18B), showed a positive hit in which particles containing fluorescent bacteria were observed, indicating the existence of an antibody-secreting B cell that secreted a molecule that bound to the cells in a manner to disrupt the outer membrane and/or induce an outer membrane stress. This B cell is then selected using a micromanipulator needle (Origio, Cat. No. C140819) for single-cell antibody cloning or mass spectrometry to identify the OM- disrupting molecule.
Example 5 : Multicolor Imaging in Acza toJ>ac &clerm
[0356] Fusion constructs containing either an RFP (mRuby2; SEQ ID NO:33) or GFP (mClover3; SEQ ID NO:40) reporter sequence operatively linked to the Tac promoter (pTac) for inducible expression of the reporter in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG) were incorporated into A. baumannii pACH106 (also called pMMB67EH, ATCC No. 37622; SEQ ID NO: 29). The resulting plasmids were individually transfected into wildtype A. baumannii (ATCC 17978). Transfected A. baumannii 17978 + pTac-mRuby2 plasmid and A baumannii 17978 + pTac-mClover3plasmid were mixed equally (1: 1) and then were incubated in Luria broth (LB) containing 500 μΜ IPTG to induce expression of the reporters. Reporter expression was monitored by epifluorescence microscopy. As shown in the micrograph images depicted in Fig. 19A-D expression of both the mRuby 2(RFP) and mClover3(GFP) reporter signals were observed in the mixed population strain, thereby demonstrating that two non- overlapping fluorescent proteins could be detected. This result demonstrates that different reporter constructs can be utilized for simultaneous and/or multiplex imaging of discrete strains.
Figure imgf000166_0001
Bacterial Strains to Outer Membrane Stress
[0357] Reporter A. baumannii cells carrying the reporter construct PAis_oo32-sfGFP (SEQ ID NO:30), PAis_2885-sfGFP (SEQ ID NO:32), or PAis_2889-sfGFP (SEQ ID NO:31) in wildtype A. baumannii strain ATCC 17978, were incubated for 30 minutes in the presence of 2-fold serial dilutions PMBN. For comparison, A. baumannii cells carrying an isopropyl β-D- l- thiogalactopyranoside (IPTG)-inducible promoter operatively linked to a gene encoding sfGFP gene (Ptac-sfGFP) in wildtype A. baumannii (ATCC 17978), were incubated for 30 minutes in the presence of 2-fold serial dilutions of IPTG. The cells were then subjected to flow cytometry to assess fluorescence in response to treatment and mean fluorescence intensity (MFI) was determined.
[0358] As shown in FIG. 20A, induction of GFP fluorescence was dose-responsive in the presence of increasing doses of PMBN from A. baumannii cells carrying the GFP reporter operatively linked to the exemplary transcriptional regulatory region of OM stress-induced genes. A similar dose-response induction of GFP fluorescence also was observed from A.
baumannii cells carrying the GFP reporter operatively linked to the control IPTG-inducible promoter. These result demonstrated that the OM-responsive promoters are capable of a dose- dependent induction of fluorescent signal in response to OM stress, such that the promoters may, in some aspects, distinguish qualitative differences in stress induction.
Example 7 : Evidence of Outer Membrane Stress Reporter Activation in the Presence of an Antibody Targeted Against the Outer Membrane of A. ύαα/η ηηίί
[0359] A. baumannii Ab307-0294 cells (a multi-drug resistant strain) carrying the exemplary PAis_2889-sfGFP reporter construct (SEQ ID NO:31) were incubated with or without 10% fetal bovine serum in the presence of media only, a non-binding IgG isotype control, or an exemplary purified monoclonal antibody that binds the OM of live A. baumannii cells. Serum, which contains active complement components, was included during the incubation because a number of bacterial pathogens, including Acinetobacter baumannii are intrinsically resistant to serum alone, but can still be killed by serum components in the presence of antibodies that activate and fix complement via the classical pathway. Because complement directly causes disruption of the bacterial OM, activation of the OM stress reporter is anticipated in the presence of antibody that can stimulate complement activation. Following incubation for 1 hour, the cells were then subjected to flow cytometry to assess fluorescence in response to treatment.
[0360] Fig. 21A-F show the flow cytometry histograms from each condition. As shown in FIG. 21F, cells treated with the exemplary OM-specific antibody in the presences of serum resulted in the induction of a fluorescent signal in cells. The induction of fluorescence was not observed in the absence of serum (FIG. 21C), nor in cells incubated in the presence of a nonspecific isotype control antibody (FIG. 21A or 21D) or media only (FIG. 21B or 21E), whether in the presence or absence of serum. Without being bound by theory, this result is consistent with the anti-OM antibody fixing or concentrating complement at the cell surface, which can disrupt the bacterial membrane. This result confirms that a non-chemical outer membrane stress, such as induced in the presence of an antibody to the OM, can induce a reporter signal from A. baumannii cells carrying an OM-stress responsive transcriptional reporter construct. Example 8 Assessment of Response of Modified A. />af////a//////K.eyi)rter Bacterial Strains with Alternative Reporter to Outer Membrane Stress
[0361] An exemplary reporter vector encoding mRuby2 regulated by a promoter containing the region upstream of A. baumannii gene A1S_2889 was introduced into wildtype A.
baumannii strain ATCC 17978. The resulting cells were grown for 30 minutes in the presence or absence of 500 μg/mL PMBN. Substantial red fluorescence was detected for bacteria grown in the presence of PMBN, whereas bacteria grown in the absence of PMBN display little to no red fluorescence.
[0362] The present invention is not intended to be limited in scope to the particular disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are intended to fall within the scope of the present disclosure.
SEQUENCES
Figure imgf000169_0001
GGGTTTTAACCATGAAAATGACGGCTAAAATTGCATTATTCAGT
CTTGTACACTTGGAACTTTGCAGAAAAATTGATGAAGACGTTCAAAACTGAT PA1 _2 89 TTCAGACACAGCAATTGACCTAGCTTGAAAAATGCTAATGGTAGCACAGATA Regulatory CAGATTCTTCATATTTTCTTTATAGTCATTACAAACAATATCAATCAAAGCC Region ATTTTATTCATGGTTTCTCCTTGACTCTCTCTTAATCTAGCTTCAG
ATAGGCTTTTTCATGGAGTATTCCTTTTTATCTTGATCTGAAGCTAGATTAA PA1 _2 90 GAGAGAGTCAAGGAGAAACCATGAATAAAATGGCTTTGATTGATATTGTTTG Regulatory TAATGACXATAAAGAAAATATGAAGAATCTGTATCTGTGCTACCATTAGCAT Region TTTTCAAGCTAGGTCAATTGCTGTGTCTGAAATCAGTTTTGAACGTCTTCAT CAATTTTTCTGCAAAGTTCCAAGTGTACAAGAAGCCCGTATC
ATATAGGCATGTATCACATGAACAACTATAGAAAAATTTAGGAGATGAAGGT J?A1S_3127 TAAAAAGCTGTAAGATGAAAATAGGACCAGCTTAATAAGAATAACTAAAAAA Regulatory TAAATGTTCTTTTTCACTTTATTATTAATTTTTATTAATCTTCACAATTTAT Region CCCTAGTCTGTTTACTCAATGTAACACTGTTGTAACGTTTTGATGAAGTGAG TTCCCAAGCTCATATTTCCTTAGCTATGATGCGATTGCGTTT
AAGAAGGAGATATACAT Synthetic
Shine-
Dalgarno
ATGGTGAGCAAGGGCGAAGAATTGTTTACTGGGGTGGTACCCATCTTGGTCG sfGFP AGCTTGACGGGGATGTAAACGGCCACAAATTTTCTGTTCGTGGCGAAGGAGA GGGCGACGCGACGAACGGAAAGTTGACCCTGAAATTTATCTGCACCACCGGA AAGCTTCCCGTACCGTGGCCCACCTTAGTTACAACACTTACATACGGTGTGC AATGCTTCTCACGTTATCCTGACCACATGAAGCGTCATGACTTCTTTAAATC GGCCATGCCCGAAGGTTATGTTCAAGAACGTACTATTTCGTTTAAGGACGAC GGGACGTATAAAACACGCGCTGAAGTAAAATTCGAGGGTGATACTTTGGTCA ATCGTATTGAATTGAAGGGGATCGACTTTAAAGAAGACGGCAATATCTTAGG ACACAAACTGGAGTATAATTTCAATTCTCACAATGTGTACATCACTGCAGAC AAACAGAAAAACGGGATTAAAGCGAACTTTAAAATCCGCCACAACGTAGAAG ACGGAAGTGTGCAGTTAGCTGATCACTATCAACAAAATACGCCCATTGGTGA TGGTCCGGTTCTTCTTCCAGACAACCACTACTTGAGTACACAAAGCGTTCTG AGCAAAGACCCGAATGAAAAACGCGATCACATGGTTCTGCTGGAGTTCGTAA CTGCTGCTGGGATCACACACGGCATGGATGAACTGTATAAATAA
CTAAGATAGATAGCTCCTCACTATCGCTTAGTAGGAAAGGATGAAGCTGATC PA1 _0032— GGCTTTATCCTTTTTTTATTTTTATATCAATATGATATAAAAATTTAAATCT SfGFP TCTAAATTTCTTCTTATTTCATAGAAAATTTTCACATTTATTATTCTTCGCC fusion TTCACGATTTATCTGTAATTTTTCACTAATCTTCAATTATCGAAATTTAATA GATCATTCGAGGTTTACGAAGAAGGAGATATACATATGGTGAGCAAGGGCGA AGAATTGTTTACTGGGGTGGTACCCATCTTGGTCGAGCTTGACGGGGATGTA AACGGCCACAAATTTTCTGTTCGTGGCGAAGGAGAGGGCGACGCGACGAACG GAAAGTTGACCCTGAAATTTATCTGCACCACCGGAAAGCTTCCCGTACCGTG GCCCACCTTAGTTACAACACTTACATACGGTGTGCAATGCTTCTCACGTTAT CCTGACCACATGAAGCGTCATGACTTCTTTAAATCGGCCATGCCCGAAGGTT ATGTTCAAGAACGTACTATTTCGTTTAAGGACGACGGGACGTATAAAACACG CGCTGAAGTAAAATTCGAGGGTGATACTTTGGTCAATCGTATTGAATTGAAG GGGATCGACTTTAAAGAAGACGGCAATATCTTAGGACACAAACTGGAGTATA ATTTCAATTCTCACAATGTGTACATCACTGCAGACAAACAGAAAAACGGGAT TAAAGCGAACTTTAAAATCCGCCACAACGTAGAAGACGGAAGTGTGCAGTTA GCTGATCACTATCAACAAAATACGCCCATTGGTGATGGTCCGGTTCTTCTTC CAGACAACCACTACTTGAGTACACAAAGCGTTCTGAGCAAAGACCCGAATGA AAAACGCGATCACATGGTTCTGCTGGAGTTCGTAACTGCTGCTGGGATCACA CACGGCATGGATGAACTGTATAAATAA CTCAATGGACAAAGTATTGATGCACAGGATTAAATAATTACAGATGAAAAGC PA TAAAATAATTTATAGCCGATCATCACATCTGGCTTGGCTATACCTGAAAAGA SfGFP TATATACAAGCCTTTTTTATTATTGAGAAAAACGCTGGATTTATCCGTAATT fusion TTAATTGAAGCATTAAATGATCTTCACAGTTTCTCTGCATTTTGCGCGTACA GTATTCAACATGAGAGACATCAGATGTAAACGAGGATGCATCATGGTGAGCA AGGGCGAAGAATTGTTTACTGGGGTGGTACCCATCTTGGTCGAGCTTGACGG GGATGTAAACGGCCACAAATTTTCTGTTCGTGGCGAAGGAGAGGGCGACGCG ACGAACGGAAAGTTGACCCTGAAATTTATCTGCACCACCGGAAAGCTTCCCG TACCGTGGCCCACCTTAGTTACAACACTTACATACGGTGTGCAATGCTTCTC ACGTTATCCTGACCACATGAAGCGTCATGACTTCTTTAAATCGGCCATGCCC GAAGGTTATGTTCAAGAACGTACTATTTCGTTTAAGGACGACGGGACGTATA AAACACGCGCTGAAGTAAAATTCGAGGGTGATACTTTGGTCAATCGTATTGA ATTGAAGGGGATCGACTTTAAAGAAGACGGCAATATCTTAGGACACAAACTG GAGTATAATTTCAATTCTCACAATGTGTACATCACTGCAGACAAACAGAAAA ACGGGATTAAAGCGAACTTTAAAATCCGCCACAACGTAGAAGACGGAAGTGT GCAGTTAGCTGATCACTATCAACAAAATACGCCCATTGGTGATGGTCCGGTT CTTCTTCCAGACAACCACTACTTGAGTACACAAAGCGTTCTGAGCAAAGACC CGAATGAAAAACGCGATCACATGGTTCTGCTGGAGTTCGTAACTGCTGCTGG GATCACACACGGCATGGATGAACTGTATAAATAA
AAGAGCAGATCGAACGCGGTCAACTGGTCTGTTTAACCCATCCAAACCGTAG PA AACGGTGATTGCTCGAAACTATGTCAAGAACGGCTTAGACGTCATCCGTGAT SfGFP CGTGAGCAGCGGGGGCAGCTGATTCGTTATAGCTCCAATCTACTCAACTCAA fusion TGATCAAGGAACGTGAAGTACGCACGCTGATTTTTAATCGTCTGAAAAGTGC TTTGTTTTAACCCTTATCTTTTAAATCTTTGAGTGAAAATCCATGGTGAGCA AGGGCGAAGAATTGTTTACTGGGGTGGTACCCATCTTGGTCGAGCTTGACGG GGATGTAAACGGCCACAAATTTTCTGTTCGTGGCGAAGGAGAGGGCGACGCG ACGAACGGAAAGTTGACCCTGAAATTTATCTGCACCACCGGAAAGCTTCCCG TACCGTGGCCCACCTTAGTTACAACACTTACATACGGTGTGCAATGCTTCTC ACGTTATCCTGACCACATGAAGCGTCATGACTTCTTTAAATCGGCCATGCCC GAAGGTTATGTTCAAGAACGTACTATTTCGTTTAAGGACGACGGGACGTATA AAACACGCGCTGAAGTAAAATTCGAGGGTGATACTTTGGTCAATCGTATTGA ATTGAAGGGGATCGACTTTAAAGAAGACGGCAATATCTTAGGACACAAACTG GAGTATAATTTCAATTCTCACAATGTGTACATCACTGCAGACAAACAGAAAA ACGGGATTAAAGCGAACTTTAAAATCCGCCACAACGTAGAAGACGGAAGTGT GCAGTTAGCTGATCACTATCAACAAAATACGCCCATTGGTGATGGTCCGGTT CTTCTTCCAGACAACCACTACTTGAGTACACAAAGCGTTCTGAGCAAAGACC CGAATGAAAAACGCGATCACATGGTTCTGCTGGAGTTCGTAACTGCTGCTGG GATCACACACGGCATGGATGAACTGTATAAATAA
TGCGGGGTGGAGCAGTCTGGTAGCTCGTCGGGCTCATAACCCGAAGGTCGTT PA1 _1224— GGTTCAAATCCGACCCCCGCATCCAAAATATTATGCTCTGTGATGTACTACT sfGFP TTTCATGATACGAAATCATGGAAAGCAATTAGCTTATAAATAACCCTATATC fusion AAGCTAAATATTAAATTCTAGACGAATTGTCCATACCTTAAATCTAGCGTTC TAATTAAATACCGTTAAATCACATGAGCTCAGTGATGAGTGAATGGTGAGCA AGGGCGAAGAATTGTTTACTGGGGTGGTACCCATCTTGGTCGAGCTTGACGG GGATGTAAACGGCCACAAATTTTCTGTTCGTGGCGAAGGAGAGGGCGACGCG ACGAACGGAAAGTTGACCCTGAAATTTATCTGCACCACCGGAAAGCTTCCCG TACCGTGGCCCACCTTAGTTACAACACTTACATACGGTGTGCAATGCTTCTC ACGTTATCCTGACCACATGAAGCGTCATGACTTCTTTAAATCGGCCATGCCC GAAGGTTATGTTCAAGAACGTACTATTTCGTTTAAGGACGACGGGACGTATA AAACACGCGCTGAAGTAAAATTCGAGGGTGATACTTTGGTCAATCGTATTGA ATTGAAGGGGATCGACTTTAAAGAAGACGGCAATATCTTAGGACACAAACTG GAGTATAATTTCAATTCTCACAATGTGTACATCACTGCAGACAAACAGAAAA ACGGGATTAAAGCGAACTTTAAAATCCGCCACAACGTAGAAGACGGAAGTGT
GCAGTTAGCTGATCACTATCAACAAAATACGCCCATTGGTGATGGTCCGGTT CTTCTTCCAGACAACCACTACTTGAGTACACAAAGCGTTCTGAGCAAAGACC CGAATGAAAAACGCGATCACATGGTTCTGCTGGAGTTCGTAACTGCTGCTGG GATCACACACGGCATGGATGAACTGTATAAATAA
GCAAGACTCAAAGTTCAGTTAGAACGTTATGAGCAGCTATTACCAAGTAATG PA CGATTAGTAAGCAAGAAGTAAGTAATGCTCAAGCTCAGTATCGTCAGGCTCT s fGFP AGCCGATGTCGCTCAAATGAAAGCATTGCTGGCCAGACAAAACTTGTAATCT fusion GCAATATGCAACAGTTCGAGCGCCTATTTCTGGGCGTATTGGGCAATCTTTT GTCACTGAAGGTGCATTGGTCGGTCAGGGCGATACCAATACGATGGTGAGCA AGGGCGAAGAATTGTTTACTGGGGTGGTACCCATCTTGGTCGAGCTTGACGG GGATGTAAACGGCCACAAATTTTCTGTTCGTGGCGAAGGAGAGGGCGACGCG ACGAACGGAAAGTTGACCCTGAAATTTATCTGCACCACCGGAAAGCTTCCCG TACCGTGGCCCACCTTAGTTACAACACTTACATACGGTGTGCAATGCTTCTC ACGTTATCCTGACCACATGAAGCGTCATGACTTCTTTAAATCGGCCATGCCC GAAGGTTATGTTCAAGAACGTACTATTTCGTTTAAGGACGACGGGACGTATA AAACACGCGCTGAAGTAAAATTCGAGGGTGATACTTTGGTCAATCGTATTGA ATTGAAGGGGATCGACTTTAAAGAAGACGGCAATATCTTAGGACACAAACTG GAGTATAATTTCAATTCTCACAATGTGTACATCACTGCAGACAAACAGAAAA ACGGGATTAAAGCGAACTTTAAAATCCGCCACAACGTAGAAGACGGAAGTGT GCAGTTAGCTGATCACTATCAACAAAATACGCCCATTGGTGATGGTCCGGTT CTTCTTCCAGACAACCACTACTTGAGTACACAAAGCGTTCTGAGCAAAGACC CGAATGAAAAACGCGATCACATGGTTCTGCTGGAGTTCGTAACTGCTGCTGG GATCACACACGGCATGGATGAACTGTATAAATAA
TTCACGTTTTAAATAATTTTCAATAATGTCGCCAATATCGTAGTCATCTTCT PA ACCACAAGAATAACTTTATCTTGGCAATCAAAAGAAAAAGAATGATC AACA SfGFP TAGAAAATCTGGCTATAGAAAGTGCTTCAACTCATCATACGCTAAATTATCC fusion GTATTTCTCCACACTTACTCCACACTTTAGTGATTATCCCTACACACTCATC AAAAATAATACGAACATCAAAAACTCACTAGGTTTGGACAGTATGGTGAGCA AGGGCGAAGAATTGTTTACTGGGGTGGTACCCATCTTGGTCGAGCTTGACGG GGATGTAAACGGCCACAAATTTTCTGTTCGTGGCGAAGGAGAGGGCGACGCG ACGAACGGAAAGTTGACCCTGAAATTTATCTGCACCACCGGAAAGCTTCCCG TACCGTGGCCCACCTTAGTTACAACACTTACATACGGTGTGCAATGCTTCTC ACGTTATCCTGACCACATGAAGCGTCATGACTTCTTTAAATCGGCCATGCCC GAAGGTTATGTTCAAGAACGTACTATTTCGTTTAAGGACGACGGGACGTATA AAACACGCGCTGAAGTAAAATTCGAGGGTGATACTTTGGTCAATCGTATTGA ATTGAAGGGGATCGACTTTAAAGAAGACGGCAATATCTTAGGACACAAACTG GAGTATAATTTCAATTCTCACAATGTGTACATCACTGCAGACAAACAGAAAA ACGGGATTAAAGCGAACTTTAAAATCCGCCACAACGTAGAAGACGGAAGTGT GCAGTTAGCTGATCACTATCAACAAAATACGCCCATTGGTGATGGTCCGGTT CTTCTTCCAGACAACCACTACTTGAGTACACAAAGCGTTCTGAGCAAAGACC CGAATGAAAAACGCGATCACATGGTTCTGCTGGAGTTCGTAACTGCTGCTGG GATCACACACGGCATGGATGAACTGTATAAATAA
ATACTGTGTTTGTTTACCTTTATAAAATTAATGGGACTTCATTTATACATTT PA CAAATTAGAATCGTCTTTTATTAGCACAGATTAGACCAAAATTACGATAATC sfGFP GCTGCTTATATAATCAATCTATACAATAAAAATAAAACTCCTTTCGCTTTTA fusion ACATTTTTGAGGAGCTGCTTACATTTTGGGCATAGTTCTTGCCTTATCCAAT ATACCAGTATAAGTATTTCAATCAGAACAAGAGGAACTCACTATGGTGAGCA AGGGCGAAGAATTGTTTACTGGGGTGGTACCCATCTTGGTCGAGCTTGACGG GGATGTAAACGGCCACAAATTTTCTGTTCGTGGCGAAGGAGAGGGCGACGCG ACGAACGGAAAGTTGACCCTGAAATTTATCTGCACCACCGGAAAGCTTCCCG TACCGTGGCCCACCTTAGTTACAACACTTACATACGGTGTGCAATGCTTCTC ACGTTATCCTGACCACATGAAGCGTCATGACTTCTTTAAATCGGCCATGCCC
GAAGGTTATGTTCAAGAACGTACTATTTCGTTTAAGGACGACGGGACGTATA AAACACGCGCTGAAGTAAAATTCGAGGGTGATACTTTGGTCAATCGTATTGA ATTGAAGGGGATCGACTTTAAAGAAGACGGCAATATCTTAGGACACAAACTG GAGTATAATTTCAATTCTCACAATGTGTACATCACTGCAGACAAACAGAAAA ACGGGATTAAAGCGAACTTTAAAATCCGCCACAACGTAGAAGACGGAAGTGT GCAGTTAGCTGATCACTATCAACAAAATACGCCCATTGGTGATGGTCCGGTT CTTCTTCCAGACAACCACTACTTGAGTACACAAAGCGTTCTGAGCAAAGACC CGAATGAAAAACGCGATCACATGGTTCTGCTGGAGTTCGTAACTGCTGCTGG GATCACACACGGCATGGATGAACTGTATAAATAA
