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WO2000065082A1 - Identification de modulateurs des proteines de la famille marr - Google Patents

Identification de modulateurs des proteines de la famille marr Download PDF

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Publication number
WO2000065082A1
WO2000065082A1 PCT/US2000/010829 US0010829W WO0065082A1 WO 2000065082 A1 WO2000065082 A1 WO 2000065082A1 US 0010829 W US0010829 W US 0010829W WO 0065082 A1 WO0065082 A1 WO 0065082A1
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marr
polypeptide
compound
marr family
family polypeptide
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PCT/US2000/010829
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English (en)
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Stuart B. Levy
Michael N. Alekshun
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Trustees Of Tufts College
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Priority to AU43671/00A priority Critical patent/AU4367100A/en
Priority to CA002370426A priority patent/CA2370426A1/fr
Publication of WO2000065082A1 publication Critical patent/WO2000065082A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics

Definitions

  • Multidrug resistance in microbes is generally attributed to the acquisition of multiple transposons and plasmids bearing genetic determinants for different mechanisms of resistance (Gold et al. 1996. N. Engl. J. Med. 335:1445).
  • descriptions of intrinsic mechanisms that confer multidrug resistance have begun to emerge. The first of these was a chromosomally encoded multiple antibiotic resistance (mar) locus in Escherichia coli (George and Levy. 1983. J. Bacteriol 155:531; George and Levy 1983. J. Bacteriol. 155:541).
  • the multiple antibiotic resistance (mar) operon o ⁇ Escherichia coli is a chromosomally encoded locus that controls an adaptational response to antibiotics and other environmental hazards (Alekshun, M. ⁇ . and Levy, S.B. 1997. Antimicrob. Agents Chemother. 10: 2067-2075).
  • This control is accomplished on the genomic level by MarA, a transcriptional activator encoded within the marRAB operon, that regulates the expression of multiple genes on the E. coli chromosome (Alekshun, M. ⁇ . and Levy, S.B. 199 '. Antimicrob. Agents Chemother. 10: 2067-2075).
  • MarR negatively regulates expression of the marRAB operon (Cohen, S.P., et al. 1993. J. Bacteriol. 175: 1484-1492.; Martin, R.G. and Rosner, J.L. 1995. Proc. Natl. Acad. Sci. 92: 5456-5460; Seoane, A.S. and Levy, S.B. 1995. J. Bacteriol 177: 3414-3419., 1995). D ⁇ A footprinting experiments suggest that MarR dimerizes at two locations, sites I and II, within the mar operator (marO) (Martin and Rosner, 1995, supra).
  • Site I is positioned among the -35 and -10 hexamers and site II spans the putative MarR ribosome binding site (reviewed in Alekshun, M. ⁇ . and Levy, S.B. 1997. Antimicrob. Agents Chemother. 10: 2067-2075).
  • MarR is a member of a newly recognized family of regulatory proteins (Alekshun, M.N. and Levy, S.B. 1997. Antimicrob. Agents Chemother. 10: 2067-2075.
  • MarR homologues have been found to control expression of multiple antibiotic resistance operons, some regulate tissue-specific adhesive properties, some control expression of a cryptic hemolysin, some regulate protease production, and some regulate sporulation. Insight into the mechanism by which the MarR family of proteins is regulated would be of great value in controlling functions in microbes that are regulated by this family of proteins, for example, antibiotic resistance and virulence, and ultimately controlling them.
  • the present invention represents an important advance in controlling microbial processes by demonstrating that multiple compounds bind to and directly affect the function of MarR as well as by elucidating the domains of MarR which are critical in mediating its function. Accordingly, the invention provides, inter alia, MarR family polypeptides and methods of their use. This new understanding of how MarR works to regulate gene transcription will be invaluable to understanding and ultimately controlling microbial processes that are regulated by this protein. Moreover, since MarR is a member of a family of proteins, deciphering the function and structure of MarR allows control of related proteins performing other essential functions in bacteria.
  • the invention pertains to a method for identifying a compound that modulates MarR family polypeptide activity or expression, by contacting a MarR family polypeptide with a compound under conditions which allow interaction of the compound with the polypeptide; and detecting the ability of the compound to modulate the activity or expression of the MarR family polypeptide to thereby identify a compound that modulates MarR family polypeptide activity or expression.
  • the ability of the compound to modulate MarR family polypeptide activity is detected. In another embodiment, the ability of the compound to modulate MarR family polypeptide expression is detected. In another aspect, the invention pertains to a method for identifying a compound that modulates the ability of a compound to modulate the ability of a MarR family polypeptide to interact with a MarR binding partner, by contacting a MarR family polypeptide with a compound under conditions which allow interaction of the compound with the polypeptide; and detecting the ability of the compound to modulate the ability of the MarR family polypeptide to interact with a MarR binding partner to thereby identify a compound that modulates the ability of a MarR family polypeptide to interact with a MarR binding partner.
  • the MarR binding partner is a DNA molecule. In another embodiment, the MarR binding partner is a polypeptide.