TATATCCAAAAAGATATTTAAATATATTTTTAGATATCTTTTAAATATTAAA P ATTTCCAACAGAAATTAGTTACACAGAAAGAATAATTATTTTTATATATTAT sfGFP TTTCAATAACTTATATTTTAAATTAAGTGTATTTTTAAAAATTGGCACACTC fusion AGTGCAATAGTACAAAGCACACAACATCATTCGGATATTGTGAATAACACCT GCCTGCAAACGCTTGGCAGGCAGGCTTTGAAAGGAAAAGAAAATGGTGAGCA AGGGCGAAGAATTGTTTACTGGGGTGGTACCCATCTTGGTCGAGCTTGACGG GGATGTAAACGGCCACAAATTTTCTGTTCGTGGCGAAGGAGAGGGCGACGCG ACGAACGGAAAGTTGACCCTGAAATTTATCTGCACCACCGGAAAGCTTCCCG TACCGTGGCCCACCTTAGTTACAACACTTACATACGGTGTGCAATGCTTCTC ACGTTATCCTGACCACATGAAGCGTCATGACTTCTTTAAATCGGCCATGCCC GAAGGTTATGTTCAAGAACGTACTATTTCGTTTAAGGACGACGGGACGTATA AAACACGCGCTGAAGTAAAATTCGAGGGTGATACTTTGGTCAATCGTATTGA ATTGAAGGGGATCGACTTTAAAGAAGACGGCAATATCTTAGGACACAAACTG GAGTATAATTTCAATTCTCACAATGTGTACATCACTGCAGACAAACAGAAAA ACGGGATTAAAGCGAACTTTAAAATCCGCCACAACGTAGAAGACGGAAGTGT GCAGTTAGCTGATCACTATCAACAAAATACGCCCATTGGTGATGGTCCGGTT CTTCTTCCAGACAACCACTACTTGAGTACACAAAGCGTTCTGAGCAAAGACC CGAATGAAAAACGCGATCACATGGTTCTGCTGGAGTTCGTAACTGCTGCTGG GATCACACACGGCATGGATGAACTGTATAAATAA
GAAACAATGGAGAGTTTTTCTTTTCGTTGTTGGTTACAATGCAAAATATAGA P GTAAAAATGAGAGAGTGCTTTTGAACGTTCTACGTGTTCCCATAGCCTTGCG sfGFP GTTATTTTTAACGGTCTTGCTTACCACGCTGTTAATTGCAACTGCAAGTTTG fusion AGTGTTCTGCACTGGACGATGCAAAAGAACTTTGCCAAATATGTAGCCGATG TTGAAATGCAAAAGCTAGATCGGCTCATTACTAATCTGGGAAATGGTGAGCA AGGGCGAAGAATTGTTTACTGGGGTGGTACCCATCTTGGTCGAGCTTGACGG GGATGTAAACGGCCACAAATTTTCTGTTCGTGGCGAAGGAGAGGGCGACGCG ACGAACGGAAAGTTGACCCTGAAATTTATCTGCACCACCGGAAAGCTTCCCG TACCGTGGCCCACCTTAGTTACAACACTTACATACGGTGTGCAATGCTTCTC ACGTTATCCTGACCACATGAAGCGTCATGACTTCTTTAAATCGGCCATGCCC GAAGGTTATGTTCAAGAACGTACTATTTCGTTTAAGGACGACGGGACGTATA AAACACGCGCTGAAGTAAAATTCGAGGGTGATACTTTGGTCAATCGTATTGA ATTGAAGGGGATCGACTTTAAAGAAGACGGCAATATCTTAGGACACAAACTG GAGTATAATTTCAATTCTCACAATGTGTACATCACTGCAGACAAACAGAAAA ACGGGATTAAAGCGAACTTTAAAATCCGCCACAACGTAGAAGACGGAAGTGT GCAGTTAGCTGATCACTATCAACAAAATACGCCCATTGGTGATGGTCCGGTT CTTCTTCCAGACAACCACTACTTGAGTACACAAAGCGTTCTGAGCAAAGACC CGAATGAAAAACGCGATCACATGGTTCTGCTGGAGTTCGTAACTGCTGCTGG GATCACACACGGCATGGATGAACTGTATAAATAA
CACTCAAACTTGCAGTTGCAATTAACAGCGTGGTAAGCAAGACCGTTAAAAA P TAACCGCAAGGCTATGGGAACACGTAGAACGTTCAAAAGCACTCTCTCATTT sfGFP TTACTCTATATTTTGCATTGTAACCAACAACGAAAAGAAAAACTCTCCATTG fusion TTTCTTCATCTTTATTATCTAATCTTTAAGTCATCTTAAAACAAACATTAAA GGGTTTTAACCATGAAAATGACGGCTAAAATTGCATTATTCAGTATGGTGAG
CAAGGGCGAAGAATTGTTTACTGGGGTGGTACCCATCTTGGTCGAGCTTGAC GGGGATGTAAACGGCCACAAATTTTCTGTTCGTGGCGAAGGAGAGGGCGACG CGACGAACGGAAAGTTGACCCTGAAATTTATCTGCACCACCGGAAAGCTTCC CGTACCGTGGCCCACCTTAGTTACAACACTTACATACGGTGTGCAATGCTTC TCACGTTATCCTGACCACATGAAGCGTCATGACTTCTTTAAATCGGCCATGC CCGAAGGTTATGTTCAAGAACGTACTATTTCGTTTAAGGACGACGGGACGTA TAAAACACGCGCTGAAGTAAAATTCGAGGGTGATACTTTGGTCAATCGTATT GAATTGAAGGGGATCGACTTTAAAGAAGACGGCAATATCTTAGGACACAAAC TGGAGTATAATTTCAATTCTCACAATGTGTACATCACTGCAGACAAACAGAA AAACGGGATTAAAGCGAACTTTAAAATCCGCCACAACGTAGAAGACGGAAGT GTGCAGTTAGCTGATCACTATCAACAAAATACGCCCATTGGTGATGGTCCGG TTCTTCTTCCAGACAACCACTACTTGAGTACACAAAGCGTTCTGAGCAAAGA CCCGAATGAAAAACGCGATCACATGGTTCTGCTGGAGTTCGTAACTGCTGCT GGGATCACACACGGCATGGATGAACTGTATAAATAA
CTTGTACACTTGGAACTTTGCAGAAAAATTGATGAAGACGTTCAAAACTGAT PA TTCAGACACAGCAATTGACCTAGCTTGAAAAATGCTAATGGTAGCACAGATA sfGFP CAGATTCTTCATATTTTCTTTATAGTCATTACAAACAATATCAATCAAAGCC fusion ATTTTATTCATGGTTTCTCCTTGACTCTCTCTTAATCTAGCTTCAGAAGAAG GAGATATACATATGGTGAGCAAGGGCGAAGAATTGTTTACTGGGGTGGTACC CATCTTGGTCGAGCTTGACGGGGATGTAAACGGCCACAAATTTTCTGTTCGT GGCGAAGGAGAGGGCGACGCGACGAACGGAAAGTTGACCCTGAAATTTATCT GCACCACCGGAAAGCTTCCCGTACCGTGGCCCACCTTAGTTACAACACTTAC ATACGGTGTGCAATGCTTCTCACGTTATCCTGACCACATGAAGCGTCATGAC TTCTTTAAATCGGCCATGCCCGAAGGTTATGTTCAAGAACGTACTATTTCGT TTAAGGACGACGGGACGTATAAAACACGCGCTGAAGTAAAATTCGAGGGTGA TACTTTGGTCAATCGTATTGAATTGAAGGGGATCGACTTTAAAGAAGACGGC AATATCTTAGGACACAAACTGGAGTATAATTTCAATTCTCACAATGTGTACA TCACTGCAGACAAACAGAAAAACGGGATTAAAGCGAACTTTAAAATCCGCCA CAACGTAGAAGACGGAAGTGTGCAGTTAGCTGATCACTATCAACAAAATACG CCCATTGGTGATGGTCCGGTTCTTCTTCCAGACAACCACTACTTGAGTACAC AAAGCGTTCTGAGCAAAGACCCGAATGAAAAACGCGATCACATGGTTCTGCT GGAGT CGTAACTGCTGCTGGGATCACACACGGCATGGATGAACTGTA AAA TAA
ATAGGCTTTTTCATGGAGTATTCCTTTTTATCTTGATCTGAAGCTAGATTAA PA GAGAGAGTCAAGGAGAAACCATGAATAAAATGGCTTTGATTGATATTGTTTG sfGFP TAATGACTATAAAGAAAATATGAAGAATCTGTATCTGTGCTACCATTAGCAT fusion TTTTCAAGCTAGGTCAATTGCTGTGTCTGAAATCAGTTTTGAACGTCTTCAT CAATTTTTCTGCAAAGTTCCAAGTGTACAAGAAGCCCGTATCATGGTGAGCA AGGGCGAAGAATTGTTTACTGGGGTGGTACCCATCTTGGTCGAGCTTGACGG GGATGTAAACGGCCACAAATTTTCTGTTCGTGGCGAAGGAGAGGGCGACGCG ACGAACGGAAAGTTGACCCTGAAATTTATCTGCACCACCGGAAAGCTTCCCG TACCGTGGCCCACCTTAGTTACAACACTTACATACGGTGTGCAATGCTTCTC ACGTTATCCTGACCACATGAAGCGTCATGACTTCTTTAAATCGGCCATGCCC GAAGGTTATGTTCAAGAACGTACTATTTCGTTTAAGGACGACGGGACGTATA AAACACGCGCTGAAGTAAAATTCGAGGGTGATACTTTGGTCAATCGTATTGA ATTGAAGGGGATCGACTTTAAAGAAGACGGCAATATCTTAGGACACAAACTG GAGTATAATTTCAATTCTCACAATGTGTACATCACTGCAGACAAACAGAAAA ACGGGATTAAAGCGAACTTTAAAATCCGCCACAACGTAGAAGACGGAAGTGT GCAGTTAGCTGATCACTATCAACAAAATACGCCCATTGGTGATGGTCCGGTT CTTCTTCCAGACAACCACTACTTGAGTACACAAAGCGTTCTGAGCAAAGACC CGAATGAAAAACGCGATCACATGGTTCTGCTGGAGTTCGTAACTGCTGCTGG GATCACACACGGCATGGATGAACTGTATAAATAA
ATATAGGCATGTATCACATGAACAACTATAGAAAAATTTAGGAGATGAAGGT P TAAAAAGCTGTAAGATGAAAATAGGACCAGCTTAATAAGAATAACTAAAAAA sfGFP TAAATGTTCTTTTTCACTTTATTATTAATTTTTATTAATCTTCACAATTTAT fusion CCCTAGTCTGTTTACTCAATGTAACACTGTTGTAACGTTTTGATGAAGTGAG TTCCCAAGCTCATATTTCCTTAGCTATGATGCGATTGCGTTTATGGTGAGCA AGGGCGAAGAATTGTTTACTGGGGTGGTACCCATCTTGGTCGAGCTTGACGG GGATGTAAACGGCCACAAATTTTCTGTTCGTGGCGAAGGAGAGGGCGACGCG ACGAACGGAAAGTTGACCCTGAAATTTATCTGCACCACCGGAAAGCTTCCCG TACCGTGGCCCACCTTAGTTACAACACTTACATACGGTGTGCAATGCTTCTC ACGTTATCCTGACCACATGAAGCGTCATGACTTCTTTAAATCGGCCATGCCC GAAGGTTATGTTCAAGAACGTACTATTTCGTTTAAGGACGACGGGACGTATA AAACACGCGCTGAAGTAAAATTCGAGGGTGATACTTTGGTCAATCGTATTGA ATTGAAGGGGATCGACTTTAAAGAAGACGGCAATATCTTAGGACACAAACTG GAGTATAATTTCAATTCTCACAATGTGTACATCACTGCAGACAAACAGAAAA ACGGGATTAAAGCGAACTTTAAAATCCGCCACAACGTAGAAGACGGAAGTGT GCAGTTAGCTGATCACTATCAACAAAATACGCCCATTGGTGATGGTCCGGTT CTTCTTCCAGACAACCACTACTTGAGTACACAAAGCGTTCTGAGCAAAGACC CGAATGAAAAACGCGATCACATGGTTCTGCTGGAGTTCGTAACTGCTGCTGG GATCACACACGGCATGGATGAACTGTATAAATAA
CCAGCCGCCCGCATTGGAGAAATTCTTCAAATTCCCGTTGCACATAGCCCGG Full length CAATTCCTTTCCCTGCTCTGCCATAAGCGCAGCGAATGCCGGGTAATACTCG pACH106 TCAACGATCTGATAGAGAAGGGTTTGCTCGGGTCGGTGGCTCTGGTAACGAC CAGTATCCCGATCCCGGCTGGCCGTCCTGGCCGCCACATGAGGCATGTTCCG CGTCCTTGCAATACTGTGTTTACATACAGTCTATCGCTTAGCGGAAAGTTCT TTTACCCTCAGCCGAAATGCCTGCCGTTGCTAGACATTGCCAGCCAGTGCCC GTCACTCCCGTACTAACTGTCACGAACCCCTGCAATAACTGTCACGCCCCCC TGCAATAACTGTCACGAACCCCTGCAATAACTGTCACGCCCCCAAACCTGCA AACCCAGCAGGGGCGGGGGCTGGCGGGGTGTTGGAAAAATCCATCCATGATT ATCTAAGAATAATCCACTAGGCGCGGTTATCAGCGCCCTTGTGGGGCGCTGC TGCCCTTGCCCAATATGCCCGGCCAGAGGCCGGATAGCTGGTCTATTCGCTG CGCTAGGCTACACACCGCCCCACCGCTGCGCGGCAGGGGGAAAGGCGGGCAA AGCCCGCTAAACCCCACACCAAACCCCGCAGAAATACGCTGGAGCGCTTTTA GCCGCTTTAGCGGCCTTTCCCCCTACCCGAAGGGTGGGGGCGCGTGTGCAGC CCCGCAGGGCCTGTCTCGGTCGATCATTCAGCCCGGCTCATCCTTCTGGCGT GGCGGCAGACCGAACAAGGCGCGGTCGTGGTCGCGTTCAAGGTACGCATCCA TTGCCGCCATGAGCCGATCCTCCGGCCACTCGCTGCTGTTCACCTTGGCCAA AATCATGGCCCCCACCAGCACCTTGCGCCTTGTTTCGTTCTTGCGCTCTTGC TGCTGTTCCCTTGCCCGCTCCCGCTGAATTTCGGCATTGATTCGCGCTCGTT GTTCTTCGAGCTTGGCCAGCCGATCCGCCGCCTTGTTGCTCCCCTTAACCAT CTTGACACCCCATTGTTAATGTGCTGTCTCGTAGGCTATCATGGAGGCACAG CGGCGGCAATCCCGACCCTACTTTGTAGGGGAGGGCGCACTTACCGGTTTCT CTTCGAGAAACTGGCCTAACGGCCACCCTTCGGGCGGTGCGCTCTCCGAGGG CCATTGCATGGAGCCGAAAAGCAAAAGCAACAGCGAGGCAGCATGGCGATTT ATCACCTTACGGCGAAAACCGGCAGCAGGTCGGGCGGCCAATCGGCCAGGGC CAAGGCCGACTACATCCAGCGCGAAGGCAAGTATGCCCGCGACATGGATGAA GTCTTGCACGCCGAATCCGGGCACATGCCGGAGTTCGTCGAGCGGCCCGCCG ACTACTGGGATGCTGCCGACCTGTATGAACGCGCCAATGGGCGGCTGTTCAA GGAGGTCGAATTTGCCCTGCCGGTCGAGCTGACCCTCGACCAGCAGAAGGCG CTGGCGTCCGAGTTCGCCCAGCACCTGACCGGTGCCGAGCGCCTGCCGTATA CGCTGGCCATCCATGCCGGTGGCGGCGAGAACCCGCACTGCCACCTGATGAT CTCCGAGCGGATCAATGACGGCATCGAGCGGCCCGCCGCTCAGTGGTTCAAG CGGTACAACGGCAAGACCCCGGAGAAGGGCGGGGCACAGAAGACCGAAGCGC TCAAGCCCAAGGCATGGCTTGAGCAGACCCGCGAGGCATGGGCCGACCATGC CAACCGGGCATTAGAGCGGGCTGGCCACGACGCCCGCATTGACCACAGAACA CTTGAGGCGCAGGGCATCGAGCGCCTGCCCGGTGTTCACCTGGGGCCGAACG TGGTGGAGATGGAAGGCCGGGGCATCCGCACCGACCGGGCAGACGTGGCCCT GAACATCGACACCGCCAACGCCCAGATCATCGACTTACAGGAATACCGGGAG GC AATAGACC AT GAACGC AATC GAC AGAGT GAAGAAAT CC AGAGGC AT C AAC GAGTTAGCGGAGCAGATCGAACCGCTGGCCCAGAGCATGGCGACACTGGCCG ACGAAGCCCGGCAGGTCATGAGCCAGACCAAGCAGGCCAGCGAGGCGCAGGC GGCGGAGTGGCTGAAAGCCCAGCGCCAGACAGGGGCGGCATGGGTGGAGCTG GCCAAAGAGTTGCGGGAGGTAGCCGCCGAGGTGAGCAGCGCCGCGCAGAGCG CCCGGAGCGCGTCGCGGGGGTGGCACTGGAAGCTATGGCTAACCGTGATGCT GGCTTCCATGATGCCTACGGTGGTGCTGCTGATCGCATCGTTGCTCTTGCTC GACCTGACGCCACTGACAACCGAGGACGGCTCGATCTGGCTGCGCTTGGTGG CCCGATGAAGAACGACAGGACTTTGCAGGCCATAGGCCGACAGCTCAAGGCC ATGGGCTGTGAGCGCTTCGATATCGGCGTCAGGGACGCACCCACCGGCCAGA TGATGAACCGGGAATGGTCAGCCGCCGAAGTGCTCCAGAACACGCCATGGCT CAAGCGGATGAATGCCCAGGGCAATGACGTGTATATCAGGCCCGCCGAGCAG GAGCGGCATGGTCTGGTGCTGGTGGACGACCTCAGCGAGTTTGACCTGGATG ACATGAAAGCCGAGGGCCGGGAGCCTGCCCTGGTAGTGGAAACCAGCCCGAA GAACTATCAGGCATGGGTCAAGGTGGCCGACGCCGCAGGCGGTGAACTTCGG GGGCAGATTGCCCGGACGCTGGCCAGCGAGTACGACGCCGACCCGGCCAGCG CCGACAGCCGCCACTATGGCCGCTTGGCGGGCTTCACCAACCGCAAGGACAA GCACACCACCCGCGCCGGTTATCAGCCGTGGGTGCTGCTGCGTGAATCCAAG GGCAAGACCGCCACCGCTGGCCCGGCGCTGGTGCAGCAGGCTGGCCAGCAGA TCGAGCAGGCCCAGCGGCAGCAGGAGAAGGCCCGCAGGCTGGCCAGCCTCGA ACTGCCCGAGCGGCAGCTTAGCCGCCACCGGCGCACGGCGCTGGACGAGTAC CGCAGCGAGATGGCCGGGCTGGTCAAGCGCTTCGGTCATGACCTCAGCAAGT GCGACTTTATCGCCGCGCAGAAGCTGGCCAGCCGGGGCCGCAGTGCCGAGGA AATCGGCAAGGCCATGGCCGAGGCCAGCCCAGCGCTGGCAGAGCGCAAGCCC GGCCACGAAGCGGATTACATCGAGCGCACCGTCAGCAAGGTCATGGGTCTGC CCAGCGTCCAGCTTGCGCGGGCCGAGCTGGCACGGGCACCGGCACCCCGCCA GCGAGGCATGGACAGGGGCGGGCCAGATTTCAGCATGTAGTGCTTGCGTTGG TACTCACGCCTGTTATACTATGAGTACTCACGCACAGAAGGGGGTTTTATGG AATACGAAAAAAGCGCTTCAGGGTCGGTCTACCTGATCAAAAGTGACAAGGG CTATTGGTTGCCCGGTGGCTTTGGTTATACGTCAAACAAGGCCGAGGCTGGC CGCTTTTCAGTCGCTGATATGGCCAGCCTTAACCTTGACGGCTGCACCTTGT CCTTGTTCCGCGAAGACAAGCCTTTCGGCCCCGGCAAGTTTCTCGGTGACTG ATATGAAAGACCAAAAGGACAAGCAGACCGGCGACCTGCTGGCCAGCCCTGA CGCTGTACGCCAAGCGCGATATGCCGAGCGCATGAAGGCCAAAGGGATGCGT CAGCGCAAGTTCTGGCTGACCGAC GACGAATACGAGGCGCTGCGCGAGTGCC TGGAAGAACTCAGAGCGGCGCAGGGCGGGGGTAGTGACCCCGCCAGCGCCTA ACCACCAACTGCCTGCAAAGGAGGCAATCAATGGCTACCCATAAGCCTATCA ATATTCTGGAGGCGTTCGCAGCAGCGCCGCCACCGCTGGACTACGTTTTGCC CAACATGGTGGCCGGTACGGTCGGGGCGCTGGTGTCGCCCGGTGGTGCCGGT AAATCCATGCTGGCCCTGCAACTGGCCGCACAGATTGCAGGCGGGCCGGATC TGCTGGAGGTGGGCGAACTGCCCACCGGCCCGGTGATCTACCTGCCCGCCGA AGACCCGCCCACCGCCATTCATCACCGCCTGCACGCCCTTGGGGCGCACCTC AGCGCCGAGGAACGGCAAGCCGTGGCTGACGGCCTGCTGATCCAGCCGCTGA TCGGCAGCCTGCCCAACATCATGGCCCCGGAGTGGTTCGACGGCCTCAAGCG CGCCGCCGAGGGCCGCCGCCTGATGGTGCTGGACACGCTGCGCCGGTTCCAC ATCGAGGAAGAAAACGCCAGCGGCCCCATGGCCCAGGTCATCGGTCGCATGG AGGCCATCGCCGCCGATACCGGGTGCTCTATCGTGTTCCTGCACCATGCCAG CAAGGGCGCGGCCATGATGGGCGCAGGCGACCAGCAGCAGGCCAGCCGGGGC AGCTCGGTACTGGTCGATAACATCCGCTGGCAGTCCTACCTGTCGAGCATGA CCAGCGCCGAGGCCGAGGAATGGGGTGTGGAC GACGACCAGCGCCGGTTCTT CGTCCGCTTCGGTGTGAGCAAGGCCAACTATGGCGCACCGTTCGCTGATCGG TGGTTCAGGCGGCATGACGGCGGGGTGCTCAAGCCCGCCGTGCTGGAGAGGC AGCGCAAGAGCAAGGGGGTGCCCCGTGGTGAAGCCTAAGAACAAGCACAGCC TCAGCCACGTCCGGCACGACCCGGCGCACTGTCTGGCCCCCGGCCTGTTCCG TGCCCTCAAGCGGGGCGAGCGCAAGCGCAGCAAGCTGGACGTGACGTATGAC TACGGCGACGGCAAGCGGATCGAGTTCAGCGGCCCGGAGCCGCTGGGCGCTG ATGATCTGCGCATCCTGCAAGGGCTGGTGGCCATGGCTGGGCCTAATGGCCT AGTGCTTGGCCCGGAACCCAAGACCGAAGGCGGACGGCAGCTCCGGCTGTTC CTGGAACCCAAGTGGGAGGCCGTCACCGCTGAATGCCATGTGGTCAAAGGTA GCTATCGGGCGCTGGCAAAGGAAATCGGGGCAGAGGTCGATAGTGGTGGGGC GCTCAAGCACATACAGGACTGCATCGAGCGCCTTTGGAAGGTATCCATCATC GCCCAGAATGGCCGCAAGCGGCAGGGGTTTCGGCTGCTGTCGGAGTACGCCA GCGACGAGGCGGACGGGCGCCTGTACGTGGCCCTGAACCCCTTGATCGCGCA GGCCGTCATGGGTGGCGGCCAGCATGTGCGCATCAGCATGGACGAGGTGCGG GCGCTGGACAGCGAAACCGCCCGCCTGCTGCACCAGCGGCTGTGTGGCTGGA TCGACCCCGGCAAAACCGGCAAGGCTTCCATAGATACCTTGTGCGGCTATGT CTGGCCGTCAGAGGCCAGTGGTTCGACCATGCGCAAGCGCCGCAAGCGGGTG CGCGAGGCGTTGCCGGAGCTGGTCGCGCTGGGCTGGACGGTAACCGAGTTCG CGGCGGGCAAGTACGACATCACCCGGCCCAAGGCGGCAGGCTGACCCCCCCC ACTCTATTGTAAACAAGACATTTTTATCTTTTATATTCAATGGCTTATTTTC CTGCTAATTGGTAATACCATGAAAAATACCATGCTCAGAAAAGGCTTAACAA TATTTTGAAAAATTGCCTACTGAGCGCTGCCGCACAGCTCCATAGGCCGCTT TCCTGGCTTTGCTTCCAGATGTATGCTCTTCTGCTCCCGAACGCCAGCAAGA CGTAGCCCAGCGCGTCGGCCAGCTTGCAATTCGCGCTAACTTACATTAATTG CGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCA TTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAG GGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACC GCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGTGGTTTGCCCCAGCAG GCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCT TCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGG ACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAG CATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAA CCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGAT TGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGA ACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGA TGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGA TGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGC AGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGC CCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGA CGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGC GCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTG GAGGTGGCAACGCCAATCAGCAAC GACTGTTTGCCCGCCAGTTGTTGTGCCA CGCGGTTGGGAATGTAATTCAGCTCCGC CATC GCCGCTTCC ACT TTTTCCCG CGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGA TAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACAT TCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAA GGTTTTGCACCATTCGATGGTGTCAACGTAAATGCCGCTTCGCCTTCGCGCG CGAATTGCAAGCTGATCCGGGCTTATCGACTGCACGGTGCACCAATGCTTCT GGCGTCAGGCAGCCATCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAATCA
CTGCATAATTCGTGTCGCTCAAGGCGCACTCCCGTTCTGGATAATGTTTTTT GCGCCGACATCATAACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAA TTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCAC ACAGGAAACAGAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTG CAGGCATGCAAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGA TACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGG CGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAA CGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACT GCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTT TTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGG AGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGC CCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATG GCCTTTTTGCGTTTCTACAAACTCTTTTGTTTATTTTTCTAAATACATTCAA ATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATATTG AAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTT TTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAG TAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGA TCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCA ATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTG ACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTT GGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTA AGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACT TACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAA CATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAA GCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAA CGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACA ATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCG GCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTG GGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTAT CGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGA CAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACC AAGTTTACTCATATATACTTTAGATTGATTTCTGAAAGCGACCAGGTGCTCG GCGTGGCAAGACTCGCAGCGAACCCGTAGAAAGCCATGCT
CCAGCCGCCCGCATTGGAGAAATTCTTCAAATTCCCGTTGCACATAGCCCGG P CAATTCCTTTCCCTGCTCTGCCATAAGCGCAGCGAATGCCGGGTAATACTCG sfGFP
TCAACGATCTGATAGAGAAGGGTTTGCTCGGGTCGGTGGCTCTGGTAACGAC (pACH106 : : PA CAGTATCCCGATCCCGGCTGGCCGTCCTGGCCGCCACATGAGGCATGTTCCG -SfGFP) CGTCCTTGCAATACTGTGTTTACATACAGTCTATCGCTTAGCGGAAAGTTCT TTTACCCTCAGCCGAAATGCCTGCCGTTGCTAGACATTGCCAGCCAGTGCCC GTCACTCCCGTACTAACTGTCACGAACCCCTGCAATAACTGTCACGCCCCCC TGCAATAACTGTCACGAACCCCTGCAATAACTGTCACGCCCCCAAACCTGCA AACCCAGCAGGGGCGGGGGCTGGCGGGGTGTTGGAAAAATCCATCC TGATT ATCTAAGAATAATCCACTAGGCGCGGTTATCAGCGCCCTTGTGGGGCGCTGC TGCCCTTGCCCAATATGCCCGGCCAGAGGCCGGATAGCTGGTCTATTCGCTG CGCTAGGCTACACACCGCCCCACCGCTGCGCGGCAGGGGGAAAGGCGGGCAA AGCCCGCTAAACCCCACACCAAACCCCGCAGAAATACGCTGGAGCGCTTTTA GCCGCTTTAGCGGCCTTTCCCCCTACCCGAAGGGTGGGGGCGCGTGTGCAGC CCCGCAGGGCCTGTCTCGGTCGATCATTCAGCCCGGCTCATCCTTCTGGCGT GGCGGCAGACCGAACAAGGCGCGGTCGTGGTCGCGTTCAAGGTACGCATCCA TTGCCGCCATGAGCCGATCCTCCGGCCACTCGCTGCTGTTCACCTTGGCCAA AATCATGGCCCCCACCAGCACCTTGCGCCTTGTTTCGTTCTTGCGCTCTTGC TGCTGTTCCCTTGCCCGCTCCCGCTGAATTTCGGCATTGATTCGCGCTCGTT GTTCTTCGAGCTTGGCCAGCCGATCCGCCGCCTTGTTGCTCCCCTTAACCAT CTTGACACCCCATTGTTAATGTGCTGTCTCGTAGGCTATCATGGAGGCACAG CGGCGGCAATCCCGACCCTACTTTGTAGGGGAGGGCGCACTTACCGGTTTCT CTTCGAGAAACTGGCCTAACGGCCACCCTTCGGGCGGTGCGCTCTCCGAGGG CCATTGCATGGAGCCGAAAAGCAAAAGCAACAGCGAGGCAGCATGGCGATTT ATCACCTTACGGCGAAAACCGGCAGCAGGTCGGGCGGCCAATCGGCCAGGGC CAAGGCCGACTACATCCAGCGCGAAGGCAAGTATGCCCGCGACATGGATGAA GTCTTGCACGCCGAATCCGGGCACATGCCGGAGTTCGTCGAGCGGCCCGCCG ACTACTGGGATGCTGCCGACCTGTATGAACGCGCCAATGGGCGGCTGTTCAA GGAGGTCGAATTTGCCCTGCCGGTCGAGCTGACCCTCGACCAGCAGAAGGCG CTGGCGTCCGAGTTCGCCCAGCACCTGACCGGTGCCGAGCGCCTGCCGTATA CGCTGGCCATCCATGCCGGTGGCGGCGAGAACCCGCACTGCCACCTGATGAT CTCCGAGCGGATCAATGACGGCATCGAGCGGCCCGCCGCTCAGTGGTTCAAG CGGTACAACGGCAAGACCCCGGAGAAGGGCGGGGCACAGAAGACCGAAGCGC TCAAGCCCAAGGCATGGCTTGAGCAGACCCGCGAGGCATGGGCCGACCATGC CAACCGGGCATTAGAGCGGGCTGGCCACGACGCCCGCATTGACCACAGAACA CTTGAGGCGCAGGGCATCGAGCGCCTGCCCGGTGTTCACCTGGGGCCGAACG TGGTGGAGATGGAAGGCCGGGGCATCCGCACCGACCGGGCAGACGTGGCCCT GAACATCGACACCGCCAACGCCCAGATCATCGACTTACAGGAATACCGGGAG GC AATAGACC AT GAACGC AATC GAC AGAGT GAAGAAAT CC AGAGGC AT C AAC GAGTTAGCGGAGCAGATCGAACCGCTGGCCCAGAGCATGGCGACACTGGCCG ACGAAGCCCGGCAGGTCATGAGCCAGACCAAGCAGGCCAGCGAGGCGCAGGC GGCGGAGTGGCTGAAAGCCCAGCGCCAGACAGGGGCGGCATGGGTGGAGCTG GCCAAAGAGTTGCGGGAGGTAGCCGCCGAGGTGAGCAGCGCCGCGCAGAGCG CCCGGAGCGCGTCGCGGGGGTGGCACTGGAAGCTATGGCTAACCGTGATGCT GGCTTCCATGATGCCTACGGTGGTGCTGCTGATCGCATCGTTGCTCTTGCTC GACCTGACGCCACTGACAACCGAGGACGGCTCGATCTGGCTGCGCTTGGTGG CCCGATGAAGAACGACAGGACTTTGCAGGCCATAGGCCGACAGCTCAAGGCC ATGGGCTGTGAGCGCTTCGATATCGGCGTCAGGGACGCACCCACCGGCCAGA TGATGAACCGGGAATGGTCAGCCGCCGAAGTGCTCCAGAACACGCCATGGCT CAAGCGGATGAATGCCCAGGGCAATGACGTGTATATCAGGCCCGCCGAGCAG GAGCGGCATGGTCTGGTGCTGGTGGACGACCTCAGCGAGTTTGACCTGGATG ACATGAAAGCCGAGGGCCGGGAGCCTGCCCTGGTAGTGGAAACCAGCCCGAA GAACTATCAGGCATGGGTCAAGGTGGCCGACGCCGCAGGCGGTGAACTTCGG GGGCAGATTGCCCGGACGCTGGCCAGCGAGTACGACGCCGACCCGGCCAGCG CCGACAGCCGCCACTATGGCCGCTTGGCGGGCTTCACCAACCGCAAGGACAA GCACACCACCCGCGCCGGTTATCAGCCGTGGGTGCTGCTGCGTGAATCCAAG GGCAAGACCGCCACCGCTGGCCCGGCGCTGGTGCAGCAGGCTGGCCAGCAGA TCGAGCAGGCCCAGCGGCAGCAGGAGAAGGCCCGCAGGCTGGCCAGCCTCGA ACTGCCCGAGCGGCAGCTTAGCCGCCACCGGCGCACGGCGCTGGACGAGTAC CGCAGCGAGATGGCCGGGCTGGTCAAGCGCTTCGGTCATGACCTCAGCAAGT GCGACTTTATCGCCGCGCAGAAGCTGGCCAGCCGGGGCCGCAGTGCCGAGGA AATCGGCAAGGCCATGGCCGAGGCCAGCCCAGCGCTGGCAGAGCGCAAGCCC GGCCACGAAGCGGATTACATCGAGCGCACCGTCAGCAAGGTCATGGGTCTGC CCAGCGTCCAGCTTGCGCGGGCCGAGCTGGCACGGGCACCGGCACCCCGCCA GCGAGGCATGGACAGGGGCGGGCCAGATTTCAGCATGTAGTGCTTGCGTTGG TACTCACGCCTGTTATACTATGAGTACTCACGCACAGAAGGGGGTTTTATGG AATACGAAAAAAGCGCTTCAGGGTCGGTCTACCTGATCAAAAGTGACAAGGG CTATTGGTTGCCCGGTGGCTTTGGTTATACGTCAAACAAGGCCGAGGCTGGC CGCTTTTCAGTCGCTGATATGGCCAGCCTTAACCTTGACGGCTGCACCTTGT CCTTGTTCCGCGAAGACAAGCCTTTCGGCCCCGGCAAGTTTCTCGGTGACTG ATATGAAAGACCAAAAGGACAAGCAGACCGGCGACCTGCTGGCCAGCCCTGA CGCTGTACGCCAAGCGCGATATGCCGAGCGCATGAAGGCCAAAGGGATGCGT CAGCGCAAGTTCTGGCTGACCGAC GACGAATACGAGGCGCTGCGCGAGTGCC TGGAAGAACTCAGAGCGGCGCAGGGCGGGGGTAGTGACCCCGCCAGCGCCTA ACCACCAACTGCCTGCAAAGGAGGCAATCAATGGCTACCCATAAGCCTATCA ATATTCTGGAGGCGTTCGCAGCAGCGCCGCCACCGCTGGACTACGTTTTGCC CAACATGGTGGCCGGTACGGTCGGGGCGCTGGTGTCGCCCGGTGGTGCCGGT AAATCCATGCTGGCCCTGCAACTGGCCGCACAGATTGCAGGCGGGCCGGATC TGCTGGAGGTGGGCGAACTGCCCACCGGCCCGGTGATCTACCTGCCCGCCGA AGACCCGCCCACCGCCATTCATCACCGCCTGCACGCCCTTGGGGCGCACCTC AGCGCCGAGGAACGGCAAGCCGTGGCTGACGGCCTGCTGATCCAGCCGCTGA TCGGCAGCCTGCCCAACATCATGGCCCCGGAGTGGTTCGACGGCCTCAAGCG CGCCGCCGAGGGCCGCCGCCTGATGGTGCTGGACACGCTGCGCCGGTTCCAC ATCGAGGAAGAAAACGCCAGCGGCCCCATGGCCCAGGTCATCGGTCGCATGG AGGCCATCGCCGCCGATACCGGGTGCTCTATCGTGTTCCTGCACCATGCCAG CAAGGGCGCGGCCATGATGGGCGCAGGCGACCAGCAGCAGGCCAGCCGGGGC AGCTCGGTACTGGTCGATAACATCCGCTGGCAGTCCTACCTGTCGAGCATGA CCAGCGCCGAGGCCGAGGAATGGGGTGTGGAC GACGACCAGCGCCGG CTT CGTCCGCTTCGGTGTGAGCAAGGCCAACTATGGCGCACCGTTCGCTGATCGG TGGTTCAGGCGGCATGACGGCGGGGTGCTCAAGCCCGCCGTGCTGGAGAGGC AGCGCAAGAGCAAGGGGGTGCCCCGTGGTGAAGCCTAAGAACAAGCACAGCC TCAGCCACGTCCGGCACGACCCGGCGCACTGTCTGGCCCCCGGCCTGTTCCG TGCCCTCAAGCGGGGCGAGCGCAAGCGCAGCAAGCTGGACGTGACGTATGAC TACGGCGACGGCAAGCGGATCGAGTTCAGCGGCCCGGAGCCGCTGGGCGCTG ATGATCTGCGCATCCTGCAAGGGCTGGTGGCCATGGCTGGGCCTAATGGCCT AGTGCTTGGCCCGGAACCCAAGACCGAAGGCGGACGGCAGCTCCGGCTGTTC CTGGAACCCAAGTGGGAGGCCGTCACCGCTGAATGCCATGTGGTCAAAGGTA GCTATCGGGCGCTGGCAAAGGAAATCGGGGCAGAGGTCGATAGTGGTGGGGC GCTCAAGCACATACAGGACTGCATCGAGCGCCTTTGGAAGGTATCCATCATC GCCCAGAATGGCCGCAAGCGGCAGGGGTTTCGGCTGCTGTCGGAGTACGCCA GCGACGAGGCGGACGGGCGCCTGTACGTGGCCCTGAACCCCTTGATCGCGCA GGCCGTCATGGGTGGCGGCCAGCATGTGCGCATCAGCATGGACGAGGTGCGG GCGCTGGACAGCGAAACCGCCCGCCTGCTGCACCAGCGGCTGTGTGGCTGGA TCGACCCCGGCAAAACCGGCAAGGCTTCCATAGATACCTTGTGCGGCTATGT CTGGCCGTCAGAGGCCAGTGGTTCGACCATGCGCAAGCGCCGCAAGCGGGTG CGCGAGGCGTTGCCGGAGCTGGTCGCGCTGGGCTGGACGGTAACCGAGTTCG CGGCGGGCAAGTACGACATCACCCGGCCCAAGGCGGCAGGCTGACCCCCCCC ACTCTATTGTAAACAAGACATTTTTATCTTTTATATTCAATGGCTTATTTTC CTGCTAATTGGTAATACCATGAAAAATACCATGCTCAGAAAAGGCTTAACAA TATTTTGAAAAATTGCCTACTGAGCGCTGCCGCACAGCTCCATAGGCCTAAG ATAGATAGCTCCTCACTATCGCTTAGTAGGAAAGGATGAAGCTGATCGGCTT TATCCTTTTTTTATTTTTATATCAATATGATATAAAAATTTAAATCTTCTAA ATTTCTTCTTATTTCATAGAAAATTTTCACATTTATTATTCTTCGCCTTCAC GATTTATCTGTAATTTTTCACTAATCTTCAATTATCGAAATTTAATAGATCA TTCGAGGTTTACGAAGAAGGAGATATACATATGGTGAGCAAGGGCGAAGAAT TGTTTACTGGGGTGGTACCCATCTTGGTCGAGCTTGACGGGGATGTAAACGG CCACAAATTTTCTGTTCGTGGCGAAGGAGAGGGCGACGCGACGAACGGAAAG TTGACCCTGAAATTTATCTGCACCACCGGAAAGCTTCCCGTACCGTGGCCCA CCTTAGTTACAACACTTACATACGGTGTGCAATGCTTCTCACGTTATCCTGA CCACATGAAGCGTCATGACTTCTTTAAATCGGCCATGCCCGAAGGTTATGTT CAAGAACGTACTATTTCGTTTAAGGACGACGGGACGTATAAAACACGCGCTG AAGTAAAATTCGAGGGTGATACTTTGGTCAATCGTATTGAATTGAAGGGGAT CGACTTTAAAGAAGACGGCAATATCTTAGGACACAAACTGGAGTATAATTTC
AATTCTCACAATGTGTACATCACTGCAGACAAACAGAAAAACGGGATTAAAG CGAACTTTAAAATCCGCCACAACGTAGAAGACGGAAGTGTGCAGTTAGCTGA TCACTATCAACAAAATACGCCCATTGGTGATGGTCCGGTTCTTCTTCCAGAC AACCACTACTTGAGTACACAAAGCGTTCTGAGCAAAGACCCGAATGAAAAAC GCGATCACATGGTTCTGCTGGAGTTCGTAACTGCTGCTGGGATCACACACGG CATGGATGAACTGTATAAATAATCAGAACGCAGAAGCGGTCTGATAAAACAG AATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACT CAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAG AGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTG GGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACA AATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGC GGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCAT CCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTGTTTATTTTTCTA AATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTT CAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCC TTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAAC GCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTAC ATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAG AACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATT ATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCT CAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATG GCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACAC TGCGGCC ACTT CTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCT TTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGG AGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGC AATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTAGCT TCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCAC TTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGC CGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAG CCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATG AACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTA ACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTCTGAAAGCGA CCAGGTGCTCGGCGTGGCAAGACTCGCAGCGAACCCGTAGAAAGCCATGCT
CCAGCCGCCCGCATTGGAGAAATTCTTCAAATTCCCGTTGCACATAGCCCGG P CAATTCCTTTCCCTGCTCTGCCATAAGCGCAGCGAATGCCGGGTAATACTCG SfGFP TCAACGATCTGATAGAGAAGGGTTTGCTCGGGTCGGTGGCTCTGGTAACGAC (ACH106: :PM CAGTATCCCGATCCCGGCTGGCCGTCCTGGCCGCCACATGAGGCATGTTCCG s_2889-sfGFP) CGTCCTTGCAATACTGTGTTTACATACAGTCTATCGCTTAGCGGAAAGTTCT TTTACCCTCAGCCGAAATGCCTGCCGTTGCTAGACATTGCCAGCCAGTGCCC GTCACTCCCGTACTAACTGTCACGAACCCCTGCAATAACTGTCACGCCCCCC TGCAATAACTGTCACGAACCCCTGCAATAACTGTCACGCCCCCAAACCTGCA AACCCAGCAGGGGCGGGGGCTGGCGGGGTGTTGGAAAAATCCATCCATGATT ATCTAAGAATAATCCACTAGGCGCGGTTATCAGCGCCCTTGTGGGGCGCTGC TGCCCTTGCCCAATATGCCCGGCCAGAGGCCGGATAGCTGGTCTATTCGCTG CGCTAGGCTACACACCGCCCCACCGCTGCGCGGCAGGGGGAAAGGCGGGCAA AGCCCGCTAAACCCCACACCAAACCCCGCAGAAATACGCTGGAGCGCTTTTA GCCGCTTTAGCGGCCTTTCCCCCTACCCGAAGGGTGGGGGCGCGTGTGCAGC CCCGCAGGGCCTGTCTCGGTCGATCATTCAGCCCGGCTCATCCTTCTGGCGT GGCGGCAGACCGAACAAGGCGCGGTCGTGGTCGCGTTCAAGGTACGCATCCA TTGCCGCCATGAGCCGATCCTCCGGCCACTCGCTGCTGTTCACCTTGGCCAA AATCATGGCCCCCACCAGCACCTTGCGCCTTGTTTCGTTCTTGCGCTCTTGC TGCTGTTCCCTTGCCCGCTCCCGCTGAATTTCGGCATTGATTCGCGCTCGTT GTTCTTCGAGCTTGGCCAGCCGATCCGCCGCCTTGTTGCTCCCCTTAACCAT CTTGACACCCCATTGTTAATGTGCTGTCTCGTAGGCTATCATGGAGGCACAG CGGCGGCAATCCCGACCCTACTTTGTAGGGGAGGGCGCACTTACCGGTTTCT CTTCGAGAAACTGGCCTAACGGCCACCCTTCGGGCGGTGCGCTCTCCGAGGG CCATTGCATGGAGCCGAAAAGCAAAAGCAACAGCGAGGCAGCATGGCGATTT ATCACCTTACGGCGAAAACCGGCAGCAGGTCGGGCGGCCAATCGGCCAGGGC CAAGGCCGACTACATCCAGCGCGAAGGCAAGTATGCCCGCGACATGGATGAA GTCTTGCACGCCGAATCCGGGCACATGCCGGAGTTCGTCGAGCGGCCCGCCG ACTACTGGGATGCTGCCGACCTGTATGAACGCGCCAATGGGCGGCTGTTCAA GGAGGTCGAATTTGCCCTGCCGGTCGAGCTGACCCTCGACCAGCAGAAGGCG CTGGCGTCCGAGTTCGCCCAGCACCTGACCGGTGCCGAGCGCCTGCCGTATA CGCTGGCCATCCATGCCGGTGGCGGCGAGAACCCGCACTGCCACCTGATGAT CTCCGAGCGGATCAATGACGGCATCGAGCGGCCCGCCGCTCAGTGGTTCAAG CGGTACAACGGCAAGACCCCGGAGAAGGGCGGGGCACAGAAGACCGAAGCGC TCAAGCCCAAGGCATGGCTTGAGCAGACCCGCGAGGCATGGGCCGACCATGC CAACCGGGCATTAGAGCGGGCTGGCCACGACGCCCGCATTGACCACAGAACA CTTGAGGCGCAGGGCATCGAGCGCCTGCCCGGTGTTCACCTGGGGCCGAACG TGGTGGAGATGGAAGGCCGGGGCATCCGCACCGACCGGGCAGACGTGGCCCT GAACATCGACACCGCCAACGCCCAGATCATCGACTTACAGGAATACCGGGAG GC AATAGACC AT GAACGC AATC GAC AGAGT GAAGAAAT CC AGAGGC AT C AAC GAGTTAGCGGAGCAGATCGAACCGCTGGCCCAGAGCATGGCGACACTGGCCG ACGAAGCCCGGCAGGTCATGAGCCAGACCAAGCAGGCCAGCGAGGCGCAGGC GGCGGAGTGGCTGAAAGCCCAGCGCCAGACAGGGGCGGCATGGGTGGAGCTG GCCAAAGAGTTGCGGGAGGTAGCCGCCGAGGTGAGCAGCGCCGCGCAGAGCG CCCGGAGCGCGTCGCGGGGGTGGCACTGGAAGCTATGGCTAACCGTGATGCT GGCTTCCATGATGCCTACGGTGGTGCTGCTGATCGCATCGTTGCTCTTGCTC GACCTGACGCCACTGACAACCGAGGACGGCTCGATCTGGCTGCGCTTGGTGG CCCGATGAAGAACGACAGGACTTTGCAGGCCATAGGCCGACAGCTCAAGGCC ATGGGCTGTGAGCGCTTCGATATCGGCGTCAGGGACGCACCCACCGGCCAGA TGATGAACCGGGAATGGTCAGCCGCCGAAGTGCTCCAGAACACGCCATGGCT CAAGCGGATGAATGCCCAGGGCAATGACGTGTATATCAGGCCCGCCGAGCAG GAGCGGCATGGTCTGGTGCTGGTGGACGACCTCAGCGAGTTTGACCTGGATG ACATGAAAGCCGAGGGCCGGGAGCCTGCCCTGGTAGTGGAAACCAGCCCGAA GAACTATCAGGCATGGGTCAAGGTGGCCGACGCCGCAGGCGGTGAACTTCGG GGGCAGATTGCCCGGACGCTGGCCAGCGAGTACGACGCCGACCCGGCCAGCG CCGACAGCCGCCACTATGGCCGCTTGGCGGGCTTCACCAACCGCAAGGACAA GCACACCACCCGCGCCGGTTATCAGCCGTGGGTGCTGCTGCGTGAATCCAAG GGCAAGACCGCCACCGCTGGCCCGGCGCTGGTGCAGCAGGCTGGCCAGCAGA TCGAGCAGGCCCAGCGGCAGCAGGAGAAGGCCCGCAGGCTGGCCAGCCTCGA ACTGCCCGAGCGGCAGCTTAGCCGCCACCGGCGCACGGCGCTGGACGAGTAC CGCAGCGAGATGGCCGGGCTGGTCAAGCGCTTCGGTCATGACCTCAGCAAGT GCGACTTTATCGCCGCGCAGAAGCTGGCCAGCCGGGGCCGCAGTGCCGAGGA AATCGGCAAGGCCATGGCCGAGGCCAGCCCAGCGCTGGCAGAGCGCAAGCCC GGCCACGAAGCGGATTACATCGAGCGCACCGTCAGCAAGGTCATGGGTCTGC CCAGCGTCCAGCTTGCGCGGGCCGAGCTGGCACGGGCACCGGCACCCCGCCA GCGAGGCATGGACAGGGGCGGGCCAGATTTCAGCATGTAGTGCTTGCGTTGG TACTCACGCCTGTTATACTATGAGTACTCACGCACAGAAGGGGGTTTTATGG AATACGAAAAAAGCGCTTCAGGGTCGGTCTACCTGATCAAAAGTGACAAGGG CTATTGGTTGCCCGGTGGCTTTGGTTATACGTCAAACAAGGCCGAGGCTGGC CGCTTTTCAGTCGCTGATATGGCCAGCCTTAACCTTGACGGCTGCACCTTGT CCTTGTTCCGCGAAGACAAGCCTTTCGGCCCCGGCAAGTTTCTCGGTGACTG ATATGAAAGACCAAAAGGACAAGCAGACCGGCGACCTGCTGGCCAGCCCTGA CGCTGTACGCCAAGCGCGATATGCCGAGCGCATGAAGGCCAAAGGGATGCGT CAGCGCAAGTTCTGGCTGACCGAC GACGAATACGAGGCGCTGCGCGAGTGCC TGGAAGAACTCAGAGCGGCGCAGGGCGGGGGTAGTGACCCCGCCAGCGCCTA ACCACCAACTGCCTGCAAAGGAGGCAATCAATGGCTACCCATAAGCCTATCA ATATTCTGGAGGCGTTCGCAGCAGCGCCGCCACCGCTGGACTACGTTTTGCC CAACATGGTGGCCGGTACGGTCGGGGCGCTGGTGTCGCCCGGTGGTGCCGGT AAATCCATGCTGGCCCTGCAACTGGCCGCACAGATTGCAGGCGGGCCGGATC TGCTGGAGGTGGGCGAACTGCCCACCGGCCCGGTGATCTACCTGCCCGCCGA AGACCCGCCCACCGCCATTCATCACCGCCTGCACGCCCTTGGGGCGCACCTC AGCGCCGAGGAACGGCAAGCCGTGGCTGACGGCCTGCTGATCCAGCCGCTGA TCGGCAGCCTGCCCAACATCATGGCCCCGGAGTGGTTCGACGGCCTCAAGCG CGCCGCCGAGGGCCGCCGCCTGATGGTGCTGGACACGCTGCGCCGGTTCCAC ATCGAGGAAGAAAACGCCAGCGGCCCCATGGCCCAGGTCATCGGTCGCATGG AGGCCATCGCCGCCGATACCGGGTGCTCTATCGTGTTCCTGCACCATGCCAG CAAGGGCGCGGCCATGATGGGCGCAGGCGACCAGCAGCAGGCCAGCCGGGGC AGCTCGGTACTGGTCGATAACATCCGCTGGCAGTCCTACCTGTCGAGCATGA CCAGCGCCGAGGCCGAGGAATGGGGTGTGGAC GACGACCAGCGCCGG CTT CGTCCGCTTCGGTGTGAGCAAGGCCAACTATGGCGCACCGTTCGCTGATCGG TGGTTCAGGCGGCATGACGGCGGGGTGCTCAAGCCCGCCGTGCTGGAGAGGC AGCGCAAGAGCAAGGGGGTGCCCCGTGGTGAAGCCTAAGAACAAGCACAGCC TCAGCCACGTCCGGCACGACCCGGCGCACTGTCTGGCCCCCGGCCTGTTCCG TGCCCTCAAGCGGGGCGAGCGCAAGCGCAGCAAGCTGGACGTGACGTATGAC TACGGCGACGGCAAGCGGATCGAGTTCAGCGGCCCGGAGCCGCTGGGCGCTG ATGATCTGCGCATCCTGCAAGGGCTGGTGGCCATGGCTGGGCCTAATGGCCT AGTGCTTGGCCCGGAACCCAAGACCGAAGGCGGACGGCAGCTCCGGCTGTTC CTGGAACCCAAGTGGGAGGCCGTCACCGCTGAATGCCATGTGGTCAAAGGTA GCTATCGGGCGCTGGCAAAGGAAATCGGGGCAGAGGTCGATAGTGGTGGGGC GCTCAAGCACATACAGGACTGCATCGAGCGCCTTTGGAAGGTATCCATCATC GCCCAGAATGGCCGCAAGCGGCAGGGGTTTCGGCTGCTGTCGGAGTACGCCA GCGACGAGGCGGACGGGCGCCTGTACGTGGCCCTGAACCCCTTGATCGCGCA GGCCGTCATGGGTGGCGGCCAGCATGTGCGCATCAGCATGGACGAGGTGCGG GCGCTGGACAGCGAAACCGCCCGCCTGCTGCACCAGCGGCTGTGTGGCTGGA TCGACCCCGGCAAAACCGGCAAGGCTTCCATAGATACCTTGTGCGGCTATGT CTGGCCGTCAGAGGCCAGTGGTTCGACCATGCGCAAGCGCCGCAAGCGGGTG CGCGAGGCGTTGCCGGAGCTGGTCGCGCTGGGCTGGACGGTAACCGAGTTCG CGGCGGGCAAGTACGACATCACCCGGCCCAAGGCGGCAGGCTGACCCCCCCC ACTCTATTGTAAACAAGACATTTTTATCTTTTATATTCAATGGCTTATTTTC CTGCTAATTGGTAATACCATGAAAAATACCATGCTCAGAAAAGGCTTAACAA TATTTTGAAAAATTGCCTACTGAGCGCTGCCGCACAGCTCCATAGGCCTTGT ACACTTGGAACTTTGCAGAAAAATTGATGAAGACGTTCAAAACTGATTTCAG ACACAGCAATTGACCTAGCTTGAAAAATGCTAATGGTAGCACAGATACAGAT TCTTCATATTTTCTTTATAGTCATTACAAACAATATCAATCAAAGCCATTTT ATTCATGGTTTCTCCTTGACTCTCTCTTAATCTAGCTTCAGAAGAAGGAGAT ATACATATGGTGAGCAAGGGCGAAGAATTGTTTACTGGGGTGGTACCCATCT TGGTCGAGCTTGACGGGGATGTAAACGGCCACAAATTTTCTGTTCGTGGCGA AGGAGAGGGCGACGCGACGAACGGAAAGTTGACCCTGAAATTTATCTGCACC ACCGGAAAGCTTCCCGTACCGTGGCCCACCTTAGTTACAACACTTACATACG GTGTGCAATGCTTCTCACGTTATCCTGACCACATGAAGCGTCATGACTTCTT TAAATCGGCCATGCCCGAAGGTTATGTTCAAGAACGTACTATTTCGTTTAAG GACGACGGGACGTATAAAACACGCGCTGAAGTAAAATTCGAGGGTGATACTT TGGTCAATCGTATTGAATTGAAGGGGATCGACTTTAAAGAAGACGGCAATAT CTTAGGACACAAACTGGAGTATAATTTCAATTCTCACAATGTGTACATCACT