  • the invention pertains to a method for identifying a compound that modulates MDR, by contacting a MarR family polypeptide with a compound under conditions which allow interaction of the compound with the polypeptide; and detecting the ability of the compound to modulate MDR to thereby identify a compound that modulates MDR.
  • the MarR family polypeptide is expressed in a cell. In another embodiment, the MarR family polypeptide is an isolated polypeptide. In another embodiment, the DNA binding activity of the MarR family polypeptide is measured by detecting transcription from a gene locus regulated by a MarR family polypeptide.
  • the MarR family polypeptide is derived from a protein selected from the group consisting of: MarR, Ecl7kd, MprA(EmrR), and MexR.
  • the MarR family polypeptide is an E. coli MarR polypeptide. In yet another embodiment, the MarR family polypeptide comprises a MarR family polypeptide helix-turn-helix domain corresponding to about amino acids 61-80 or about 97-116 of MarR.
  • the polypeptide comprises an amino acid sequence corresponding to about amino acid 1 to about amino acid 41 of MarR. In yet another embodiment, the polypeptide comprises an amino acid sequence corresponding to about amino acid 41 to about amino acid 144 of MarR. In still another embodiment, the step of detecting the MarR family polypeptide activity comprises detecting transcription from a marRAB responsive promoter.
  • the step of detecting comprises detecting the ability of the compound to modulate the binding of MarR to marO.
  • the marRAB responsive promoter is Pmarll
  • the marRAB responsive promoter is linked to a reporter gene.
  • the reporter gene is selected from the group consisting of lacZ, phoA, or green fluorescence protein.
  • the step of detecting comprises detecting the amount of reporter gene product produced by the cell. In another embodiment, the step of assaying comprises detecting the amount of RNA produced by the cell.
  • the step of detecting comprises detecting the activity of the reporter gene product.
  • the step of detecting comprises detecting the ability of an antibody to bind to the reporter gene product.
  • the invention pertains to a method for identifying a compound that modulates MDR, comprising: screening a library of bacteriophage displaying on their surface a MarR polypeptide, the polypeptide sequence being specified by a nucleic acid molecule contained within the bacteriophage, for the ability to bind a compound to obtain those compounds having affinity for the MarR polypeptide; said method by contacting the phage which displays the MarR polypeptide with a compound so that the polypeptide can form a complex with a compound having an affinity for the polypeptide; contacting the complex of the polypeptide and bound compound with an agent that dissociates the bacteriophage from the compound; and identifying the compounds that bound to the polypeptide to thereby identify a compound that modulates MDR.
  • the compound is an antibiotic compound. In another embodiment, the compound is non-antibiotic compound. In another embodiment, the compound is a candidate disinfectant or antiseptic compound. In yet another embodiment, the compound is derived from a library of compounds.
  • Figure 1 shows the genetic organization of the Escherichia coli mar locus. Expression of the two transcriptional units, containing marC and marRAB, is under the control of independent promoters (Pmarf and Pmarjj) located within a centrally positioned promoter/operator region (marP/O).
  • the repressor MarR negatively controls marRAB expression by binding to marO.
  • MarA activates transcription o ⁇ marRAB by binding to Pmarff.
  • MarB has unknown function as does MarC which encodes a putative inner membrane protein with multiple transmembrane spanning helices.
  • FIG. 2 illustrates the structures of some chemical inducers that directly affect
  • Figure 3 shows a map of the plasmid pSup-Test.
  • Expression o ⁇ ccdB is positively controlled by the marRAB promoter (Pmarjj), 196 bp upstream of ccdB, and negatively regulated by MarR binding to the mar operator (marO) and LacR to the lac operator (lacO).
  • the sequence o ⁇ marO is given and within it sites I and II, the known MarR binding regions, are underlined. The locations of the two Sspl recognition sequences are indicated and the Sspl restriction site in marO is boldfaced.
  • Figure 4 shows locations of several mutations in MarR.
  • the thick horizontal line represents full length MarR and the scale above it depicts residues in the protein.
  • the full-length sequence of MarR is available (Cohen, S. P., et al. 1993. J. Bacteriol. 175: 1484-1492.).
  • Solid dots and asterisks on the line designate nonsense and Pro to Ser missense mutations, respectively.
  • Vertical bars above the line indicate mutations that result in moderately active MarR; vertical bars below the line mark positions of negative complementing traHs-dominant mutations.
  • Broken bars below the line depict mutations that result in near wild type repressor activities.
  • the first 41 amino acids are presumed to participate in the oligomerization of the repressor.
  • HTH putative helix-turn-helix
  • Figure 5 compares sequences of the two putative helix-turn-helix (HTH) motifs in MarR comprising residues 61-80 (MarR-M) in the middle and 97-1 16 (MarR-C) in the C-terminus of the full length protein with other known HTH structures.