GCAGACAAACAGAAAAACGGGATTAAAGCGAACTTTAAAATCCGCCACAACG TAGAAGACGGAAGTGTGCAGTTAGCTGATCACTATCAACAAAATACGCCCAT TGGTGATGGTCCGGTTCTTCTTCCAGACAACCACTACTTGAGTACACAAAGC GTTCTGAGCAAAGACCCGAATGAAAAACGCGATCACATGGTTCTGCTGGAGT TCGTAACTGCTGCTGGGATCACACACGGCATGGATGAACTGTATAAATAATC AGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCG GTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCG ATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAA TAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTT GTCGGTGAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAAC GTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTG CCAGGCATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTT TCTACAAACTCTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCT CATGAGACAATAACCCTGAT AATGCTTCAATAATATTGAAAAAGGAAGAGT ATGAGTATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTT GCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGA AGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGT AAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTT TTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGA GCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCA CCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCA GTGCTGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAAC GATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCAT GTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACG ACGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACT ATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGG ATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTG GCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTAT CATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTAC ACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGA TAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATA TATACTTTAGATTGATTTCTGAAAGCGACCAGGTGCTCGGCGTGGCAAGACT CGCAGCGAACCCGTAGAAAGCCATGCT
CCAGCCGCCCGCATTGGAGAAATTCTTCAAATTCCCGTTGCACATAGCCCGG PAlS_2885 CAATTCCTTTCCCTGCTCTGCCATAAGCGCAGCGAATGCCGGGTAATACTCG sfGFP
TCAACGATCTGATAGAGAAGGGTTTGCTCGGGTCGGTGGCTCTGGTAACGAC (pACH106 : : PA CAGTATCCCGATCCCGGCTGGCCGTCCTGGCCGCCACATGAGGCATGTTCCG ls_2885-sfGFP) CGTCCTTGCAATACTGTGTTTACATACAGTCTATCGCTTAGCGGAAAGTTCT TTTACCCTCAGCCGAAATGCCTGCCGTTGCTAGACATTGCCAGCCAGTGCCC GTCACTCCCGTACTAACTGTCACGAACCCCTGCAATAACTGTCACGCCCCCC TGCAATAACTGTCACGAACCCCTGCAATAACTGTCACGCCCCCAAACCTGCA AACCCAGCAGGGGCGGGGGCTGGCGGGGTGTTGGAAAAATCCATCC TGATT ATCTAAGAATAATCCACTAGGCGCGGTTATCAGCGCCCTTGTGGGGCGCTGC TGCCCTTGCCCAATATGCCCGGCCAGAGGCCGGATAGCTGGTCTATTCGCTG CGCTAGGCTACACACCGCCCCACCGCTGCGCGGCAGGGGGAAAGGCGGGCAA AGCCCGCTAAACCCCACACCAAACCCCGCAGAAATACGCTGGAGCGCTTTTA GCCGCTTTAGCGGCCTTTCCCCCTACCCGAAGGGTGGGGGCGCGTGTGCAGC CCCGCAGGGCCTGTCTCGGTCGATCATTCAGCCCGGCTCATCCTTCTGGCGT GGCGGCAGACCGAACAAGGCGCGGTCGTGGTCGCGTTCAAGGTACGCATCCA TTGCCGCCATGAGCCGATCCTCCGGCCACTCGCTGCTGTTCACCTTGGCCAA AATCATGGCCCCCACCAGCACCTTGCGCCTTGTTTCGTTCTTGCGCTCTTGC TGCTGTTCCCTTGCCCGCTCCCGCTGAATTTCGGCATTGATTCGCGCTCGTT GTTCTTCGAGCTTGGCCAGCCGATCCGCCGCCTTGTTGCTCCCCTTAACCAT CTTGACACCCCATTGTTAATGTGCTGTCTCGTAGGCTATCATGGAGGCACAG CGGCGGCAATCCCGACCCTACTTTGTAGGGGAGGGCGCACTTACCGGTTTCT CTTCGAGAAACTGGCCTAACGGCCACCCTTCGGGCGGTGCGCTCTCCGAGGG CCATTGCATGGAGCCGAAAAGCAAAAGCAACAGCGAGGCAGCATGGCGATTT ATCACCTTACGGCGAAAACCGGCAGCAGGTCGGGCGGCCAATCGGCCAGGGC CAAGGCCGACTACATCCAGCGCGAAGGCAAGTATGCCCGCGACATGGATGAA GTCTTGCACGCCGAATCCGGGCACATGCCGGAGTTCGTCGAGCGGCCCGCCG ACTACTGGGATGCTGCCGACCTGTATGAACGCGCCAATGGGCGGCTGTTCAA GGAGGTCGAATTTGCCCTGCCGGTCGAGCTGACCCTCGACCAGCAGAAGGCG CTGGCGTCCGAGTTCGCCCAGCACCTGACCGGTGCCGAGCGCCTGCCGTATA CGCTGGCCATCCATGCCGGTGGCGGCGAGAACCCGCACTGCCACCTGATGAT CTCCGAGCGGATCAATGACGGCATCGAGCGGCCCGCCGCTCAGTGGTTCAAG CGGTACAACGGCAAGACCCCGGAGAAGGGCGGGGCACAGAAGACCGAAGCGC TCAAGCCCAAGGCATGGCTTGAGCAGACCCGCGAGGCATGGGCCGACCATGC CAACCGGGCATTAGAGCGGGCTGGCCACGACGCCCGCATTGACCACAGAACA CTTGAGGCGCAGGGCATCGAGCGCCTGCCCGGTGTTCACCTGGGGCCGAACG TGGTGGAGATGGAAGGCCGGGGCATCCGCACCGACCGGGCAGACGTGGCCCT GAACATCGACACCGCCAACGCCCAGATCATCGACTTACAGGAATACCGGGAG GC AATAGACC AT GAACGC AATC GAC AGAGT GAAGAAAT CC AGAGGC AT C AAC GAGTTAGCGGAGCAGATCGAACCGCTGGCCCAGAGCATGGCGACACTGGCCG ACGAAGCCCGGCAGGTCATGAGCCAGACCAAGCAGGCCAGCGAGGCGCAGGC GGCGGAGTGGCTGAAAGCCCAGCGCCAGACAGGGGCGGCATGGGTGGAGCTG GCCAAAGAGTTGCGGGAGGTAGCCGCCGAGGTGAGCAGCGCCGCGCAGAGCG CCCGGAGCGCGTCGCGGGGGTGGCACTGGAAGCTATGGCTAACCGTGATGCT GGCTTCCATGATGCCTACGGTGGTGCTGCTGATCGCATCGTTGCTCTTGCTC GACCTGACGCCACTGACAACCGAGGACGGCTCGATCTGGCTGCGCTTGGTGG CCCGATGAAGAACGACAGGACTTTGCAGGCCATAGGCCGACAGCTCAAGGCC ATGGGCTGTGAGCGCTTCGATATCGGCGTCAGGGACGCACCCACCGGCCAGA TGATGAACCGGGAATGGTCAGCCGCCGAAGTGCTCCAGAACACGCCATGGCT CAAGCGGATGAATGCCCAGGGCAATGACGTGTATATCAGGCCCGCCGAGCAG GAGCGGCATGGTCTGGTGCTGGTGGACGACCTCAGCGAGTTTGACCTGGATG ACATGAAAGCCGAGGGCCGGGAGCCTGCCCTGGTAGTGGAAACCAGCCCGAA GAACTATCAGGCATGGGTCAAGGTGGCCGACGCCGCAGGCGGTGAACTTCGG GGGCAGATTGCCCGGACGCTGGCCAGCGAGTACGACGCCGACCCGGCCAGCG CCGACAGCCGCCACTATGGCCGCTTGGCGGGCTTCACCAACCGCAAGGACAA GCACACCACCCGCGCCGGTTATCAGCCGTGGGTGCTGCTGCGTGAATCCAAG GGCAAGACCGCCACCGCTGGCCCGGCGCTGGTGCAGCAGGCTGGCCAGCAGA TCGAGCAGGCCCAGCGGCAGCAGGAGAAGGCCCGCAGGCTGGCCAGCCTCGA ACTGCCCGAGCGGCAGCTTAGCCGCCACCGGCGCACGGCGCTGGACGAGTAC CGCAGCGAGATGGCCGGGCTGGTCAAGCGCTTCGGTCATGACCTCAGCAAGT GCGACTTTATCGCCGCGCAGAAGCTGGCCAGCCGGGGCCGCAGTGCCGAGGA AATCGGCAAGGCCATGGCCGAGGCCAGCCCAGCGCTGGCAGAGCGCAAGCCC GGCCACGAAGCGGATTACATCGAGCGCACCGTCAGCAAGGTCATGGGTCTGC CCAGCGTCCAGCTTGCGCGGGCCGAGCTGGCACGGGCACCGGCACCCCGCCA GCGAGGCATGGACAGGGGCGGGCCAGATTTCAGCATGTAGTGCTTGCGTTGG TACTCACGCCTGTTATACTATGAGTACTCACGCACAGAAGGGGGTTTTATGG AATACGAAAAAAGCGCTTCAGGGTCGGTCTACCTGATCAAAAGTGACAAGGG CTATTGGTTGCCCGGTGGCTTTGGTTATACGTCAAACAAGGCCGAGGCTGGC CGCTTTTCAGTCGCTGATATGGCCAGCCTTAACCTTGACGGCTGCACCTTGT CCTTGTTCCGCGAAGACAAGCCTTTCGGCCCCGGCAAGTTTCTCGGTGACTG ATATGAAAGACCAAAAGGACAAGCAGACCGGCGACCTGCTGGCCAGCCCTGA CGCTGTACGCCAAGCGCGATATGCCGAGCGCATGAAGGCCAAAGGGATGCGT CAGCGCAAGTTCTGGCTGACCGAC GACGAATACGAGGCGCTGCGCGAGTGCC TGGAAGAACTCAGAGCGGCGCAGGGCGGGGGTAGTGACCCCGCCAGCGCCTA ACCACCAACTGCCTGCAAAGGAGGCAATCAATGGCTACCCATAAGCCTATCA ATATTCTGGAGGCGTTCGCAGCAGCGCCGCCACCGCTGGACTACGTTTTGCC CAACATGGTGGCCGGTACGGTCGGGGCGCTGGTGTCGCCCGGTGGTGCCGGT AAATCCATGCTGGCCCTGCAACTGGCCGCACAGATTGCAGGCGGGCCGGATC TGCTGGAGGTGGGCGAACTGCCCACCGGCCCGGTGATCTACCTGCCCGCCGA AGACCCGCCCACCGCCATTCATCACCGCCTGCACGCCCTTGGGGCGCACCTC AGCGCCGAGGAACGGCAAGCCGTGGCTGACGGCCTGCTGATCCAGCCGCTGA TCGGCAGCCTGCCCAACATCATGGCCCCGGAGTGGTTCGACGGCCTCAAGCG CGCCGCCGAGGGCCGCCGCCTGATGGTGCTGGACACGCTGCGCCGGTTCCAC ATCGAGGAAGAAAACGCCAGCGGCCCCATGGCCCAGGTCATCGGTCGCATGG AGGCCATCGCCGCCGATACCGGGTGCTCTATCGTGTTCCTGCACCATGCCAG CAAGGGCGCGGCCATGATGGGCGCAGGCGACCAGCAGCAGGCCAGCCGGGGC AGCTCGGTACTGGTCGATAACATCCGCTGGCAGTCCTACCTGTCGAGCATGA CCAGCGCCGAGGCCGAGGAATGGGGTGTGGAC GACGACCAGCGCCGG CTT CGTCCGCTTCGGTGTGAGCAAGGCCAACTATGGCGCACCGTTCGCTGATCGG TGGTTCAGGCGGCATGACGGCGGGGTGCTCAAGCCCGCCGTGCTGGAGAGGC AGCGCAAGAGCAAGGGGGTGCCCCGTGGTGAAGCCTAAGAACAAGCACAGCC TCAGCCACGTCCGGCACGACCCGGCGCACTGTCTGGCCCCCGGCCTGTTCCG TGCCCTCAAGCGGGGCGAGCGCAAGCGCAGCAAGCTGGACGTGACGTATGAC TACGGCGACGGCAAGCGGATCGAGTTCAGCGGCCCGGAGCCGCTGGGCGCTG ATGATCTGCGCATCCTGCAAGGGCTGGTGGCCATGGCTGGGCCTAATGGCCT AGTGCTTGGCCCGGAACCCAAGACCGAAGGCGGACGGCAGCTCCGGCTGTTC CTGGAACCCAAGTGGGAGGCCGTCACCGCTGAATGCCATGTGGTCAAAGGTA GCTATCGGGCGCTGGCAAAGGAAATCGGGGCAGAGGTCGATAGTGGTGGGGC GCTCAAGCACATACAGGACTGCATCGAGCGCCTTTGGAAGGTATCCATCATC GCCCAGAATGGCCGCAAGCGGCAGGGGTTTCGGCTGCTGTCGGAGTACGCCA GCGACGAGGCGGACGGGCGCCTGTACGTGGCCCTGAACCCCTTGATCGCGCA GGCCGTCATGGGTGGCGGCCAGCATGTGCGCATCAGCATGGACGAGGTGCGG GCGCTGGACAGCGAAACCGCCCGCCTGCTGCACCAGCGGCTGTGTGGCTGGA TCGACCCCGGCAAAACCGGCAAGGCTTCCATAGATACCTTGTGCGGCTATGT CTGGCCGTCAGAGGCCAGTGGTTCGACCATGCGCAAGCGCCGCAAGCGGGTG CGCGAGGCGTTGCCGGAGCTGGTCGCGCTGGGCTGGACGGTAACCGAGTTCG CGGCGGGCAAGTACGACATCACCCGGCCCAAGGCGGCAGGCTGACCCCCCCC ACTCTATTGTAAACAAGACATTTTTATCTTTTATATTCAATGGCTTATTTTC CTGCTAATTGGTAATACCATGAAAAATACCATGCTCAGAAAAGGCTTAACAA TATTTTGAAAAATTGCCTACTGAGCGCTGCCGCACAGCTCCATAGGCCACTC AAACTTGCAGTTGCAATTAACAGCGTGGTAAGCAAGACCGTTAAAAATAACC GC AAGGCTATGGGAACACGTAGAACGTTCAAAAGC ACT CTCTC ATT TT TACT CTATATTTTGCATTGTAACCAACAACGAAAAGAAAAACTCTCCATTGTTTCT TCATCTTTATTATCTAATCTTTAAGTCATCTTAAAACAAACATTAAAGGGTT TTAACCATGAAAATGACGGCTAAAATTGCATTATTCAGTATGGTGAGCAAGG GCGAAGAATTGTTTACTGGGGTGGTACCCATCTTGGTCGAGCTTGACGGGGA TGTAAACGGCCACAAATTTTCTGTTCGTGGCGAAGGAGAGGGCGACGCGACG AACGGAAAGTTGACCCTGAAATTTATCTGCACCACCGGAAAGCTTCCCGTAC CGTGGCCCACCTTAGTTACAACACTTACATACGGTGTGCAATGCTTCTCACG TTATCCTGACCACATGAAGCGTCATGACTTCTTTAAATCGGCCATGCCCGAA GGTTATGTTCAAGAACGTACTATTTCGTTTAAGGACGACGGGACGTATAAAA CACGCGCTGAAGTAAAATTCGAGGGTGATACTTTGGTCAATCGTATTGAATT GAAGGGGATCGACTTTAAAGAAGACGGCAATATCTTAGGACACAAACTGGAG
TATAATTTCAATTCTCACAATGTGTACATCACTGCAGACAAACAGAAAAACG GGATTAAAGCGAACTTTAAAATCCGCCACAACGTAGAAGACGGAAGTGTGCA GTTAGCTGATCACTATCAACAAAATACGCCCATTGGTGATGGTCCGGTTCTT CTTCCAGACAACCACTACTTGAGTACACAAAGCGTTCTGAGCAAAGACCCGA ATGAAAAACGCGATCACATGGTTCTGCTGGAGTTCGTAACTGCTGCTGGGAT CACACACGGCATGGATGAACTGTATAAATAATCAGAACGCAGAAGCGGTCTG ATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCA TGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCC CCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTC GAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTG AGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCG GAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCA GAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTGTTT ATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGA TAAATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCC GTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCA CCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGA GTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTC GCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGG CGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATA CACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATC TTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAG TGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAG CTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTT GGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGAT GCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTT ACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTG CAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAA ATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCA GATGGTAAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAA CTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAA GCATTGGTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTC TGAAAGCGACCAGGTGCTCGGCGTGGCAAGACTCGCAGCGAACCCGTAGAAA GCCATGCT
ATGGTGAGCAAGGGCGAAGAGCTGATTAAGGAGAATATGCGCATGAAAGTTG mRuby 2 TTATGGAGGGAAGCGTTAACGGACATCAGTTTAAATGCACCGGCGAGGGCGA GGGTAATCCTTACATGGGCACGCAGACAATGCGCATTAAGGTCATCGAGGGG GGCCCCCTTCCGTTTGCCTTCGATATTTTAGCTACATCTTTCATGTATGGTT CCCGCACCTTTATTAAGTATCCCAAGGGTATTCCCGACTTCTTTAAGCAGTC CTTCCCGGAGGGGTTTACG GGGAGCGTGTCACGCGCTATGAGGATGGTGGA GTTGTCACTGTAATGCAAGATACATCCCTTGAAGACGGGTGTTTAGTTTACC ATGTTCAAGTGCGCGGAGTTAATTTCCCAAGTAACGGCCCAGTAATGCAGAA GAAAACTAAAGGCTGGGAACCCAACACTGAGATGATGTACCCCGCAGACGGT GGGTTACGCGGGTACACGCACATGGCCTTGAAAGTCGATGGTGGTGGGCACT TATCCTGTAGCTTTGTCACCACCTACCGTAGCAAAAAGACTGTAGGAAACAT CAAAATGCCCGGCATTCATGCAGTAGACCATCGTTTAGAACGCCTTGAGGAG AGTGATAACGAGATGTTTGTAGTACAACGTGAACACGCTGTCGCGAAATTTG CCGGTCTTGGTGGAGGTATGGATGAACTGTATAAATAA
CCTAAGATAGATAGCTCCTCACTATCGCTTAGTAGGAAAGGATGAAGCTGAT PA1 _0032~ CGGCTTTATCCTTTTTTTATTTTTATATCAATATGATATAAAAATTTAAATC mRuby2 TTCTAAATTTCTTCTTATTTCATAGAAAATTTTCACATTTATTATTCTTCGC fusion CTTCACGATTTATCTGTAATTTTTCACTAATCTTCAATTATCGAAATTTAAT
AGATCATTCGAGGTTTACGAAGAAGGAGATATACATATGGTGAGCAAGGGCG AAGAGCTGATTAAGGAGAATATGCGCATGAAAGTTGTTATGGAGGGAAGCGT TAACGGACATCAGTTTAAATGCACCGGCGAGGGCGAGGGTAATCCTTACATG GGCACGCAGACAATGCGCATTAAGGTCATCGAGGGGGGCCCCCTTCCGTTTG CCTTCGATATTTTAGCTACATCTTTCATGTATGGTTCCCGCACCTTTATTAA GTATCCCAAGGGTATTCCCGACTTCTTTAAGCAGTCCTTCCCGGAGGGGTTT ACGTGGGAGCGTGTCACGCGCTATGAGGATGGTGGAGTTGTCACTGTAATGC AAGATACATCCCTTGAAGACGGGTGTTTAGTTTACCATGTTCAAGTGCGCGG AGTTAATTTCCCAAGTAACGGCCCAGTAATGCAGAAGAAAACTAAAGGCTGG GAACCCAACACTGAGATGATGTACCCCGCAGACGGTGGGTTACGCGGGTACA CGCACATGGCCTTGAAAGTCGATGGTGGTGGGCACTTATCCTGTAGCTTTGT CACCACCTACCGTAGCAAAAAGACTGTAGGAAACATCAAAATGCCCGGCATT CATGCAGTAGACCATCGTTTAGAACGCCTTGAGGAGAGTGATAACGAGATGT TTGTAGTACAACGTGAACACGCTGTCGCGAAATTTGCCGGTCTTGGTGGAGG TATGGATGAACTGTATAAATAA
CTTGTACACTTGGAACTTTGCAGAAAAATTGATGAAGACGTTCAAAACTGAT PA TTCAGACACAGCAATTGACCTAGCTTGAAAAATGCTAATGGTAGCACAGATA mRuby2 CAGATTCTTCATATTTTCTTTATAGTCATTACAAACAATATCAATCAAAGCC fusion ATTTTATTCATGGTTTCTCCTTGACTCTCTCTTAATCTAGCTTCAGAAGAAG GAGATATACATATGGTGAGCAAGGGCGAAGAGCTGATTAAGGAGAATATGCG CATGAAAGTTGTTATGGAGGGAAGCGTTAACGGACATCAGTTTAAATGCACC GGCGAGGGCGAGGGTAATCCTTACATGGGCACGCAGACAATGCGCATTAAGG TCATCGAGGGGGGCCCCCTTCCGTTTGCCTTCGATATTTTAGCTACATCTTT CATGTATGGTTCCCGCACCTTTATTAAGTATCCCAAGGGTATTCCCGACTTC TTTAAGCAGTCCTTCCCGGAGGGGTTTACGTGGGAGCGTGTCACGCGCTATG AGGATGGTGGAGTTGTCACTGTAATGCAAGATACATCCCTTGAAGACGGGTG TTTAGTTTACCATGTTCAAGTGCGCGGAGTTAATTTCCCAAGTAACGGCCCA GTAATGCAGAAGAAAACTAAAGGCTGGGAACCCAACACTGAGATGATGTACC CCGCAGACGGTGGGTTACGCGGGTACACGCACATGGCCTTGAAAGTCGATGG TGGTGGGCACTTATCCTGTAGCTTTGTCACCACCTACCGTAGCAAAAAGACT GTAGGAAACATCAAAATGCCCGGCATTCATGCAGTAGACCATCGTTTAGAAC GCCTTGAGGAGAGTGATAACGAGATGTTTGTAGTACAACGTGAACACGCTGT CGCGAAATTTGCCGGTCTTGGTGGAGGTATGGATGAACTGTATAAATAA
CACTCAAACTTGCAGTTGCAATTAACAGCGTGGTAAGCAAGACCGTTAAAAA PA TAACCGCAAGGCTATGGGAACACGTAGAACGTTCAAAAGCACTCTCTCATTT mRuby2 TTACTCTATATTTTGCATTGTAACCAACAACGAAAAGAAAAACTCTCCATTG fusion TTTCTTCATCTTTATTATCTAATCTTTAAGTCATCTTAAAACAAACATTAAA GGGTTTTAACCATGAAAATGACGGCTAAAATTGCATTATTCAGTATGGTGAG CAAGGGCGAAGAGCTGATTAAGGAGAATATGCGCATGAAAGTTGTTATGGAG GGAAGCGTTAACGGACATCAGTTTAAATGCACCGGCGAGGGCGAGGGTAATC CTTACATGGGCACGCAGACAATGCGCATTAAGGTCATCGAGGGGGGCCCCCT TCCGTTTGCCTTCGATATTTTAGCTACATCTTTCATGTATGGTTCCCGCACC TTTATTAAGTATCCCAAGGGTATTCCCGACTTCTTTAAGCAGTCCTTCCCGG AGGGGTTTACGTGGGAGCGTGTCACGCGCTATGAGGATGGTGGAGTTGTCAC TGTAATGCAAGATACATCCCTTGAAGACGGGTGTTTAGTTTACCATGTTCAA GTGCGCGGAGTTAATTTCCCAAGTAACGGCCCAGTAATGCAGAAGAAAACTA AAGGCTGGGAACCCAACACTGAGATGATGTACCCCGCAGACGGTGGGTTACG CGGGTACACGCACATGGCCTTGAAAGTCGATGGTGGTGGGCACTTATCCTGT AGCTTTGTCACCACCTACCGTAGCAAAAAGACTGTAGGAAACATCAAAATGC CCGGCATTCATGCAGTAGACCATCGTTTAGAACGCCTTGAGGAGAGTGATAA CGAGATGTTTGTAGTACAACGTGAACACGCTGTCGCGAAATTTGCCGGTCTT GGTGGAGGTATGGATGAACTGTATAAATAA
CCAGCCGCCCGCATTGGAGAAATTCTTCAAATTCCCGTTGCACATAGCCCGG PA CAATTCCTTTCCCTGCTCTGCCATAAGCGCAGCGAATGCCGGGTAATACTCG mRuby2 TCAACGATCTGATAGAGAAGGGTTTGCTCGGGTCGGTGGCTCTGGTAACGAC (pACH106 : : PA CAGTATCCCGATCCCGGCTGGCCGTCCTGGCCGCCACATGAGGCATGTTCCG CGTCCTTGCAATACTGTGTTTACATACAGTCTATCGCTTAGCGGAAAGTTCT mRuby2 ) TTTACCCTCAGCCGAAATGCCTGCCGTTGCTAGACATTGCCAGCCAGTGCCC GTCACTCCCGTACTAACTGTCACGAACCCCTGCAATAACTGTCACGCCCCCC TGCAATAACTGTCACGAACCCCTGCAATAACTGTCACGCCCCCAAACCTGCA AACCCAGCAGGGGCGGGGGCTGGCGGGGTGTTGGAAAAATCCATCCATGATT ATCTAAGAATAATCCACTAGGCGCGGTTATCAGCGCCCTTGTGGGGCGCTGC TGCCCTTGCCCAATATGCCCGGCCAGAGGCCGGATAGCTGGTCTATTCGCTG CGCTAGGCTACACACCGCCCCACCGCTGCGCGGCAGGGGGAAAGGCGGGCAA AGCCCGCTAAACCCCACACCAAACCCCGCAGAAATACGCTGGAGCGCTTTTA GCCGCTTTAGCGGCCTTTCCCCCTACCCGAAGGGTGGGGGCGCGTGTGCAGC CCCGCAGGGCCTGTCTCGGTCGATCATTCAGCCCGGCTCATCCTTCTGGCGT GGCGGCAGACCGAACAAGGCGCGGTCGTGGTCGCGTTCAAGGTACGCATCCA TTGCCGCCATGAGCCGATCCTCCGGCCACTCGCTGCTGTTCACCTTGGCCAA AATCATGGCCCCCACCAGCACCTTGCGCCTTGTTTCGTTCTTGCGCTCTTGC TGCTGTTCCCTTGCCCGCTCCCGCTGAATTTCGGCATTGATTCGCGCTCGTT GTTCTTCGAGCTTGGCCAGCCGATCCGCCGCCTTGTTGCTCCCCTTAACCAT CTTGACACCCCATTGTTAATGTGCTGTCTCGTAGGCTATCATGGAGGCACAG CGGCGGCAATCCCGACCCTACTTTGTAGGGGAGGGCGCACTTACCGGTTTCT CTTCGAGAAACTGGCCTAACGGCCACCCTTCGGGCGGTGCGCTCTCCGAGGG CCATTGCATGGAGCCGAAAAGCAAAAGCAACAGCGAGGCAGCATGGCGATTT ATCACCTTACGGCGAAAACCGGCAGCAGGTCGGGCGGCCAATCGGCCAGGGC CAAGGCCGACTACATCCAGCGCGAAGGCAAGTATGCCCGCGACATGGATGAA GTCTTGCACGCCGAATCCGGGCACATGCCGGAGTTCGTCGAGCGGCCCGCCG ACTACTGGGATGCTGCCGACCTGTATGAACGCGCCAATGGGCGGCTGTTCAA GGAGGTCGAATTTGCCCTGCCGGTCGAGCTGACCCTCGACCAGCAGAAGGCG CTGGCGTCCGAGTTCGCCCAGCACCTGACCGGTGCCGAGCGCCTGCCGTATA CGCTGGCCATCCATGCCGGTGGCGGCGAGAACCCGCACTGCCACCTGATGAT CTCCGAGCGGATCAATGACGGCATCGAGCGGCCCGCCGCTCAGTGGTTCAAG CGGTACAACGGCAAGACCCCGGAGAAGGGCGGGGCACAGAAGACCGAAGCGC TCAAGCCCAAGGCATGGCTTGAGCAGACCCGCGAGGCATGGGCCGACCATGC CAACCGGGCATTAGAGCGGGCTGGCCACGACGCCCGCATTGACCACAGAACA CTTGAGGCGCAGGGCATCGAGCGCCTGCCCGGTGTTCACCTGGGGCCGAACG TGGTGGAGATGGAAGGCCGGGGCATCCGCACCGACCGGGCAGACGTGGCCCT GAACATCGACACCGCCAACGCCCAGATCATCGACTTACAGGAATACCGGGAG GCAATAGACCATGAACGCAATCGACAGAGTGAAGAAATCCAGAGGCATCAAC GAGTTAGCGGAGCAGATCGAACCGCTGGCCCAGAGCATGGCGACACTGGCCG ACGAAGCCCGGCAGGTCATGAGCCAGACCAAGCAGGCCAGCGAGGCGCAGGC GGCGGAGTGGCTGAAAGCCCAGCGCCAGACAGGGGCGGCATGGGTGGAGCTG GCCAAAGAGTTGCGGGAGGTAGCCGCCGAGGTGAGCAGCGCCGCGCAGAGCG CCCGGAGCGCGTCGCGGGGGTGGCACTGGAAGCTATGGCTAACCGTGATGCT GGCTTCCATGATGCCTACGGTGGTGCTGCTGATCGCATCGTTGCTCTTGCTC GACCTGACGCCACTGACAACCGAGGACGGCTCGATCTGGCTGCGCTTGGTGG CCCGATGAAGAACGACAGGACTTTGCAGGCCATAGGCCGACAGCTCAAGGCC ATGGGCTGTGAGCGCTTCGATATCGGCGTCAGGGACGCACCCACCGGCCAGA TGATGAACCGGGAATGGTCAGCCGCCGAAGTGCTCCAGAACACGCCATGGCT CAAGCGGATGAATGCCCAGGGCAATGACGTGTATATCAGGCCCGCCGAGCAG GAGCGGCATGGTCTGGTGCTGGTGGACGACCTCAGCGAGTTTGACCTGGATG ACATGAAAGCCGAGGGCCGGGAGCCTGCCCTGGTAGTGGAAACCAGCCCGAA GAACTATCAGGCATGGGTCAAGGTGGCCGACGCCGCAGGCGGTGAACTTCGG GGGCAGATTGCCCGGACGCTGGCCAGCGAGTACGACGCCGACCCGGCCAGCG CCGACAGCCGCCACTATGGCCGCTTGGCGGGCTTCACCAACCGCAAGGACAA GCACACCACCCGCGCCGGTTATCAGCCGTGGGTGCTGCTGCGTGAATCCAAG GGCAAGACCGCCACCGCTGGCCCGGCGCTGGTGCAGCAGGCTGGCCAGCAGA TCGAGCAGGCCCAGCGGCAGCAGGAGAAGGCCCGCAGGCTGGCCAGCCTCGA ACTGCCCGAGCGGCAGCTTAGCCGCCACCGGCGCACGGCGCTGGACGAGTAC CGCAGCGAGATGGCCGGGCTGGTCAAGCGCTTCGGTCATGACCTCAGCAAGT GCGACTTTATCGCCGCGCAGAAGCTGGCCAGCCGGGGCCGCAGTGCCGAGGA AATCGGCAAGGCCATGGCCGAGGCCAGCCCAGCGCTGGCAGAGCGCAAGCCC GGCCACGAAGCGGATTACATCGAGCGCACCGTCAGCAAGGTCATGGGTCTGC CCAGCGTCCAGCTTGCGCGGGCCGAGCTGGCACGGGCACCGGCACCCCGCCA GCGAGGCATGGACAGGGGCGGGCCAGATTTCAGCATGTAGTGCTTGCGTTGG TACTCACGCCTGTTATACTATGAGTACTCACGCACAGAAGGGGGTTTTATGG AATACGAAAAAAGCGCTTCAGGGTCGGTCTACCTGATCAAAAGTGACAAGGG CTATTGGTTGCCCGGTGGCTTTGGTTATACGTCAAACAAGGCCGAGGCTGGC CGCTTTTCAGTCGCTGATATGGCCAGCCTTAACCTTGACGGCTGCACCTTGT CCTTGTTCCGCGAAGACAAGCCTTTCGGCCCCGGCAAGTTTCTCGGTGACTG ATATGAAAGACCAAAAGGACAAGCAGACCGGCGACCTGCTGGCCAGCCCTGA CGCTGTACGCCAAGCGCGATATGCCGAGCGCATGAAGGCCAAAGGGATGCGT CAGCGCAAGTTCTGGCTGACCGAC GACGAATACGAGGCGCTGCGCGAGTGCC TGGAAGAACTCAGAGCGGCGCAGGGCGGGGGTAGTGACCCCGCCAGCGCCTA ACCACCAACTGCCTGCAAAGGAGGCAATCAATGGCTACCCATAAGCCTATCA ATATTCTGGAGGCGTTCGCAGCAGCGCCGCCACCGCTGGACTACGTTTTGCC CAACATGGTGGCCGGTACGGTCGGGGCGCTGGTGTCGCCCGGTGGTGCCGGT AAATCCATGCTGGCCCTGCAACTGGCCGCACAGATTGCAGGCGGGCCGGATC TGCTGGAGGTGGGCGAACTGCCCACCGGCCCGGTGATCTACCTGCCCGCCGA AGACCCGCCCACCGCCATTCATCACCGCCTGCACGCCCTTGGGGCGCACCTC AGCGCCGAGGAACGGCAAGCCGTGGCTGACGGCCTGCTGATCCAGCCGCTGA TCGGCAGCCTGCCCAACATCATGGCCCCGGAGTGGTTCGACGGCCTCAAGCG CGCCGCCGAGGGCCGCCGCCTGATGGTGCTGGACACGCTGCGCCGGTTCCAC ATCGAGGAAGAAAACGCCAGCGGCCCCATGGCCCAGGTCATCGGTCGCATGG AGGCCATCGCCGCCGATACCGGGTGCTCTATCGTGTTCCTGCACCATGCCAG CAAGGGCGCGGCCATGATGGGCGCAGGCGACCAGCAGCAGGCCAGCCGGGGC AGCTCGGTACTGGTCGATAACATCCGCTGGCAGTCCTACCTGTCGAGCATGA CCAGCGCCGAGGCCGAGGAATGGGGTGTGGAC GACGACCAGCGCCGGTTCTT CGTCCGCTTCGGTGTGAGCAAGGCCAACTATGGCGCACCGTTCGCTGATCGG TGGTTCAGGCGGCATGACGGCGGGGTGCTCAAGCCCGCCGTGCTGGAGAGGC AGCGCAAGAGCAAGGGGGTGCCCCGTGGTGAAGCCTAAGAACAAGCACAGCC TCAGCCACGTCCGGCACGACCCGGCGCACTGTCTGGCCCCCGGCCTGTTCCG TGCCCTCAAGCGGGGCGAGCGCAAGCGCAGCAAGCTGGACGTGACGTATGAC TACGGCGACGGCAAGCGGATCGAGTTCAGCGGCCCGGAGCCGCTGGGCGCTG ATGATCTGCGCATCCTGCAAGGGCTGGTGGCCATGGCTGGGCCTAATGGCCT AGTGCTTGGCCCGGAACCCAAGACCGAAGGCGGACGGCAGCTCCGGCTGTTC CTGGAACCCAAGTGGGAGGCCGTCACCGCTGAATGCCATGTGGTCAAAGGTA GCTATCGGGCGCTGGCAAAGGAAATCGGGGCAGAGGTCGATAGTGGTGGGGC GCTCAAGCACATACAGGACTGCATCGAGCGCCTTTGGAAGGTATCCATCATC GCCCAGAATGGCCGCAAGCGGCAGGGGTTTCGGCTGCTGTCGGAGTACGCCA GCGACGAGGCGGACGGGCGCCTGTACGTGGCCCTGAACCCCTTGATCGCGCA GGCCGTCATGGGTGGCGGCCAGCATGTGCGCATCAGCATGGACGAGGTGCGG GCGCTGGACAGCGAAACCGCCCGCCTGCTGCACCAGCGGCTGTGTGGCTGGA TCGACCCCGGCAAAACCGGCAAGGCTTCCATAGATACCTTGTGCGGCTATGT CTGGCCGTCAGAGGCCAGTGGTTCGACCATGCGCAAGCGCCGCAAGCGGGTG CGCGAGGCGTTGCCGGAGCTGGTCGCGCTGGGCTGGACGGTAACCGAGTTCG CGGCGGGCAAGTACGACATCACCCGGCCCAAGGCGGCAGGCTGACCCCCCCC ACTCTATTGTAAACAAGACATTTTTATCTTTTATATTCAATGGCTTATTTTC CTGCTAATTGGTAATACCATGAAAAATACCATGCTCAGAAAAGGCTTAACAA TATTTTGAAAAATTGCCTACTGAGCGCTGCCGCACAGCTCCATAGGCCTAAG ATAGATAGCTCCTCACTATCGCTTAGTAGGAAAGGATGAAGCTGATCGGCTT TATCCTTTTTTTATTTTTATATCAATATGATATAAAAATTTAAATCTTCTAA ATTTCTTCTTATTTCATAGAAAATTTTCACATTTATTATTCTTCGCCTTCAC GATTTATCTGTAATTTTTCACTAATCTTCAATTATCGAAATTTAATAGATCA TTCGAGGTTTACGAAGAAGGAGATATACATATGGTGAGCAAGGGCGAAGAGC TGATTAAGGAGAATATGCGCATGAAAGTTGTTATGGAGGGAAGCGTTAACGG ACATCAGTTTAAATGCACCGGCGAGGGCGAGGGTAATCCTTACATGGGCACG CAGACAATGCGCATTAAGGTCATCGAGGGGGGCCCCCTTCCGTTTGCCTTCG ATATTTTAGCTACATCTTTCATGTATGGTTCCCGCACCTTTATTAAGTATCC CAAGGGTATTCCCGACTTCTTTAAGCAGTCCTTCCCGGAGGGGTTTACGTGG GAGCGTGTCACGCGCTATGAGGATGGTGGAGTTGTCACTGTAATGCAAGATA CATCCCTTGAAGACGGGTGTTTAGTTTACCATGTTCAAGTGCGCGGAGTTAA TTTCCCAAGTAACGGCCCAGTAATGCAGAAGAAAACTAAAGGCTGGGAACCC AACACTGAGATGATGTACCCCGCAGACGGTGGGTTACGCGGGTACACGCACA TGGCCTTGAAAGTCGATGGTGGTGGGCACTTATCCTGTAGCTTTGTCACCAC CTACCGTAGCAAAAAGACTGTAGGAAACATCAAAATGCCCGGCATTCATGCA GTAGACCATCGTTTAGAACGCCTTGAGGAGAGTGATAACGAGATGTTTGTAG TACAACGTGAACACGCTGTCGCGAAATTTGCCGGTCTTGGTGGAGGTATGGA TGAACTGTATAAATAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTG CCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAG TGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGG GAACTGCCAGGCATCAAATAAAAC GAAAGGCTCAGTCGAAAGACTGGGCCTT TCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAATCCG CCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGTGGCGGGCAG GACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGCCATCCTGAC GGATGGCCTTTTTGCGTTTCTACAAACTCTTTTGTTTATTTTTCTAAATACA TTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAA TATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCCCTTATTC CCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGGT GAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAA CTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTT TTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCG TGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAAT GACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGGATGGCATGA CAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACTGCGGC CAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTG CACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGA ATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCAATGGC AACAACGTTGCGCAAACT AT TAACTGGCGAAC TACT TACT CTAGCTTCCCGG CAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGC GCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGA GCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCC CGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAA ATAGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTC AGACCAAGTTTACTCATATATACTTTAGATTGATTTCTGAAAGCGACCAGGT GCTCGGCGTGGCAAGACTCGCAGCGAACCCGTAGAAAGCCATGCT
CCAGCCGCCCGCATTGGAGAAATTCTTCAAATTCCCGTTGCACATAGCCCGG PAlS_2889 CAATTCCTTTCCCTGCTCTGCCATAAGCGCAGCGAATGCCGGGTAATACTCG mRuby2 TCAACGATCTGATAGAGAAGGGTTTGCTCGGGTCGGTGGCTCTGGTAACGAC (pACH106 : : PA CAGTATCCCGATCCCGGCTGGCCGTCCTGGCCGCCACATGAGGCATGTTCCG 1S_2889— CGTCCTTGCAATACTGTGTTTACATACAGTCTATCGCTTAGCGGAAAGTTCT mRuby2 ) TTTACCCTCAGCCGAAATGCCTGCCGTTGCTAGACATTGCCAGCCAGTGCCC GTCACTCCCGTACTAACTGTCACGAACCCCTGCAATAACTGTCACGCCCCCC TGCAATAACTGTCACGAACCCCTGCAATAACTGTCACGCCCCCAAACCTGCA AACCCAGCAGGGGCGGGGGCTGGCGGGGTGTTGGAAAAATCCATCCATGATT ATCTAAGAATAATCCACTAGGCGCGGTTATCAGCGCCCTTGTGGGGCGCTGC TGCCCTTGCCCAATATGCCCGGCCAGAGGCCGGATAGCTGGTCTATTCGCTG CGCTAGGCTACACACCGCCCCACCGCTGCGCGGCAGGGGGAAAGGCGGGCAA AGCCCGCTAAACCCCACACCAAACCCCGCAGAAATACGCTGGAGCGCTTTTA GCCGCTTTAGCGGCCTTTCCCCCTACCCGAAGGGTGGGGGCGCGTGTGCAGC CCCGCAGGGCCTGTCTCGGTCGATCATTCAGCCCGGCTCATCCTTCTGGCGT GGCGGCAGACCGAACAAGGCGCGGTCGTGGTCGCGTTCAAGGTACGCATCCA TTGCCGCCATGAGCCGATCCTCCGGCCACTCGCTGCTGTTCACCTTGGCCAA AATCATGGCCCCCACCAGCACCTTGCGCCTTGTTTCGTTCTTGCGCTCTTGC TGCTGTTCCCTTGCCCGCTCCCGCTGAATTTCGGCATTGATTCGCGCTCGTT GTTCTTCGAGCTTGGCCAGCCGATCCGCCGCCTTGTTGCTCCCCTTAACCAT CTTGACACCCCATTGTTAATGTGCTGTCTCGTAGGCTATCATGGAGGCACAG CGGCGGCAATCCCGACCCTACTTTGTAGGGGAGGGCGCACTTACCGGTTTCT CTTCGAGAAACTGGCCTAACGGCCACCCTTCGGGCGGTGCGCTCTCCGAGGG CCATTGCATGGAGCCGAAAAGCAAAAGCAACAGCGAGGCAGCATGGCGATTT ATCACCTTACGGCGAAAACCGGCAGCAGGTCGGGCGGCCAATCGGCCAGGGC CAAGGCCGACTACATCCAGCGCGAAGGCAAGTATGCCCGCGACATGGATGAA GTCTTGCACGCCGAATCCGGGCACATGCCGGAGTTCGTCGAGCGGCCCGCCG ACTACTGGGATGCTGCCGACCTGTATGAACGCGCCAATGGGCGGCTGTTCAA GGAGGTCGAATTTGCCCTGCCGGTCGAGCTGACCCTCGACCAGCAGAAGGCG CTGGCGTCCGAGTTCGCCCAGCACCTGACCGGTGCCGAGCGCCTGCCGTATA CGCTGGCCATCCATGCCGGTGGCGGCGAGAACCCGCACTGCCACCTGATGAT CTCCGAGCGGATCAATGACGGCATCGAGCGGCCCGCCGCTCAGTGGTTCAAG CGGTACAACGGCAAGACCCCGGAGAAGGGCGGGGCACAGAAGACCGAAGCGC TCAAGCCCAAGGCATGGCTTGAGCAGACCCGCGAGGCATGGGCCGACCATGC CAACCGGGCATTAGAGCGGGCTGGCCACGACGCCCGCATTGACCACAGAACA CTTGAGGCGCAGGGCATCGAGCGCCTGCCCGGTGTTCACCTGGGGCCGAACG TGGTGGAGATGGAAGGCCGGGGCATCCGCACCGACCGGGCAGACGTGGCCCT GAACATCGACACCGCCAACGCCCAGATCATCGACTTACAGGAATACCGGGAG GCAATAGACCATGAACGCAATCGACAGAGTGAAGAAATCCAGAGGCATCAAC GAGTTAGCGGAGCAGATCGAACCGCTGGCCCAGAGCATGGCGACACTGGCCG ACGAAGCCCGGCAGGTCATGAGCCAGACCAAGCAGGCCAGCGAGGCGCAGGC GGCGGAGTGGCTGAAAGCCCAGCGCCAGACAGGGGCGGCATGGGTGGAGCTG GCCAAAGAGTTGCGGGAGGTAGCCGCCGAGGTGAGCAGCGCCGCGCAGAGCG CCCGGAGCGCGTCGCGGGGGTGGCACTGGAAGCTATGGCTAACCGTGATGCT GGCTTCCATGATGCCTACGGTGGTGCTGCTGATCGCATCGTTGCTCTTGCTC GACCTGACGCCACTGACAACCGAGGACGGCTCGATCTGGCTGCGCTTGGTGG CCCGATGAAGAACGACAGGACTTTGCAGGCCATAGGCCGACAGCTCAAGGCC ATGGGCTGTGAGCGCTTCGATATCGGCGTCAGGGACGCACCCACCGGCCAGA TGATGAACCGGGAATGGTCAGCCGCCGAAGTGCTCCAGAACACGCCATGGCT CAAGCGGATGAATGCCCAGGGCAATGACGTGTATATCAGGCCCGCCGAGCAG GAGCGGCATGGTCTGGTGCTGGTGGACGACCTCAGCGAGTTTGACCTGGATG ACATGAAAGCCGAGGGCCGGGAGCCTGCCCTGGTAGTGGAAACCAGCCCGAA GAACTATCAGGCATGGGTCAAGGTGGCCGACGCCGCAGGCGGTGAACTTCGG GGGCAGATTGCCCGGACGCTGGCCAGCGAGTACGACGCCGACCCGGCCAGCG CCGACAGCCGCCACTATGGCCGCTTGGCGGGCTTCACCAACCGCAAGGACAA GCACACCACCCGCGCCGGTTATCAGCCGTGGGTGCTGCTGCGTGAATCCAAG GGCAAGACCGCCACCGCTGGCCCGGCGCTGGTGCAGCAGGCTGGCCAGCAGA TCGAGCAGGCCCAGCGGCAGCAGGAGAAGGCCCGCAGGCTGGCCAGCCTCGA ACTGCCCGAGCGGCAGCTTAGCCGCCACCGGCGCACGGCGCTGGACGAGTAC CGCAGCGAGATGGCCGGGCTGGTCAAGCGCTTCGGTCATGACCTCAGCAAGT GCGACTTTATCGCCGCGCAGAAGCTGGCCAGCCGGGGCCGCAGTGCCGAGGA AATCGGCAAGGCCATGGCCGAGGCCAGCCCAGCGCTGGCAGAGCGCAAGCCC GGCCACGAAGCGGATTACATCGAGCGCACCGTCAGCAAGGTCATGGGTCTGC CCAGCGTCCAGCTTGCGCGGGCCGAGCTGGCACGGGCACCGGCACCCCGCCA GCGAGGCATGGACAGGGGCGGGCCAGATTTCAGCATGTAGTGCTTGCGTTGG TACTCACGCCTGTTATACTATGAGTACTCACGCACAGAAGGGGGTTTTATGG AATACGAAAAAAGCGCTTCAGGGTCGGTCTACCTGATCAAAAGTGACAAGGG CTATTGGTTGCCCGGTGGCTTTGGTTATACGTCAAACAAGGCCGAGGCTGGC CGCTTTTCAGTCGCTGATATGGCCAGCCTTAACCTTGACGGCTGCACCTTGT CCTTGTTCCGCGAAGACAAGCCTTTCGGCCCCGGCAAGTTTCTCGGTGACTG ATATGAAAGACCAAAAGGACAAGCAGACCGGCGACCTGCTGGCCAGCCCTGA CGCTGTACGCCAAGCGCGATATGCCGAGCGCATGAAGGCCAAAGGGATGCGT CAGCGCAAGTTCTGGCTGACCGAC GACGAATACGAGGCGCTGCGCGAGTGCC TGGAAGAACTCAGAGCGGCGCAGGGCGGGGGTAGTGACCCCGCCAGCGCCTA ACCACCAACTGCCTGCAAAGGAGGCAATCAATGGCTACCCATAAGCCTATCA ATATTCTGGAGGCGTTCGCAGCAGCGCCGCCACCGCTGGACTACGTTTTGCC CAACATGGTGGCCGGTACGGTCGGGGCGCTGGTGTCGCCCGGTGGTGCCGGT AAATCCATGCTGGCCCTGCAACTGGCCGCACAGATTGCAGGCGGGCCGGATC TGCTGGAGGTGGGCGAACTGCCCACCGGCCCGGTGATCTACCTGCCCGCCGA AGACCCGCCCACCGCCATTCATCACCGCCTGCACGCCCTTGGGGCGCACCTC AGCGCCGAGGAACGGCAAGCCGTGGCTGACGGCCTGCTGATCCAGCCGCTGA TCGGCAGCCTGCCCAACATCATGGCCCCGGAGTGGTTCGACGGCCTCAAGCG CGCCGCCGAGGGCCGCCGCCTGATGGTGCTGGACACGCTGCGCCGGTTCCAC ATCGAGGAAGAAAACGCCAGCGGCCCCATGGCCCAGGTCATCGGTCGCATGG AGGCCATCGCCGCCGATACCGGGTGCTCTATCGTGTTCCTGCACCATGCCAG CAAGGGCGCGGCCATGATGGGCGCAGGCGACCAGCAGCAGGCCAGCCGGGGC AGCTCGGTACTGGTCGATAACATCCGCTGGCAGTCCTACCTGTCGAGCATGA CCAGCGCCGAGGCCGAGGAATGGGGTGTGGAC GACGACCAGCGCCGGTTCTT CGTCCGCTTCGGTGTGAGCAAGGCCAACTATGGCGCACCGTTCGCTGATCGG TGGTTCAGGCGGCATGACGGCGGGGTGCTCAAGCCCGCCGTGCTGGAGAGGC AGCGCAAGAGCAAGGGGGTGCCCCGTGGTGAAGCCTAAGAACAAGCACAGCC TCAGCCACGTCCGGCACGACCCGGCGCACTGTCTGGCCCCCGGCCTGTTCCG TGCCCTCAAGCGGGGCGAGCGCAAGCGCAGCAAGCTGGACGTGACGTATGAC TACGGCGACGGCAAGCGGATCGAGTTCAGCGGCCCGGAGCCGCTGGGCGCTG ATGATCTGCGCATCCTGCAAGGGCTGGTGGCCATGGCTGGGCCTAATGGCCT AGTGCTTGGCCCGGAACCCAAGACCGAAGGCGGACGGCAGCTCCGGCTGTTC CTGGAACCCAAGTGGGAGGCCGTCACCGCTGAATGCCATGTGGTCAAAGGTA GCTATCGGGCGCTGGCAAAGGAAATCGGGGCAGAGGTCGATAGTGGTGGGGC GCTCAAGCACATACAGGACTGCATCGAGCGCCTTTGGAAGGTATCCATCATC GCCCAGAATGGCCGCAAGCGGCAGGGGTTTCGGCTGCTGTCGGAGTACGCCA GCGACGAGGCGGACGGGCGCCTGTACGTGGCCCTGAACCCCTTGATCGCGCA GGCCGTCATGGGTGGCGGCCAGCATGTGCGCATCAGCATGGACGAGGTGCGG GCGCTGGACAGCGAAACCGCCCGCCTGCTGCACCAGCGGCTGTGTGGCTGGA TCGACCCCGGCAAAACCGGCAAGGCTTCCATAGATACCTTGTGCGGCTATGT CTGGCCGTCAGAGGCCAGTGGTTCGACCATGCGCAAGCGCCGCAAGCGGGTG CGCGAGGCGTTGCCGGAGCTGGTCGCGCTGGGCTGGACGGTAACCGAGTTCG CGGCGGGCAAGTACGACATCACCCGGCCCAAGGCGGCAGGCTGACCCCCCCC ACTCTATTGTAAACAAGACATTTTTATCTTTTATATTCAATGGCTTATTTTC CTGCTAATTGGTAATACCATGAAAAATACCATGCTCAGAAAAGGCTTAACAA TATTTTGAAAAATTGCCTACTGAGCGCTGCCGCACAGCTCCATAGGCCTTGT ACACTTGGAACTTTGCAGAAAAATTGATGAAGACGTTCAAAACTGATTTCAG ACACAGCAATTGACCTAGCTTGAAAAATGCTAATGGTAGCACAGATACAGAT TCTTCATATTTTCTTTATAGTCATTACAAACAATATCAATCAAAGCCATTTT ATTCATGGTTTCTCCTTGACTCTCTCTTAATCTAGCTTCAGAAGAAGGAGAT ATACATATGGTGAGCAAGGGCGAAGAGCTGATTAAGGAGAATATGCGCATGA AAGTTGTTATGGAGGGAAGCGTTAACGGACATCAGTTTAAATGCACCGGCGA GGGCGAGGGTAATCCTTACATGGGCACGCAGACAATGCGCATTAAGGTCATC GAGGGGGGCCCCCTTCCGTTTGCCTTCGATATTTTAGCTACATCTTTCATGT ATGGTTCCCGCACCTTTATTAAGTATCCCAAGGGTATTCCCGACTTCTTTAA GCAGTCCTTCCCGGAGGGGTTTACGTGGGAGCGTGTCACGCGCTATGAGGAT GGTGGAGTTGTCACTGTAATGCAAGATACATCCCTTGAAGACGGGTGTTTAG TTTACCATGTTCAAGTGCGCGGAGTTAATTTCCCAAGTAACGGCCCAGTAAT GCAGAAGAAAACTAAAGGCTGGGAACCCAACACTGAGATGATGTACCCCGCA GACGGTGGGTTACGCGGGTACACGCACATGGCCTTGAAAGTCGATGGTGGTG GGCACTTATCCTGTAGCTTTGTCACCACCTACCGTAGCAAAAAGACTGTAGG AAACATCAAAATGCCCGGCATTCATGCAGTAGACCATCGTTTAGAACGCCTT GAGGAGAGTGATAACGAGATGTTTGTAGTACAACGTGAACACGCTGTCGCGA AATTTGCCGGTCTTGGTGGAGGTATGGATGAACTGTATAAATAATCAGAACG CAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTC CCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTA GTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAAC GAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGT GAACGCTCTCCTGAGTAGGACAAATCCGCCGGGAGCGGATTTGAACGTTGCG AAGCAACGGCCCGGAGGGTGGCGGGCAGGACGCCCGCCATAAACTGCCAGGC ATCAAATTAAGCAGAAGGCCATCCTGACGGATGGCCTTTTTGCGTTTCTACA AACTCTTTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAG ACAATAACCCTGATAAATGCTTCAATAATATTGAAAAAGGAAGAGTAT GAGT ATTCAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTC CTGTTTTTGCTCACCCAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCA GTTGGGTGCACGAGTGGGTTACATCGAACTGGATCTCAACAGCGGTAAGATC CTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATGAGCACTTTTAAAG TTCTGCTATGTGGCGCGGTATTATCCCGTGTTGACGCCGGGCAAGAGCAACT CGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTC AC AGAAAAGC AT CT TACGGATGGC ATGACAGT AAGAGAAT T ATGCAGT GCTG CCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGG AGGACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACT CGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGAC GAGC GTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAAC TGGCGAACTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAG GCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGT TTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCGGTATCATTGC AGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACAC GACG GGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTG CCTCACTGATTAAGCATTGGTAACTGTCAGACCAAGTTTACTCATATATACT TTAGATTGATTTCTGAAAGCGACCAGGTGCTCGGCGTGGCAAGACTCGCAGC GAACCCGTAGAAAGCCATGCT
CCAGCCGCCCGCATTGGAGAAATTCTTCAAATTCCCGTTGCACATAGCCCGG PAlS_2885 CAATTCCTTTCCCTGCTCTGCCATAAGCGCAGCGAATGCCGGGTAATACTCG mRuby2 TCAACGATCTGATAGAGAAGGGTTTGCTCGGGTCGGTGGCTCTGGTAACGAC (pACH106 : : PA CAGTATCCCGATCCCGGCTGGCCGTCCTGGCCGCCACATGAGGCATGTTCCG 1S_2885— CGTCCTTGCAATACTGTGTTTACATACAGTCTATCGCTTAGCGGAAAGTTCT mRuby2 ) TTTACCCTCAGCCGAAATGCCTGCCGTTGCTAGACATTGCCAGCCAGTGCCC GTCACTCCCGTACTAACTGTCACGAACCCCTGCAATAACTGTCACGCCCCCC TGCAATAACTGTCACGAACCCCTGCAATAACTGTCACGCCCCCAAACCTGCA AACCCAGCAGGGGCGGGGGCTGGCGGGGTGTTGGAAAAATCCATCCATGATT ATCTAAGAATAATCCACTAGGCGCGGTTATCAGCGCCCTTGTGGGGCGCTGC TGCCCTTGCCCAATATGCCCGGCCAGAGGCCGGATAGCTGGTCTATTCGCTG CGCTAGGCTACACACCGCCCCACCGCTGCGCGGCAGGGGGAAAGGCGGGCAA AGCCCGCTAAACCCCACACCAAACCCCGCAGAAATACGCTGGAGCGCTTTTA GCCGCTTTAGCGGCCTTTCCCCCTACCCGAAGGGTGGGGGCGCGTGTGCAGC CCCGCAGGGCCTGTCTCGGTCGATCATTCAGCCCGGCTCATCCTTCTGGCGT GGCGGCAGACCGAACAAGGCGCGGTCGTGGTCGCGTTCAAGGTACGCATCCA TTGCCGCCATGAGCCGATCCTCCGGCCACTCGCTGCTGTTCACCTTGGCCAA AATCATGGCCCCCACCAGCACCTTGCGCCTTGTTTCGTTCTTGCGCTCTTGC TGCTGTTCCCTTGCCCGCTCCCGCTGAATTTCGGCATTGATTCGCGCTCGTT GTTCTTCGAGCTTGGCCAGCCGATCCGCCGCCTTGTTGCTCCCCTTAACCAT CTTGACACCCCATTGTTAATGTGCTGTCTCGTAGGCTATCATGGAGGCACAG CGGCGGCAATCCCGACCCTACTTTGTAGGGGAGGGCGCACTTACCGGTTTCT CTTCGAGAAACTGGCCTAACGGCCACCCTTCGGGCGGTGCGCTCTCCGAGGG CCATTGCATGGAGCCGAAAAGCAAAAGCAACAGCGAGGCAGCATGGCGATTT ATCACCTTACGGCGAAAACCGGCAGCAGGTCGGGCGGCCAATCGGCCAGGGC CAAGGCCGACTACATCCAGCGCGAAGGCAAGTATGCCCGCGACATGGATGAA GTCTTGCACGCCGAATCCGGGCACATGCCGGAGTTCGTCGAGCGGCCCGCCG ACTACTGGGATGCTGCCGACCTGTATGAACGCGCCAATGGGCGGCTGTTCAA GGAGGTCGAATTTGCCCTGCCGGTCGAGCTGACCCTCGACCAGCAGAAGGCG CTGGCGTCCGAGTTCGCCCAGCACCTGACCGGTGCCGAGCGCCTGCCGTATA CGCTGGCCATCCATGCCGGTGGCGGCGAGAACCCGCACTGCCACCTGATGAT CTCCGAGCGGATCAATGACGGCATCGAGCGGCCCGCCGCTCAGTGGTTCAAG CGGTACAACGGCAAGACCCCGGAGAAGGGCGGGGCACAGAAGACCGAAGCGC TCAAGCCCAAGGCATGGCTTGAGCAGACCCGCGAGGCATGGGCCGACCATGC CAACCGGGCATTAGAGCGGGCTGGCCACGACGCCCGCATTGACCACAGAACA CTTGAGGCGCAGGGCATCGAGCGCCTGCCCGGTGTTCACCTGGGGCCGAACG TGGTGGAGATGGAAGGCCGGGGCATCCGCACCGACCGGGCAGACGTGGCCCT GAACATCGACACCGCCAACGCCCAGATCATCGACTTACAGGAATACCGGGAG GCAATAGACCATGAACGCAATCGACAGAGTGAAGAAATCCAGAGGCATCAAC GAGTTAGCGGAGCAGATCGAACCGCTGGCCCAGAGCATGGCGACACTGGCCG ACGAAGCCCGGCAGGTCATGAGCCAGACCAAGCAGGCCAGCGAGGCGCAGGC GGCGGAGTGGCTGAAAGCCCAGCGCCAGACAGGGGCGGCATGGGTGGAGCTG GCCAAAGAGTTGCGGGAGGTAGCCGCCGAGGTGAGCAGCGCCGCGCAGAGCG CCCGGAGCGCGTCGCGGGGGTGGCACTGGAAGCTATGGCTAACCGTGATGCT GGCTTCCATGATGCCTACGGTGGTGCTGCTGATCGCATCGTTGCTCTTGCTC GACCTGACGCCACTGACAACCGAGGACGGCTCGATCTGGCTGCGCTTGGTGG CCCGATGAAGAACGACAGGACTTTGCAGGCCATAGGCCGACAGCTCAAGGCC ATGGGCTGTGAGCGCTTCGATATCGGCGTCAGGGACGCACCCACCGGCCAGA TGATGAACCGGGAATGGTCAGCCGCCGAAGTGCTCCAGAACACGCCATGGCT CAAGCGGATGAATGCCCAGGGCAATGACGTGTATATCAGGCCCGCCGAGCAG GAGCGGCATGGTCTGGTGCTGGTGGACGACCTCAGCGAGTTTGACCTGGATG ACATGAAAGCCGAGGGCCGGGAGCCTGCCCTGGTAGTGGAAACCAGCCCGAA GAACTATCAGGCATGGGTCAAGGTGGCCGACGCCGCAGGCGGTGAACTTCGG GGGCAGATTGCCCGGACGCTGGCCAGCGAGTACGACGCCGACCCGGCCAGCG CCGACAGCCGCCACTATGGCCGCTTGGCGGGCTTCACCAACCGCAAGGACAA GCACACCACCCGCGCCGGTTATCAGCCGTGGGTGCTGCTGCGTGAATCCAAG GGCAAGACCGCCACCGCTGGCCCGGCGCTGGTGCAGCAGGCTGGCCAGCAGA TCGAGCAGGCCCAGCGGCAGCAGGAGAAGGCCCGCAGGCTGGCCAGCCTCGA ACTGCCCGAGCGGCAGCTTAGCCGCCACCGGCGCACGGCGCTGGACGAGTAC CGCAGCGAGATGGCCGGGCTGGTCAAGCGCTTCGGTCATGACCTCAGCAAGT GCGACTTTATCGCCGCGCAGAAGCTGGCCAGCCGGGGCCGCAGTGCCGAGGA AATCGGCAAGGCCATGGCCGAGGCCAGCCCAGCGCTGGCAGAGCGCAAGCCC GGCCACGAAGCGGATTACATCGAGCGCACCGTCAGCAAGGTCATGGGTCTGC CCAGCGTCCAGCTTGCGCGGGCCGAGCTGGCACGGGCACCGGCACCCCGCCA GCGAGGCATGGACAGGGGCGGGCCAGATTTCAGCATGTAGTGCTTGCGTTGG TACTCACGCCTGTTATACTATGAGTACTCACGCACAGAAGGGGGTTTTATGG AATACGAAAAAAGCGCTTCAGGGTCGGTCTACCTGATCAAAAGTGACAAGGG CTATTGGTTGCCCGGTGGCTTTGGTTATACGTCAAACAAGGCCGAGGCTGGC CGCTTTTCAGTCGCTGATATGGCCAGCCTTAACCTTGACGGCTGCACCTTGT CCTTGTTCCGCGAAGACAAGCCTTTCGGCCCCGGCAAGTTTCTCGGTGACTG ATATGAAAGACCAAAAGGACAAGCAGACCGGCGACCTGCTGGCCAGCCCTGA CGCTGTACGCCAAGCGCGATATGCCGAGCGCATGAAGGCCAAAGGGATGCGT CAGCGCAAGTTCTGGCTGACCGAC GACGAATACGAGGCGCTGCGCGAGTGCC TGGAAGAACTCAGAGCGGCGCAGGGCGGGGGTAGTGACCCCGCCAGCGCCTA ACCACCAACTGCCTGCAAAGGAGGCAATCAATGGCTACCCATAAGCCTATCA ATATTCTGGAGGCGTTCGCAGCAGCGCCGCCACCGCTGGACTACGTTTTGCC CAACATGGTGGCCGGTACGGTCGGGGCGCTGGTGTCGCCCGGTGGTGCCGGT AAATCCATGCTGGCCCTGCAACTGGCCGCACAGATTGCAGGCGGGCCGGATC TGCTGGAGGTGGGCGAACTGCCCACCGGCCCGGTGATCTACCTGCCCGCCGA AGACCCGCCCACCGCCATTCATCACCGCCTGCACGCCCTTGGGGCGCACCTC AGCGCCGAGGAACGGCAAGCCGTGGCTGACGGCCTGCTGATCCAGCCGCTGA TCGGCAGCCTGCCCAACATCATGGCCCCGGAGTGGTTCGACGGCCTCAAGCG CGCCGCCGAGGGCCGCCGCCTGATGGTGCTGGACACGCTGCGCCGGTTCCAC ATCGAGGAAGAAAACGCCAGCGGCCCCATGGCCCAGGTCATCGGTCGCATGG AGGCCATCGCCGCCGATACCGGGTGCTCTATCGTGTTCCTGCACCATGCCAG CAAGGGCGCGGCCATGATGGGCGCAGGCGACCAGCAGCAGGCCAGCCGGGGC AGCTCGGTACTGGTCGATAACATCCGCTGGCAGTCCTACCTGTCGAGCATGA CCAGCGCCGAGGCCGAGGAATGGGGTGTGGAC GACGACCAGCGCCGGTTCTT CGTCCGCTTCGGTGTGAGCAAGGCCAACTATGGCGCACCGTTCGCTGATCGG TGGTTCAGGCGGCATGACGGCGGGGTGCTCAAGCCCGCCGTGCTGGAGAGGC AGCGCAAGAGCAAGGGGGTGCCCCGTGGTGAAGCCTAAGAACAAGCACAGCC TCAGCCACGTCCGGCACGACCCGGCGCACTGTCTGGCCCCCGGCCTGTTCCG TGCCCTCAAGCGGGGCGAGCGCAAGCGCAGCAAGCTGGACGTGACGTATGAC TACGGCGACGGCAAGCGGATCGAGTTCAGCGGCCCGGAGCCGCTGGGCGCTG ATGATCTGCGCATCCTGCAAGGGCTGGTGGCCATGGCTGGGCCTAATGGCCT AGTGCTTGGCCCGGAACCCAAGACCGAAGGCGGACGGCAGCTCCGGCTGTTC CTGGAACCCAAGTGGGAGGCCGTCACCGCTGAATGCCATGTGGTCAAAGGTA GCTATCGGGCGCTGGCAAAGGAAATCGGGGCAGAGGTCGATAGTGGTGGGGC GCTCAAGCACATACAGGACTGCATCGAGCGCCTTTGGAAGGTATCCATCATC GCCCAGAATGGCCGCAAGCGGCAGGGGTTTCGGCTGCTGTCGGAGTACGCCA GCGACGAGGCGGACGGGCGCCTGTACGTGGCCCTGAACCCCTTGATCGCGCA GGCCGTCATGGGTGGCGGCCAGCATGTGCGCATCAGCATGGACGAGGTGCGG GCGCTGGACAGCGAAACCGCCCGCCTGCTGCACCAGCGGCTGTGTGGCTGGA TCGACCCCGGCAAAACCGGCAAGGCTTCCATAGATACCTTGTGCGGCTATGT CTGGCCGTCAGAGGCCAGTGGTTCGACCATGCGCAAGCGCCGCAAGCGGGTG CGCGAGGCGTTGCCGGAGCTGGTCGCGCTGGGCTGGACGGTAACCGAGTTCG CGGCGGGCAAGTACGACATCACCCGGCCCAAGGCGGCAGGCTGACCCCCCCC ACTCTATTGTAAACAAGACATTTTTATCTTTTATATTCAATGGCTTATTTTC CTGCTAATTGGTAATACCATGAAAAATACCATGCTCAGAAAAGGCTTAACAA TATTTTGAAAAATTGCCTACTGAGCGCTGCCGCACAGCTCCATAGGCCACTC AAACTTGCAGTTGCAATTAACAGCGTGGTAAGCAAGACCGTTAAAAATAACC GC AAGGCTATGGGAACACGTAGAACGTTCAAAAGC ACT CTCTC ATT TT TACT CTATATTTTGCATTGTAACCAACAACGAAAAGAAAAACTCTCCATTGTTTCT TCATCTTTATTATCTAATCTTTAAGTCATCTTAAAACAAACATTAAAGGGTT TTAACCATGAAAATGACGGCTAAAATTGCATTATTCAGTATGGTGAGCAAGG GCGAAGAGCTGATTAAGGAGAATATGCGCATGAAAGTTGTTATGGAGGGAAG CGTTAACGGACATCAGTTTAAATGCACCGGCGAGGGCGAGGGTAATCCTTAC ATGGGCACGCAGACAATGCGCATTAAGGTCATCGAGGGGGGCCCCCTTCCGT TTGCCTTCGATATTTTAGCTACATCTTTCATGTATGGTTCCCGCACCTTTAT TAAGTATCCCAAGGGTATTCCCGACTTCTTTAAGCAGTCCTTCCCGGAGGGG TTTACGTGGGAGCGTGTCACGCGCTATGAGGATGGTGGAGTTGTCACTGTAA TGCAAGATACATCCCTTGAAGACGGGTGTTTAGTTTACCATGTTCAAGTGCG CGGAGTTAATTTCCCAAGTAACGGCCCAGTAATGCAGAAGAAAACTAAAGGC TGGGAACCCAACACTGAGATGATGTACCCCGCAGACGGTGGGTTACGCGGGT ACACGCACATGGCCTTGAAAGTCGATGGTGGTGGGCACTTATCCTGTAGCTT TGTCACCACCTACCGTAGCAAAAAGACTGTAGGAAACATCAAAATGCCCGGC ATTCATGCAGTAGACCATCGTTTAGAACGCCTTGAGGAGAGTGATAAC GAGA TGTTTGTAGTACAACGTGAACACGCTGTCGCGAAATTTGCCGGTCTTGGTGG AGGT AT GG AT GAAC T GT AT AAAT AAT C AGAAC GC AGAAGC GGT C TG AT AAAA CAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGA ACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGC GAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGA CTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGG ACAAATCCGCCGGGAGCGGATTTGAACGTTGCGAAGCAACGGCCCGGAGGGT GGCGGGCAGGACGCCCGCCATAAACTGCCAGGCATCAAATTAAGCAGAAGGC CATCCTGACGGATGGCCTTTTTGCGTTTCTACAAACTCTTTTGTTTATTTTT CTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAAATG CTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCG CCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGA AACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGT TACATCGAACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCG AAGAACGTTTTCCAATGATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGT ATTATCCCGTGTTGACGCCGGGCAAGAGCAACTCGGTCGCCGCATACACTAT TCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCATCTTACGG ATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAA CACTGCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACC GCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAAC CGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGT AGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTTACTCTA GCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGAC C ACT TCTGCGCTCGGCCCTTCCGGCTGGCTGGTTT ATT GCTGAT AA CTGG AGCCGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGT AAGCCCTCCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGG AT GAAC GAAATAGACAGATCGCTGAGAT AGGT GCCTCACTGATTAAGC ATT G GTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTCTGAAAG CGACCAGGTGCTCGGCGTGGCAAGACTCGCAGCGAACCCGTAGAAAGCCATG
CT
ATGGTGAGCAAGGGCGAAGAGTTGTTCACAGGTGTCGTGCCAATTCTGGTAG mClover3 AACTTGACGGAGATGTAAATGGCCACAAGTTCAGTGTGCGCGGGGAAGGTGA AGGCGACGCTACGAATGGGAAATTAACACTTAAATTCATTTGTACTACAGGG AAGTTACCCGTACCCTGGCCGACCCTGGTGACCACTTTCGGATACGGAGTTG CGTGTTTCAGCCGCTACCCTGATCACATGAAGCAGCATGATTTCTTTAAATC CGCAATGCCTGAAGGCTACGTGCAAGAGCGCACAATTTCGTTCAAAGACGAC GGCACCTACAAGACGCGTGCTGAGGTCAAATTTGAGGGCGATACGCTGGTCA ATCGCATTGAGTTAAAAGGGATCGATTTTAAAGAGGATGGTAACATCTTAGG GCATAAGTTAGAATACAACTTCAATTCACATTATGTCTATATTACCGCAGAC AAGCAAAAAAATTGTATCAAAGCGAATTTCAAAATTCGTCACAATGTAGAAG ATGGATCGGTCCAGTTGGCGGACCACTACCAACAAAACACTCCTATCGGGGA TGGGCCGGTGTTGTTGCCGGATAACCACTATTTATCACATCAGTCTAAGTTA AGTAAAGACCCGAACGAAAAACGTGATCACATGGTTCTTCTTGAATTTGTTA CAGCAGCCGGAATCACCCACGGTATGGACGAATTGTATAAATAA

Claims

CLAIMS WHAT IS CLAIMED:
1. A reporter polynucleotide comprising a sequence comprising a regulatory region of an outer membrane (OM) stress-responsive gene of an Acinetobacter bacterium operably linked to a sequence encoding a reporter molecule, wherein the OM stress-responsive gene is modulated in response to a stress to the outer membrane of the Acinetobacter bacterium.
2. The reporter polynucleotide of claim 1, wherein the Acinetobacter bacterium is Acinetobacter apis, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii, Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter gerneri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus, Acinetobacter harbinensis, Acinetobacter indicus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter kookii, Acinetobacter Iwoffii, Acinetobacter nectaris, Acinetobacter nosocomialis, Acinetobacter parvus, Acinetobacter pakistanensis, Acinetobacter pittii, Acinetobacter puyangensis, Acinetobacter qingfengensis, Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii, Acinetobacter soli, Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis, ox Acinetobacter venetianus, optionally wherein the Acinetobacter bacterium is Acinetobacter baumannii.
3. The reporter polynucleotide of claim 1 or claim 2, wherein the Acinetobacter bacterium is ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA-1605, ATCC BAA-1709, ATCC BAA-1710, ATCC BAA- 1789, ATCC BAA-1790, ATCC BAA-1791, ATCC BAA-1792, ATCC BAA-1793, ATCC BAA-1794, ATCC BAA-1795, ATCC BAA-1796, ATCC BAA-1797, ATCC BAA-1798, ATCC BAA-1799, ATCC BAA-1800, ATCC BAA-1878, ATCC BAA-2093, ATCC BAA-747, SDF, AYE, ACICU, AB0057, AB307-0294, 1656-2, MDR-ZJ06, TCDC-AB0715, MDR-TJ, TYTH-1, D1279779, BJAB07104, BJAB0715, BJAB0868, ZW85-1, AbH120-A2, AB030, AB031, AC29, LAC-4, Ab307-0294, 1656-2, ID 403, AABA041, AABA046, or a modified strain thereof, optionally wherein the Acinetobacter bacterium is ATCC 17978, Ab307-0294, AABA041, or AABA046.
4. The reporter polynucleotide of any of claims 1-3, wherein:
the stress to the outer membrane of the Acinetobacter bacterium is or is caused by depletion of BamA or treatment with polymyxin B nonapeptide (PMBN); or
the stress to the outer membrane of the Acinetobacter bacterium is or is caused by depletion of BamA and treatment with PMBN.
5. The reporter polynucleotide of any of claims 1-4, wherein the OM stress- responsive gene is upregulated or downregulated in response to the stress.
6. The reporter polynucleotide of any of claims 1-5, wherein the OM stress- responsive gene is upregulated in response to the stress at least or about at least 2-fold, 3 -fold, 4- fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more in response to the stress.
7. The reporter polynucleotide of any of claims 1-6, wherein the OM stress- responsive gene is upregulated in response to the stress, and wherein the OM stress-responsive gene is A1S_0012, A1S_0023, A1S_0027, A1S_0028, A1SJ3029, A1S_0030, A1S_0031, A1S_0032, A1S_0033, A1S_0037, A1S_0040, A1S_0041, A1S_0044, A1S_0066, A1S_0092, A1SJ3093, A1SJ3109, A1S_0110, A1S_0112, A1S_0113, A1S_0114, A1S_0115, A1S_0116, A1S_0117, A1S_0118, A1S_0126, A1S_0158, A1S_0170, A1S_0175, A1S_0178, A1S_0189, A1S_0224, A1S_0245, A1S_0256, A1S_0276, A1S_0293, A1S_0301, A1S_0309, A1S_0310, A1S_0332, A1SJ3333, A1S_0363, A1S_0372, A1S_0376, A1S_0391, A1S_0392, A1S_0401, A1S_0441, A1S_0462, A1S_0463, A1S_0464, A1S_0465, A1S_0466, A1S_0494, A1S_0508, A1S_0509, A1S_0510, A1S_0511, A1S_0512, A1S_0514, A1S_0516, A1S_0518, A1S_0519, A1S_0520, A1S_0521, A1S_0522, A1S_0523, A1S_0527, A1S_0535, A1S_0536, A1S_0537, A1S_0538, A1S_0547, A1S_0559, A1S_0561, A1S_0562, A1S_0563, A1S_0564, A1S_0566, A1S_0567, A1S_0568, A1S_0570, A1S_0624, A1S_0630, A1S_0631, A1S_0633, A1S_0634, A1S_0640, A1S_0641, A1S_0642, A1S_0643, A1S_0644, A1S_0645, A1S_0646, A1S_0647, A1S_ _0650, A1S_ _0663 A1S_ _0664, A1S_ _0665 A1S_ _0666, A1S_ _0667 A1S_ _0669, A1S_ _0670,
A1S_ _0671, A1S_ _0673 A1S_ _0677, A1S_ _0680 A1S_ _0683, A1S_ _0714 A1S_ _0717, A1S_ _0718,
A1S_ _0719, A1S_ _0736 A1S_ _0738, A1S_ _0739 A1S_ _0749, A1S_ _0770 A1S_ _0772, A1S_ _0779,
A1S_ _0780, A1S_ _0781 A1S_ _0800, A1S_ _0804 A1S_ _0830, A1S_ _0831 A1S_ _0832, A1S_ _0834,
A1S_ _0835, A1S_ _0861 A1S_ _0884, A1S_ _0889 A1S_ _0929, A1S_ _0930 A1S_ _0931, A1S_ _0932,
A1S_ _0935, A1S_ _0945 A1S_ _0959, A1S_ _0980 A1S_ _1003, A1S_ _1009 A1S_ _1010, A1S_ _1027,
A1S_ _1028, A1S_ _1030 A1S_ _1031, A1S_ _1049 A1S_ _1081, A1S_ _1106 A1S_ _1107, A1S_ _1120,
A1S_ _1121, A1S_ _1123 A1S_ _1132, A1S_ _1133 A1S_ _1134, A1S_ _1139 A1S_ _1143, A1S_ _1145,
A1S_ _1146, A1S_ _1148 A1S_ _1149, A1S_ _1150 A1S_ _1151, A1S_ _1152 A1S_ _1153, A1S_ _1155,
A1S_ _1156, A1S_ _1157 A1S_ _1158, A1S_ _1159 A1S_ _1160, A1S_ _1161 A1S_ _1162, A1S_ _1163,
A1S_ _1164, A1S_ _1165 A1S_ _1167, A1S_ _1171 A1S_ _1172, A1S_ _1173 A1S_ _1180, A1S_ _1184,
A1S_ _1186, A1S_ _1198 A1S_ _1202, A1S_ _1203 A1S_ _1223, A1S_ _1224 A1S_ _1225, A1S_ _1230,
A1S_ _1236, A1S_ _1237 A1S_ _1248, A1S_ _1255 A1S_ _1274, A1S_ _1286 A1S_ _1359, A1S_ _1360,
A1S_ _1361, A1S_ _1362 A1S_ _1363, A1S_ _1383 A1S_ _1384, A1S_ _1385 A1S_ _1386, A1S_ _1387,
A1S_ _1393, A1S_ _1404 A1S_ _1407, A1S_ _1422 A1S_ _1454, A1S_ _1472 A1S_ _1481, A1S_ _1494,
A1S_ _1512, A1S_ _1515 A1S_ _1526, A1S_ _1535 A1S_ _1539, A1S_ _1566 A1S_ _1567, A1S_ _1569,
A1S_ _1583, A1S_ _1584 A1S_ _1585, A1S_ _1589 A1S_ _1590, A1S_ _1593 A1S_ _1595, A1S_ _1596,
A1S_ _1617, A1S_ _1630 A1S_ _1644, A1S_ _1645 A1S_ _1647, A1S_ _1648 A1S_ _1649, A1S_ _1651,
A1S_ _1655, A1S_ _1658 A1S_ _1662, A1S_ _1666 A1S_ _1667, A1S_ _1669 A1S_ _1677, A1S_ _1680,
A1S_ _1681, A1S_ _1687 A1S_ _1735, A1S_ _1741 A1S_ _1743, A1S_ _1744 A1S_ _1750, A1S_ _1751,
A1S_ _1752, A1S_ _1760 A1S_ _1762, A1S_ _1767 A1S_ _1778, A1S_ _1813 A1S_ _1827, A1S_ _1829,
A1S_ _1831, A1S_ _1843 A1S_ _1876, A1S_ _1909 A1S_ _1928, A1S_ _1929 A1S_ _1934, A1S_ _1952,
A1S_ _1955, A1S_ _1956 A1S_ _1957, A1S_ _1959 A1S_ _1960, A1S_ _1961 A1S_ _1962, A1S_ _1963,
A1S_ _1979, A1S_ _1986 A1S_ _1987, A1S_ _1988 A1S_ _2006, A1S_ _2026 A1S_ _2033, A1S_ _2034,
A1S_ _2035, A1S_ _2036 A1S_ _2038, A1S_ _2039 A1S_ _2061, A1S_ _2074 A1S_ _2079, A1S_ _2082,
A1S_ _2092, A1S_ _2093 A1S_ _2106, A1S_ _2139 A1S_ _2140, A1S_ _2141 A1S_ _2142, A1S_ _2146,
A1S_ _2157, A1S_ _2158 A1S_ _2160, A1S_ _2161 A1S_ _2162, A1S_ _2178 A1S_ _2179, A1S_ _2183,
A1S_ _2186, A1S_ _2195 A1S_ _2230, A1S_ _2247 A1S_ _2252, A1S_ _2257 A1S_ _2258, A1S_ _2259,
A1S_ _2262, A1S_ _2271 A1S_ _2272, A1S_ _2273 A1S_ _2283, A1S_ _2285 A1S_ _2298, A1S_ _2311,
A1S_ _2315, A1S_ _2325 A1S_ _2326, A1S_ _2330 A1S_ _2331, A1S_ _2366 A1S_ _2367, A1S_ _2382,
A1S_ _2387, A1S_ _2389 A1S_ _2395, A1S_ _2396 A1S_ _2414, A1S_ _2434 A1S_ _2445, A1S_ _2446, A1S_ _2447, A1S_ _2448 A1S_ _2454, A1S_ _2455 A1S_ _2456, A1S_ _2458 A1S_ _2459, A1S_ _2463,
A1S_ _2480, A1S_ _2489 A1S_ _2503, A1S_ _2504 A1S_ _2508, A1S_ _2542 A1S_ _2543, A1S_ _2552,
A1S_ _2553, A1S_ _2555 A1S_ _2557, A1S_ _2558 A1S_ _2573, A1S_ _2577 A1S_ _2578, A1S_ _2580,
A1S_ _2586, A1S_ _2588 A1S_ _2593, A1S_ _2611 A1S_ _2612, A1S_ _2613 A1S_ _2624, A1S_ _2650,
A1S_ _2651, A1S_ _2654 A1S_ _2656, A1S_ _2660 A1S_ _2664, A1S_ _2668 A1S_ _2675, A1S_ _2676,
A1S_ _2677, A1S_ _2678 A1S_ _2684, A1S_ _2705 A1S_ _2729, A1S_ _2734 A1S_ _2756, A1S_ _2768,
A1S_ _2786, A1S_ _2798 A1S_ _2801, A1S_ _2807 A1S_ _2826, A1S_ _2827 A1S_ _2828, A1S_ _2839,
A1S_ _2863, A1S_ _2882 A1S_ _2883, A1S_ _2884 A1S_ _2885, A1S_ _2889 A1S_ _2892, A1S_ _2893,
A1S_ _2942, A1S_ _2943 A1S_ _2953, A1S_ _2959 A1S_ _2960, A1S_ _2968 A1S_ _2976, A1S_ _2992,
A1S_ _3011, A1S_ _3026 A1S_ _3027, A1S_ _3034 A1S_ _3035, A1S_ _3047 A1S_ _3048, A1S_ _3099,
A1S_ _3100, A1S_ _3101 A1S_ _3104, A1S_ _3105 A1S_ _3114, A1S_ _3115 A1S_ _3116, A1S_ _3117,
A1S_ _3124, A1S_ _3125 A1S_ _3126, A1S_ _3127 A1S_ _3139, A1S_ _3146 A1S_ _3147, A1S_ _3175,
A1S_ _3206, A1S_ _3224 A1S_ _3253, A1S_ _3259 A1S_ _3280, A1S_ _3281 A1S_ _3295, A1S_ _3317,
A1S_ _3326, A1S_ _3339 A1S_ _3360, A1S_ _3361 A1S_ _3367, A1S_ _3368 A1S_ _3371, A1S_ _3375,
A1S_ _3376, A1S_ _3392 A1S_ _3411, A1S_ _3412 A1S_ _3463, A1S_ _3466 A1S_ _3468, A1S_ _3469,
A1S_ _3471, A1S_ _3479 A1S_ _3480, A1S_ _3486 A1S_ _3492, A1S_ _3493 A1S_ _3494, A1S_ _3499,
A1S_ _3510, A1S_ _3512 A1S_ _3518, A1S_ _3522 A1S_ _3523, A1S_ _3533 A1S_ _3534, A1S_ _3535,
A1S_ _3539, A1S_ _3540 A1S_ _3541, A1S_ _3542 A1S_ _3543, A1S_ _3544 A1S_ _3545, A1S_ _3546,
A1S_ _3548, A1S_ _3552 A1S_ _3553, A1S_ _3558 A1S_ _3559, A1S_ _3562 A1S_ _3563, A1S_ _3567,
A1S_ _3570, A1S_ _3577 A1S_ _3580, A1S_ _3580 A1S_ _3585, A1S_ _3586 A1S_ _3594, A1S_ _3595,
A1S_ _3596, A1S_ _3601 A1S_ _3602, A1S_ _3603 A1S_ _3604, A1S_ _3605 A1S_ _3606, A1S_ _3607,
A1S_ _3608, A1S_ _3609 A1S_ _3610, A1S_ _3611 A1S_ _3612, A1S_ _3613 A1S_ _3614, A1S_ _3617,
A1S_ _3618, A1S_ _3621 A1S_ _3630, A1S_ _3632 A1S_ _3634, A1S_ _3635 A1S_ _3636, A1S_ _3637,
A1S_ _3642, A1S_ _3645 A1S_ _3649, A1S_ _3654 A1S_ _3658, A1S_ _3661 A1S_ _3662, A1S_ _3666,
A1S_ _3682, A1S_ _3686 A1S_ _3687, A1S_ _3688 A1S_ _3694, A1S_ _3695 A1S_ _3697, A1S_ _3704,
A1S_ _3707, A1S_ _3708 A1S_ _3712, A1S_ _3716 A1S_ _3725, A1S_ _3726 A1S_ _3727, A1S_ _3728,
A1S_ _3736, A1S_ _3738 A1S_ _3739, A1S_ _3740 A1S_ _3750, A1S_ _3752 A1S_ _3760, A1S_ _3768,
A1S_ _3769, A1S_ _3770 A1S_ _3771, A1S_ _3772 A1S_ _3773, A1S_ _3776 A1S_ _3777, A1S_ _3778,
A1S_ _3782, A1S_ _3783 A1S_ _3786, A1S_ _3789 A1S_ _3790, A1S_ _3791 A1S_ _3792, A1S_ _3797,
A1S_ _3810, A1S_ _3818 A1S_ _3820, A1S_ _3835 A1S_ _3837, A1S_ _3840 A1S_ _3842, A1S_ _3844, A1S_3862, A1S_3864, A1S_3865, A1S_3866, A1S_3867, A1S_3868, A1S_3873, A1S_3875, A1S_3879, A1S_3889, A1S_3900, A1S_3901, A1S_3902, A1S_3908, or A1S_3911.
8. The reporter polynucleotide of any of claims 1-7, wherein the OM stress- responsive gene is upregulated in response to the stress, and wherein the OM stress-responsive gene is A1S_0032, A1S_0033, A1S_0113, A1S_1224, A1S_1751, A1S_1752, A1S_2093, A1S_2271, A1S_2884, A1S_2885, A1S_2889, or A1S_3127, optionally wherein the OM stress- responsive gene is A1S_0032, A1S_2885, or A1S_2889.
9. The reporter polynucleotide of any of claims l-5,wherein the OM stress- responsive gene is downregulated in response to the stress at least or about at least 2-fold, 3- fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold or more in response to the stress.
10. The reporter polynucleotide of iy of claims 1-5 and 9, wherein the OM stress- responsive gene is downregulated in response the stress, and wherein the OM stress- responsive gene is A IS. _0009, A1S. _0010, A1S 0025, A1S_0027, A1S_0038, A1S_0067,
A1S_ _0070, A1S_ _0071, A1S_ _0073, A1S_ _0076, A1S_ _0077, A1S_ _0079, A1S_ _0087, A1S_ _0090,
A1S_ _0091, A1S_ _0095, A1S_ _0096, A1S_ _0097, A1S_ _0098, A1S_ _0099, A1S_ _0103, A1S_ _0104,
A1S_ _0105, A1S_ _0106, A1S_ _0107, A1S_ _0108, A1S_ _0109, A1S_ _0121, A1S_ _0128, A1S_ _0129,
A1S_ _0141, A1S_ _0148, A1S_ _0150, A1S_ _0151, A1S_ _0152, A1S_ _0153, A1S_ _0154, A1S_ _0155,
A1S_ _0156, A1S_ _0157, A1S_ _0177, A1S_ _0184, A1S_ _0200, A1S_ _0201, A1S_ _0209, A1S_ _0218,
A1S_ _0238, A1S_ _0239, A1S_ _0253, A1S_ _0257, A1S_ _0258, A1S_ _0269, A1S_ _0270, A1S_ _0279,
A1S_ _0286, A1S_ _0292, A1S_ _0302, A1S_ _0303, A1S_ _0304, A1S_ _0321, A1S_ _0322, A1S_ _0323,
A1S_ _0347, A1S_ _0365, A1S_ _0369, A1S_ _0370, A1S_ _0388, A1S_ _0408, A1S_ _0410, A1S_ _0427,
A1S_ _0429, A1S_ _0447, A1S_ _0448, A1S_ _0474, A1S_ _0480, A1S_ _0481, A1S_ _0482, A1S_ _0486,
A1S_ _0490, A1S_ _0491, A1S_ _0498, A1S_ _0526, A1S_ _0533, A1S_ _0534, A1S_ _0548, A1S_ _0549,
A1S_ _0566, A1S_ _0567, A1S_ _0568, A1S_ _0591, A1S_ _0594, A1S_ _0624, A1S_ _0625, A1S_ _0626,
A1S_ _0627, A1S_ _0629, A1S_ _0630, A1S_ _0631, A1S_ _0632, A1S_ _0633, A1S_ _0634, A1S_ _0635,
A1S_ _0639, A1S_ _0640, A1S_ _0641, A1S_ _0642, A1S_ _0643, A1S_ _0644, A1S_ _0645, A1S_ _0646,
A1S_ _0647, A1S_ _0649, A1S_ _0650, A1S_ _0651, A1S_ _0690, A1S_ _0691, A1S_ _0692, A1S_ _0695,
A1S_ _0698, A1S_ _0721, A1S_ _0731, A1S_ _0732, A1S_ _0771, A1S_ _0785, A1S_ _0786, A1S_ _0787, A1S_ _0788, A1S_ _0818 A1S_ _0822, A1S_ _0846 A1S_ _0849, A1S_ _0850 A1S_ .0851, A1S_ _0852,
A1S_ _0853, A1S_ _0854 A1S_ _0855, A1S_ _0869 A1S_ _0877, A1S_ _0882 A1S_ _0883, A1S_ _0890,
A1S_ .0891, A1S_ _0901 A1S_ .0910, A1S_ _0911 A1S_ .0912, A1S_ _0913 A1S_ _0960, A1S_ _0965,
A1S_ _0973, A1S_ _0984 A1S_ _0996, A1S_ _0997 A1S_ _0999, A1S_ _1000 A1S_ _1004, A1S_ _1008,
A1S_ .1021, A1S_ _1026 A1S_ .1044, A1S_ _1063 A1S_ .1072, A1S_ _1079 A1S_ _1080, A1S_ _1088,
A1S_ _1089, A1S_ _1091 A1S_ .1092, A1S_ _1093 A1S_ .1094, A1S_ .1109 A1S_ .1113, A1S_ .1139,
A1S_ .1142, A1S_ _1182 A1S_ .1193, A1S_ .1195 A1S_ .1199, A1S_ _1227 A1S_ _1257, A1S_ .1258,
A1S_ .1261, A1S_ _1264 A1S_ .1265, A1S_ _1266 A1S_ .1267, A1S_ _1268 A1S_ _1269, A1S_ .1270,
A1S_ .1281, A1S_ _1317 A1S_ .1318, A1S_ _1319 A1S_ .1327, A1S_ _1334 A1S_ _1335, A1S_ _1336,
A1S_ _1337, A1S_ _1338 A1S_ .1339, A1S_ _1340 A1S_ .1341, A1S_ _1342 A1S_ _1343, A1S_ .1344,
A1S_ _1345, A1S_ _1346 A1S_ .1347, A1S_ _1348 A1S_ .1349, A1S_ _1356 A1S_ _1366, A1S_ .1367,
A1S_ _1368, A1S_ _1369 A1S_ .1370, A1S_ _1372 A1S_ .1373, A1S_ _1374 A1S_ _1375, A1S_ .1376,
A1S_ _1377, A1S_ _1378 A1S_ .1379, A1S_ _1380 A1S_ .1396, A1S_ _1397 A1S_ _1428, A1S_ .1442,
A1S_ _1443, A1S_ _1450 A1S_ .1466, A1S_ _1467 A1S_ .1469, A1S_ _1470 A1S_ _1476, A1S_ .1490,
A1S_ .1491, A1S_ _1492 A1S_ .1493, A1S_ _1498 A1S_ .1499, A1S_ _1505 A1S_ .1510, A1S_ .1523,
A1S_ _1528, A1S_ _1530 A1S_ .1532, A1S_ _1543 A1S_ .1579, A1S_ _1583 A1S_ .1601, A1S_ _1608,
A1S_ _1609, A1S_ _1610 A1S_ .1611, A1S_ _1612 A1S_ .1613, A1S_ _1637 A1S_ _1638, A1S_ .1639,
A1S_ _1655, A1S_ _1692 A1S_ .1698, A1S_ _1699 A1S_ .1700, A1S_ .1701 A1S_ .1703, A1S_ .1705,
A1S_ .1717, A1S_ _1719 A1S_ .1724, A1S_ _1729 A1S_ .1730, A1S_ .1731 A1S_ .1732, A1S_ .1734,
A1S_ _1735, A1S_ _1736 A1S_ .1737, A1S_ _1738 A1S_ .1742, A1S_ _1745 A1S_ .1754, A1S_ .1756,
A1S_ _1758, A1S_ _1775 A1S_ .1776, A1S_ _1790 A1S_ .1791, A1S_ _1792 A1S_ .1794, A1S_ .1795,
A1S_ _1796, A1S_ _1797 A1S_ _1805, A1S_ _1806 A1S_ .1811, A1S_ _1830 A1S_ .1834, A1S_ _1835,
A1S_ _1836, A1S_ _1837 A1S_ _1838, A1S_ _1839 A1S_ .1840, A1S_ .1841 A1S_ .1854, A1S_ _1855,
A1S_ _1856, A1S_ _1857 A1S_ _1858, A1S_ _1859 A1S_ _1860, A1S_ .1861 A1S_ .1862, A1S_ _1863,
A1S_ _1864, A1S_ _1865 A1S_ _1866, A1S_ _1879 A1S_ _1880, A1S_ _1887 A1S_ .1908, A1S_ .1924,
A1S_ _1925, A1S_ _1926 A1S_ .1935, A1S_ _1940 A1S_ .1942, A1S_ _1948 A1S_ .1951, A1S_ .1984,
A1S_ _1996, A1S_ _2041 A1S_ _2042, A1S_ _2052 A1S_ _2053, A1S_ _2068 A1S_ _2072, A1S_ .2081,
A1S_ _2084, A1S_ _2098 A1S_ .2100, A1S_ _2101 A1S_ .2102, A1S_ _2148 A1S_ .2149, A1S_ .2150,
A1S_ _2163, A1S_ _2166 A1S_ .2167, A1S_ _2190 A1S_ .2191, A1S_ _2202 A1S_ _2203, A1S_ _2207,
A1S_ _2209, A1S_ _2218 A1S_ .2221, A1S_ _2225 A1S_ _2232, A1S_ _2234 A1S_ _2248, A1S_ _2279,
A1S_ _2280, A1S_ _2288 A1S_ _2289, A1S_ _2340 A1S_ .2341, A1S_ _2342 A1S_ _2348, A1S_ _2353, A1S_ _2354, A1S_ _2415 A1S_ _2416, A1S_ _2417 A1S_ _2418, A1S_ _2419 A1S_ _2424, A1S_ _2425,
A1S_ _2431, A1S_ _2435 A1S_ _2443, A1S_ _2449 A1S_ _2450, A1S_ _2451 A1S_ _2452, A1S_ _2475,
A1S_ _2501, A1S_ _2509 A1S_ _2510, A1S_ _2514 A1S_ _2531, A1S_ _2532 A1S_ _2533, A1S_ _2535,
A1S_ _2601, A1S_ _2602 A1S_ _2633, A1S_ _2662 A1S_ _2670, A1S_ _2671 A1S_ _2672, A1S_ _2688,
A1S_ _2692, A1S_ _2694 A1S_ _2695, A1S_ _2696 A1S_ _2701, A1S_ _2711 A1S_ _2722, A1S_ _2724,
A1S_ _2738, A1S_ _2740 A1S_ _2741, A1S_ _2748 A1S_ _2753, A1S_ _2755 A1S_ _2758, A1S_ _2761,
A1S_ _2762, A1S_ _2769 A1S_ _2773, A1S_ _2774 A1S_ _2785, A1S_ _2788 A1S_ _2789, A1S_ _2793,
A1S_ _2809, A1S_ _2814 A1S_ _2815, A1S_ _2820 A1S_ _2823, A1S_ _2847 A1S_ _2848, A1S_ _2849,
A1S_ _2852, A1S_ _2860 A1S_ _2904, A1S_ _2905 A1S_ _2906, A1S_ _2911 A1S_ _2913, A1S_ _2919,
A1S_ _2924, A1S_ _2928 A1S_ _2939, A1S_ _2946 A1S_ _2956, A1S_ _3013 A1S_ _3014, A1S_ _3025,
A1S_ _3040, A1S_ _3043 A1S_ _3049, A1S_ _3050 A1S_ _3051, A1S_ _3074 A1S_ _3084, A1S_ _3H0,
A1S_ _3120, A1S_ _3121 A1S_ _3122, A1S_ _3128 A1S_ _3129, A1S_ _3130 A1S_ _3131, A1S_ _3132,
A1S_ _3133, A1S_ _3134 A1S_ _3135, A1S_ _3144 A1S_ _3174, A1S_ _3180 A1S_ _3195, A1S_ _3207,
A1S_ _3222, A1S_ _3224 A1S_ _3225, A1S_ _3231 A1S_ _3232, A1S_ _3236 A1S_ _3238, A1S_ _3248,
A1S_ _3250, A1S_ _3268 A1S_ _3269, A1S_ _3273 A1S_ _3278, A1S_ _3290 A1S_ _3297, A1S_ _3298,
A1S_ _3300, A1S_ _3301 A1S_ _3309, A1S_ _3338 A1S_ _3342, A1S_ _3355 A1S_ _3364, A1S_ _3377,
A1S_ _3397, A1S_ _3398 A1S_ _3402, A1S_ _3403 A1S_ _3404, A1S_ _3405 A1S_ _3406, A1S_ _3407,
A1S_ _3410, A1S_ _3413 A1S_ _3414, A1S_ _3415 A1S_ _3416, A1S_ _3418 A1S_ _3431, A1S_ _3450,
A1S_ _3451, A1S_ _3458 A1S_ _3460, A1S_ _3481 A1S_ _3487, A1S_ _3491 A1S_ _3494, A1S_ _3498,
A1S_ _3506, A1S_ _3508 A1S_ _3509, A1S_ _3514 A1S_ _3518, A1S_ _3519 A1S_ _3520, A1S_ _3521,
A1S_ _3522, A1S_ _3523 A1S_ _3524, A1S_ _3526 A1S_ _3528, A1S_ _3530 A1S_ _3531, A1S_ _3532,
A1S_ _3533, A1S_ _3534 A1S_ _3535, A1S_ _3537 A1S_ _3538, A1S_ _3539 A1S_ _3540, A1S_ _3541,
A1S_ _3542, A1S_ _3543 A1S_ _3544, A1S_ _3545 A1S_ _3546, A1S_ _3547 A1S_ _3548, A1S_ _3549,
A1S_ _3550, A1S_ _3552 A1S_ _3553, A1S_ _3554 A1S_ _3568, A1S_ _3569 A1S_ _3578, A1S_ _3582,
A1S_ _3586, A1S_ _3587 A1S_ _3591, A1S_ _3597 A1S_ _3599, A1S_ _3600 A1S_ _3602, A1S_ _3611,
A1S_ _3619, A1S_ _3621 A1S_ _3624, A1S_ _3629 A1S_ _3633, A1S_ _3640 A1S_ _3641, A1S_ _3644,
A1S_ _3647, A1S_ _3651 A1S_ _3652, A1S_ _3659 A1S_ _3663, A1S_ _3667 A1S_ _3673, A1S_ _3679,
A1S_ _3701, A1S_ _3707 A1S_ _3709, A1S_ _3713 A1S_ _3715, A1S_ _3717 A1S_ _3732, A1S_ _3735,
A1S_ _3738, A1S_ _3740 A1S_ _3741, A1S_ _3742 A1S_ _3759, A1S_ _3774 A1S_ _3779, A1S_ _3787,
A1S_ _3788, A1S_ _3794 A1S_ _3801, A1S_ _3802 A1S_ _3806, A1S_ _3809 A1S_ _3811, A1S_ _3813,
A1S_ _3814, A1S_ _3816 A1S_ _3817, A1S_ _3823 A1S_ _3829, A1S_ _3831 A1S_ _3832, A1S_ _3836, A1S_3840, A1S_3846, A1S_3857, A1S_3862, A1S_3868, A1S_3870, A1S_3880, A1S_3884, A1S_3886, A1S_3887, A1S_3891, A1S_3894, A1S_3898, A1S_3907, A1S_3908, A1S_3909, A1S_3912, A1S_3914, or A1S_3915.
11. The reporter polynucleotide of any of claims 1-5 and 9-10, wherein the OM stress-responsive gene is downregulated in response to the stress, and wherein the OM stress- responsive gene is A1SJ3103, A1SJ3645, A1S_1266, A1S_1268, A1S_1335, A1S_1336, A1S_1337, A1S_1338, A1S_1339, A1S_1340, A1S_1341, A1S_1342, A1S_1343, A1S_1344, A1S_1345, A1S_1791, A1S_1792, A1S_1794, A1S_1796, A1S_1835, A1S_1836, A1S_1837, A1S_1838, A1S_1839, A1S_2449, A1S_2450, A1S_2452, A1S_3540, A1S_3541, A1S_3542, A1S_3543, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, A1S_3809, or
A1S_3908, optionally wherein the OM stress-responsive gene is A1S_1336, A1S_1836, A1S_1838, A1S_3586, A1S_3663, A1S_3707, A1S_3738, A1S_3806, or A1S_3809.
12. The reporter polynucleotide of any of claims 1-11, wherein the regulatory region comprises a contiguous sequence of nucleotides within 500 base pairs upstream or 5' of the open reading frame (ORF) of the OM stress-responsive gene, optionally wherein the contiguous sequence of nucleotides comprises at least or at least about 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 400 or more base pairs.
13. The reporter polynucleotide of any of claims 1-12, wherein the regulatory region is or comprises a promoter.
14. The reporter polynucleotide of any of claims 1-13, wherein the regulatory region comprises a sequence to further promote translation of the encoded reporter molecule, optionally wherein the sequence to further promote translation is or comprises a bacterial ribosome binding site.
15. The reporter polynucleotide of claim 14, wherein the ribosome binding site is a Shine-Dalgarno sequence, optionally wherein the Shine-Dalgarno sequence is native to the regulatory region of the OM stress-responsive gene or the Shine-Dalgarno sequence is synthetic and/or heterologous to the regulatory region of the OM stress-responsive gene, optionally wherein the Shine-Dalgarno sequence comprises the sequence set forth in SEQ ID NO: 14 or a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 14.
16. The reporter polynucleotide of any of claims 1-15, wherein the regulatory region comprises the sequence set forth in any of SEQ ID NOS: 1-13 or a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NOs: 1-13.
17. The reporter polynucleotide of any of claims 1-16, wherein the reporter molecule is a fluorescent protein, a luminescent protein, a chromoprotein, or an enzyme.
18. The reporter polynucleotide of any of claims 1-17, wherein the reporter molecule is Sirius, SBFP2, Azurite, mAzurite, EBFP2, moxBFP, mKalamal, mTagBFP2, Aquamarine, ECFP, Cerulean, mCerulean, mCerulean3, moxCerulean3, SCFP3A, mTurquoise2, CyPet, AmCyanl, MiCy (Midoriishi-Cyan), iLOV, AcGFPl, sfGFP, moxGFP, mEmerald, EGFP, mEGFP, AzamiGreen, cfSGFP2, ZsGreen, SGFP2, Clover, mClover2, mClover3, EYFP, Topaz, mTopaz, mVenus, mox Venus, SYFP2, mCitrine, YPet, ZsYellowl, mPapayal, mKusabira- Orange (mKO), mOrange, mOrange2, mK02, TurboRFP, tdTomato, mScarlet-H, mNectarine, mRuby2, eqFP611, DsRed2, mApple, mScarlet, mStrawberry, FusionRed, mRFPl, mCherry, mCherry2, mCrimson3, HcRedl, dKatushka, mKatel.3, mPlum, mRaspberry, TagRFP675, mNeptune, mCardinal, mMaroon, TagRFP657, smURFP, miRFP670, iRFP670, iRFP682, miRFP703, iRFP702, miRFP709, mIFP 683, IFP2.0, iRFP, iSplit, iRFP720, T-sapphire, mT- sapphire, mAmetrine, LSSmOrange, mKeima Red, dKeima Red, LSSmKatel, LSSmKate2, Phamret, PA-sfGFP, mPA-Emerald, PA-GFP, PATagRFP, PAmCherryl, PAmCherry2, PAmCherry3, PAm ate, PAiRFPl, PAiRFP2, Dendra2, mEos3.2, mEos4a, dEos 505, tdEos 505, mKikGR, Kaede 508, PSmOrange, PSmOrange2, rsTagRFP, rsEmerald, rsGFPl, Dronpa3, mGeos-M, amilGFP, amilCP, North American firefly luciferase, Genji-botaru luciferase, Italian firefly luciferase, Heike luciferase, East European firefly luciferase, Pennsylvania firefly luciferase, Click beetle luciferase, Railroad worm luciferase, Renilla luciferase, Rluc8, Green Renilla luciferase, Gaussia luciferase, Gaussia-Dura luciferase, Cypridina luciferase, Vargula luciferase, Metridia lucif erase, OLuc, bacterial luciferase (LuxAB), chloramphenicol acetyltransferase (CAT), β-galactosidase, alkaline phosphatase, β-glucuronidase, β -lactamase, neomycin phosphotransferase, or a modified version thereof.
19. The reporter polynucleotide of any of claims 1-18, wherein the reporter molecule is sfGFP, mClover3, or mRuby2, optionally wherein the sequence encoding the reporter molecule comprises the sequence set forth in SEQ ID NO: 15, 33, or 40 or a sequence with at least or at least about 85%, 90%, 95%, 99%, or more sequence identity to SEQ ID NO: 15, 33, or 40.
20. The reporter polynucleotide of any of claims 1-19, wherein the reporter polynucleotide comprises the sequence set forth in any of SEQ ID NOs: 16-28 or 34-36 or a sequence with at least or at least about 85%, 90%, 95%, 99%, or more sequence identity to any of SEQ ID NOs: 16-28 or 34-36.
21. A reporter vector comprising the reporter polynucleotide of any of claims 1-20.
22. The reporter vector of claim 21, wherein the reporter polynucleotide is a first reporter polynucleotide and the reporter vector further comprises a second reporter
polynucleotide of any of claims 1-20.
23. The reporter vector of claim 22, wherein the first reporter polynucleotide and the second reporter polynucleotide are different, optionally wherein the regulatory region of the first reporter polynucleotide and the regulatory region of the second reporter polynucleotide are from different OM stress-responsive genes and/or wherein the reporter molecule encoded by the first reporter polynucleotide and the reporter molecule encoded by the second reporter polynucleotide do not exhibit an overlapping emission and absorption spectra and/or are distinguishably detectable.
24. The reporter vector of any of claims 21-23, wherein the reporter vector is capable of being expressed in a host microorganism, optionally a Gram-negative bacterium.
25. The reporter vector of claim 24, wherein the host microorganism is
Acinetobacter, Bdellovibrio, Burkholderia, Chlamydia, Enterobacter, Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella, Neisseria, Pantoea,
Pseudomonas, Salmonella, Shigella, Stenotrophomonas, Vibrio, or Yersinia, optionally wherein the host microorganism is Acinetobacter.
26. The reporter vector of claim 24 or claim 25, wherein the host microorganism is Acinetobacter apis, Acinetobacter baumannii, Acinetobacter baylyi, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii, Acinetobacter calcoaceticus, Acinetobacter gandensis, Acinetobacter gerneri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus, Acinetobacter harbinensis, Acinetobacter indicus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter kookii, Acinetobacter Iwoffii, Acinetobacter nectaris, Acinetobacter nosocomialis, Acinetobacter parvus, Acinetobacter pakistanensis, Acinetobacter pittii, Acinetobacter puyangensis, Acinetobacter qingfengensis, Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii, Acinetobacter soli, Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis, or Acinetobacter venetianus, optionally wherein the host microorganism is Acinetobacter baumannii.
27. The reporter vector of any of claims 24-26, wherein the host microorganism is ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA-1605, ATCC BAA-1709, ATCC BAA-1710, ATCC BAA- 1789, ATCC BAA-1790, ATCC BAA- 1791, ATCC BAA- 1792, ATCC BAA-1793, ATCC BAA-1794, ATCC BAA-1795, ATCC BAA-1796, ATCC BAA-1797, ATCC BAA- 1798, ATCC BAA- 1799, ATCC BAA-1800, ATCC BAA-1878, ATCC BAA-2093, ATCC BAA-747, SDF, AYE, ACICU, AB0057, AB307-0294, 1656-2, MDR-ZJ06, TCDC-AB0715, MDR-TJ, TYTH-1, D1279779, BJAB07104, BJAB0715, BJAB0868, ZW85- 1, AbH120-A2, AB030, AB031, AC29, LAC -4, Ab307-0294, 1656-2, ID 403, AABA041, AABA046, or a modified strain thereof, optionally the host microorganism is ATCC 17978, Ab307-0294, AABA041, or AABA046.
28. The reporter vector of any of claims 21-27, wherein the reporter polynucleotide, optionally the first reporter polynucleotide and second reporter polynucleotide, is comprised in a backbone vector, and the backbone vector is pACH106, pWH1266, or pET-RA.
29. The reporter vector of claim 28, wherein a nucleotide sequence comprising the reporter polynucleotide, optionally the first reporter polynucleotide and the second reporter polynucleotide, is inserted into or replaces a portion of the nucleotide sequence of the backbone vector.
30. The reporter vector of claim 28 or claim 29, wherein the backbone vector comprises the sequence of nucleotides set forth in SEQ ID NO: 29 and a nucleotide sequence comprising the reporter polynucleotide replaces nucleotides 5,715-7,395 of the backbone vector.
31. The reporter vector of any of claims 21 and 24-30, wherein the reporter vector comprises the sequence set forth in any of SEQ ID NOs: 30-32 or a sequence with at least or at least about 85%, 90%, 95%, 99% or more sequence identity to any of SEQ ID NOs: 30-32.
32. A reporter microorganism comprising one or more reporter polynucleotide of any of claims 1-20 or one or more reporter vector of any of claims 21-31, optionally wherein the reporter microorganisms comprise a first reporter polynucleotide and a second reporter polynucleotide.
33. The reporter microorganism of claim 32, wherein the regulatory region of the first reporter polynucleotide and the regulatory region of the second reporter polynucleotide are from different OM stress-responsive genes, wherein the reporter molecule encoded by the first reporter polynucleotide is different from the reporter molecule encoded by the second reporter polynucleotide, and/or wherein the reporter molecule encoded by the first reporter polynucleotide and the reporter molecule encoded by the second reporter polynucleotide do not exhibit an overlapping emission and absorption spectra and/or are distinguishably detectable.
34. The reporter microorganism of claim 32 or claim 33, wherein the first reporter polynucleotide and second reporter polynucleotide are comprised in the same reporter vector or the first reporter polynucleotide and second reporter polynucleotide are comprised in different reporter vectors.
35. The reporter microorganism of any of claims 32-34, wherein the reporter microorganism is a Gram-negative bacterium.
36. The reporter microorganism of any of claims 32-35, wherein the reporter microorganism is Acinetobacter, Bdellovibrio, Burkholderia, Chlamydia, Enterobacter, Escherichia, Francisella, Haemophilus, Helicobacter, Klebsiella, Legionella, Moraxella, Neisseria, Pantoea, Pseudomonas, Salmonella, Shigella, Stenotrophomonas, Vibrio, or Yersinia, optionally wherein the reporter microorganism is Acinetobacter.
37. The reporter microorganism of any of claims 32-36, wherein the reporter microorganism is Acinetobacter apis, Acinetobacter baumannii, Acinetobacter baylyi,
Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter bohemicus, Acinetobacter boissieri, Acinetobacter bouvetii, Acinetobacter brisouii, Acinetobacter calcoaceticus,
Acinetobacter gandensis, Acinetobacter gerneri, Acinetobacter guangdongensis, Acinetobacter guillouiae, Acinetobacter gyllenbergii, Acinetobacter haemolyticus, Acinetobacter harbinensis, Acinetobacter indicus, Acinetobacter johnsonii, Acinetobacter junii, Acinetobacter kookii, Acinetobacter Iwoffii, Acinetobacter nectaris, Acinetobacter nosocomialis, Acinetobacter parvus, Acinetobacter pakistanensis, Acinetobacter pittii, Acinetobacter puyangensis,
Acinetobacter qingfengensis, Acinetobacter radioresistens, Acinetobacter rudis, Acinetobacter schindleri, Acinetobacter seifertii, Acinetobacter soli, Acinetobacter tandoii, Acinetobacter tjernbergiae, Acinetobacter towneri, Acinetobacter ursingii, Acinetobacter variabilis, or Acinetobacter venetianus, optionally wherein the reporter microorganism is Acinetobacter baumannii.
38. The reporter microorganism of any of claims 32-37, wherein the reporter microorganism is ATCC 15149, ATCC 15150, ATCC 15151, ATCC 15308, ATCC 15473, ATCC 17904, ATCC 17945, ATCC 17957, ATCC 17959, ATCC 17961, ATCC 17978, ATCC 19003, ATCC 19187, ATCC 19568, ATCC 19606, ATCC 27224, ATCC 43498, ATCC 49466, ATCC 51432, ATCC 9955, ATCC BAA- 1605, ATCC BAA- 1709, ATCC BAA- 1710, ATCC BAA-1789, ATCC BAA-1790, ATCC BAA- 1791, ATCC BAA-1792, ATCC BAA-1793, ATCC BAA-1794, ATCC BAA-1795, ATCC BAA-1796, ATCC BAA-1797, ATCC BAA- 1798, ATCC BAA-1799, ATCC BAA-1800, ATCC BAA-1878, ATCC BAA-2093, ATCC BAA-747, SDF, AYE, ACICU, AB0057, AB307-0294, 1656-2, MDR-ZJ06, TCDC-AB0715, MDR-TJ, TYTH-1, D1279779, BJAB07104, BJAB0715, BJAB0868, ZW85-1, AbH120-A2, AB030, AB031, AC29, LAC-4, Ab307-0294, 1656-2, ID 403, AABA041, AABA046, or a modified strain thereof, optionally wherein the reporter microorganism is ATCC 17978, Ab307- 0294, AABA041, or AABA046.
39. A plurality of reporter microorganisms comprising two or more reporter microorganisms of any of claims 32-38, optionally comprising 2, 3, 4, 5, or more different reporter microorganisms.
40. The plurality of reporter microorganisms of claim 40, wherein each of at least two reporter microorganisms in the plurality comprise a different reporter polynucleotide in which comprises the same regulatory region of an OM-responsive gene but that is operatively linked to a different reporter molecule, optionally wherein the different reporter molecules do not exhibit overlapping emission and absorption spectra and/or are distinguishably detectable.
41. The plurality of reporter microorganisms of claim 40, wherein the at least two reporter microorganisms are derived from the same host strain or wherein each of the at least two reporter microorganisms is derived from a different host strain.
42. A composition comprising the microorganism of any of claims 32-38 or the plurality of microorganisms of any of claims 39-41.
43. The composition of claim 42, further comprising one or more components capable of activating the complement pathway.
44. The composition of claim 41 or claim 42, further comprising serum, optionally human serum, rabbit serum, bovine serum, or mouse serum.
45. The composition of claim 44, wherein the concentration of serum is at least or at least about 2.5% (vol/vol), 5.0% (vol/vol), 7.5% (vol/vol), 10.0% (vol/vol), 15.0% (vol/vol), 20% (vol/vol), or 25% (vol/vol), optionally wherein the concentration of serum is from or from about 2.5% (vol/vol) to 25% (vol/vol), 2.5% (vol/vol) to 15% (vol/vol), 2.5% (vol/vol) to 10% (vol/vol), 2.5% (vol/vol) to 5.0% (vol/vol), 5.0% (vol/vol) to 25% (vol/vol), 5.0% (vol/vol) to 15% (vol/vol), 5.0% (vol/vol) to 10% (vol/vol), 10.0% (vol/vol) to 25% (vol/vol), 10.0%
(vol/vol) to 15% (vol/vol), or 15% (vol/vol) to 25% (vol/vol).
46. The composition of any of claims 42-45, comprising an agent, optionally a candidate antibacterial agent.
47. The composition of claim 46, wherein the agent is a small molecule compound, a peptide or a protein, optionally wherein the agent is an antibody or antigen-binding fragment thereof.
48. A microdroplet comprising the microorganism of any of claims 32-38, the plurality of microorganisms of any of claims 39-41, or the composition of any of claims 42-47.
49. A microdroplet comprising the composition of any of claims 46-48 and a cell that produces or secretes the agent.
50. The microdroplet of claim 49, wherein the cell is an antibody-producing cell.
51. The microdroplet of claim 49 or claim 50, wherein the cell is a mammalian cell or a microorganism, optionally a fungal or bacterial cell.
52. The microdroplet of any of claims 49-51 , wherein the cell is a B cell, a plasma cell or a plasmablast.
53. The microdroplet of any of claims 48-52, wherein the microdroplet comprises agarose, carrageenan, alginate, alginate-polylysine, collagen, cellulose, methylcellulose, gelatin, chitosan, extracellular matrix, dextran, starch, inulin, heparin, hyaluronan, fibrin, polyvinyl alcohol, poly(N-vinyl-2-pyrrolidone), polyethylene glycol, poly(hydroxyethyl methacrylate), acrylate polymers and sodium polyacrylate, polydimethyl siloxane, cis- polyisoprene,
Puramatrix™, poly-divenylbenzene, polyurethane, polyacrylamide or combinations thereof, optionally wherein the microdroplet comprises agarose.
54. The microdroplet of any of claims 48-53, wherein the microdroplet comprises growth media.
55. The microdroplet of any of claims 48-54, wherein the microdroplet comprises serum.
56. A composition comprising a microdroplet of any of claims 48-55 or a plurality of the microdroplets of any of claims 48-55.
57. A kit comprising:
the reporter polynucleotide of any of claims 1-20, the reporter vector of any of claims 21-31, the reporter microorganism of any of claims 32-38, the plurality of reporter
microorganisms of any of claims 39-41, the composition of any of claims 42-47 and 56, or the microdroplet of any of claims 48-55; and
instructions for use.
58. A method of assessing outer membrane (OM) stress of a microorganism, comprising:
(a) exposing the reporter microorganism of any of claims 32-38, the plurality of reporter microorganisms of any of claims 39-41, or the composition of any of claims 42-47 to a condition that is known to cause or suspected of causing stress to the outer membrane; and
(b) detecting the presence, absence, or level of a detectable signal from the reporter molecule(s).
59. The method of claim 58, wherein the method further comprises (c) determining if there a change in the level of the detectable signal from the reporter molecule(s) compared to in the absence of exposing the reporter microorganism to the condition, wherein a change in the level of the detectable signal indicates the condition causes OM stress to the microorganism.
60. The method of claim 58 or claim 59, wherein the exposing is carried out in suspension, in an array, and/or in a microdroplet.
61. The method of any of claims 58-60, wherein the condition is treatment with an agent or a combination of agents.
62. A method of screening an agent, comprising:
(a) contacting the reporter microorganism of any of claims 32-38, the plurality of reporter microorganisms of any of claims 39-41, or the composition of any of claims 42-47 with an agent; and
(b) detecting the presence, absence, or level of a detectable signal from the reporter molecule(s).
63. The method of claim 62, wherein the method further comprises (c) identifying an agent that causes a change in the level of the detectable signal from the reporter molecule compared to in the absence of the contacting of the agent.
64. A method of screening an agent, comprising:
(a) contacting an agent with a first reporter microorganism of any of claims 32-38; (b) contacting the agent with at least one additional reporter microorganism of any of claims 32-38, wherein the at least one additional reporter microorganism is not the same as the first reporter microorganism; and
(c) detecting the presence, absence, or level of a detectable signal from the reporter molecule from the first and/or at least one additional reporter microorganism.
65. The method of claim 64, wherein the contacting in (a) and (b) is carried out separately.
66. The method of claim 64, wherein the contacting in (a) and (b) is carried out together.
67. The method of claims 64 or 66, wherein the first microorganism, the at least one additional reporter microorganism, and the agent are encapsulated together in a microdroplet.
68. The method of any of claims 64-67, wherein:
the first and the at least one additional reporter microorganism comprise a different reporter polynucleotide in which comprises the same regulatory region of an OM-responsive gene but that is operatively linked to a different reporter molecule that does not exhibit overlapping emission and absorption spectra and/or are distinguishably detectable; or
the first and the at least one additional microorganism comprise a different reporter polynucleotide in which comprises a different regulatory region of an OM-responsive gene and is operatively linked to a different reporter molecule that does not exhibit overlapping emission and absorption spectra and/or are distinguishably detectable.
69. The method of any of claims 64-68, wherein the first and the at least one additional microorganism are derived from the same host strain, or wherein the first and the at least one additional microorganism are derived from a different host strain.
70. The method of any of claims 62-69, wherein the contacting is carried out in suspension, in an array, or in a microdroplet.
71. The method of any of claims 62-70, wherein the contacting is carried out for at least or about at least 5 minutes, 10 minutes, 30 minutes, 60 minutes, 2 hours or 3 hours.
72. A method of screening an agent comprising:
(a) encapsulating in a microdroplet: (i) the microorganism of any of claims 32-38, a plurality of reporter microorganisms of any of claims 39-41, or the composition of any of claims 42-47; and (ii) an agent, optionally a cell, wherein the cell produces an agent; and
(b) detecting, in the microdroplet, the presence, absence, or level of a detectable signal from the reporter molecule(s).
73. The method of any of claims 61-72, wherein step (a) is repeated with a plurality of agents.
74. The method of any of claims 61-73, wherein:
if there is a change in the presence, absence, or level of the detectable signal, the microorganism of the reporter microorganism is identified as potentially not being resistant to the agent; and
if there is not a change in the presence, absence, or level of the detectable signal, the microorganism of the reporter microorganism is identified as potentially being resistant to the agent.
75. The method of any of claims 58-74, wherein the reporter microorganism of (a) is encapsulated in a microdroplet with a cell.
76. The method of any of claims 61-75, wherein the agent is a small molecule compound, a peptide, or a protein, optionally wherein the agent is an antibiotic and/or an antibody or antigen-binding fragment thereof.
77. The method of any of claims 72-76, wherein the agent is an antibody and the cell is an antibody-producing cell.
78. The method of any of claims 12-11 , wherein the cell is a mammalian cell or a microorganism, optionally a fungal or bacterial cell.
79. The method of any of claims 72-78, wherein the cell is a B cell, a plasma cell, or a plasmablast.
80. The method of any of claims 75-79, wherein the method comprises identifying a microdroplet comprising a reporter microorganism in which there is a change in the level of the detectable signal from the reporter molecule compared to in the absence of exposing the reporter microorganism to the agent.
81. The method of any of claims 72-80, the method comprising isolating the cell from the microdroplet comprising the reporter microorganism, optionally the isolating is carried out using a micromanipulator or an automated sorter.
82. The method of any of claims 75-81, further comprising identifying the agent produced by the cell, optionally wherein identifying comprises determining the sequence of the agent, optionally using single cell PCR and nucleic acid sequencing.
83. The method of any of claims 58-82, comprising prior to (a) introducing the reporter polynucleotide of any of claims 1-20 or reporter vector of any of claims 21-31 into a host microorganism.
84. An agent identified by the method of any of claims 61-83.
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CN110283745A (en) * 2019-06-27 2019-09-27 浙江工业大学 Hospital acinetobacter calcoaceticus FK2 and its application in degradable organic pollutant
CN110283745B (en) * 2019-06-27 2021-05-11 浙江工业大学 Hospital Acinetobacter FK2 and Its Application in Degrading Organic Pollutants
CN110184368A (en) * 2019-07-15 2019-08-30 四川农业大学 A kind of specific primer and methods and applications detecting towneri acinetobacter calcoaceticus
CN110184368B (en) * 2019-07-15 2022-05-06 四川农业大学 Specific primer for detecting towneri acinetobacter, method and application
CN110628666A (en) * 2019-08-26 2019-12-31 中国农业科学院烟草研究所 A Tobacco Common Mosaic Virus Biocontrol Bacteria and Its Application
EP3819637A1 (en) * 2019-11-06 2021-05-12 Velabs Therapeutics GmbH Microfluidic droplet screening method and system
WO2022082961A1 (en) * 2020-10-20 2022-04-28 深圳大学 N-acyl homoserine lactone degrading enzyme and application thereof
CN114891694A (en) * 2022-06-10 2022-08-12 四川中烟工业有限责任公司 A kind of Acinetobacter indian and use thereof
CN114891694B (en) * 2022-06-10 2024-03-12 四川中烟工业有限责任公司 Acinetobacter indicum and application thereof
CN116478953A (en) * 2023-06-14 2023-07-25 四川大学华西医院 Acinetobacter baumannii DlaT recombinant protein, preparation method and application
CN116478953B (en) * 2023-06-14 2023-09-12 四川大学华西医院 Acinetobacter baumannii DlaT recombinant protein, preparation method and application
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