  • the MarA, TrpR, Fis, TetR, and ⁇ resolvase crystal structures have been previously characterized (Pabo, C. O. and Sauer, R. T. 1984. Ann. Rev. Biochem. 53: 293-321.; Pabo, C. O. and Sauer, R. T. 1992. Annu. Rev. Biochem.
  • Figure 6 shows diagrams of the two helix-turn-helix motifs in MarR representing amino acids 61-80 (MarR-M) and 97-116 (MarR-C) of the full length protein. Residues that are well conserved among known HTH motifs are circled. In the tram-dominant mutants, wild type residues were changed to the amino acids in the rectangles.
  • Figure 7 shows the mar operator (marO) sequence (not drawn to scale).
  • MarR protects two regions (half-sites) in marO , sites I and II (Martin, R. G. and Rosner, J. L. 1995. Proc. Natl. Acad. Sci. 92: 5456-5460.) or direct repeats (DR)-l and 1' (Cohen, S. et al. 1993. J. Bacteriol. 175: 1484-1492.), from nuclease digestion (Martin and Rosner, 1995, supra) that extend (indicated by the brackets) beyond the sequences shown here.
  • Sites I and II have a dyad axis of symmetry (indicated by the broken lined boxes) and are composed of two sub-elements (represented by arrows above or below those sequences). In this figure, the top strand of the double helix (5'-3') is contained within solid boxes and its complement (3'-5') is not.
  • Figure 8 shows the sequence of Pmarff/marO.
  • the locations of the -35/- 10 hexamers sequences and MarR ribosome binding site are indicated and the Sspl restriction enzyme recognition sequence in site I of marO is in boldfaced font.
  • Figure 9 shows the locations of the MarR superrepressor mutations identified in this study.
  • the designations in parenthesis refer to the single letter code for the wild type residue and is followed by the location of that amino acid in the full length MarR and the single letter code for the mutation isolated at this point.
  • the numbers in parenthesis represent the number of independent isolates at that site.
  • the checkered box indicates the region of amino acid homology among the MarR family members.
  • Figure 10 shows MarR repressor activity assayed in the PmarfjJacZ reporter strain E. coli SPC105 (marR + ) without or with salicylate (hatched bar). The lower the LacZ activity, the stronger the activity of MarR mutants and decreased response to salicylate.
  • the present invention represents an advance in controlling processes in microbes regulated by the MarR family of proteins, e.g., their ability to grow, cause infection, and to resist drugs.
  • the examples presented herein represent the first demonstration that multiple, structurally unrelated compounds can interact directly with MarR and affect its function.
  • Prior to the instant invention only sodium salicylate had been found to directly affect the activity of MarR.
  • Radioactive salicylic acid was demonstrated to bind MarR using equilibrium dialysis assays, while other compounds, e.g., chloramphenicol and tetracycline, were not (Martin, R.G. and Rosner. 1995. Supra).
  • MDR multidrug resistance
  • the instant examples show that structurally diverse compounds can interact with MarR.
  • critical domains of MarR are identified herein. Accordingly, the present invention provides, wter alia, assays for identifying compounds that interact with MarR and, thus, lead to the development of MDR in bacteria.
  • MarR family polypeptide as used herein includes molecules related to MarR, e.g., having certain shared structural and functional features. MarR family polypeptides share amino acid sequence similarity with MarR. MarR family members, in addition to having similarity to MarR, all bind to DNA and regulate transcription. While some MarR family members negatively control transcription (e.g., MarR), others have positive/activator functions (e.g., Sly A, BadR, NhhD, and MexR). MarR family polypeptides comprise DNA and protein binding domains. In addition, MarR family polypeptides can interact with a variety of structurally unrelated compounds that regulate their activity.
  • MarR family members are taught in the art and can be found, e.g., in Sulavik et al. (1995. Molecular Medicine. 1 :436) or Miller and Sulavik. (1996.
  • MarR family polypeptides are "structurally related" to one or more of the MarR molecules set forth in the Table above. This structural relatedness can be demonstrated by sequence similarity between two MarR family nucleotide sequences or between the amino acid sequences of two MarR family polypeptides.
  • the term "MarR family polypeptide” includes polypeptides specified by MarR family genes. In isolating or identifying other MarR family molecules, sequence similarity can be shown, e.g., by generating alignments as described in more detail below.
  • a MarR family polypeptide is MarR.
  • Other preferred MarR family polypeptides include: EmrR, Ecl7kD, and MexR.
  • nucleic acid molecule(s) includes polyribonucleotides or polydeoxribonucleotides, which may be unmodified RNA or DNA or modified RNA or DNA.
  • nucleic acid molecule(s) include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded, or triple-stranded regions, or a mixture of single- and double-stranded regions.
  • nucleic acid molecule refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • nucleic acid molecule also includes DNAs or RNAs as described above that contain one or more modified bases.
  • DNAs or RNAs with backbones modified for stability or for other reasons are “nucleic acid molecule(s)" as that term is intended herein.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples are nucleic acid molecules as the term is used herein.
  • nucleic acid molecule(s) as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of nucleic acid molecules, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, for example, simple and complex cells.
  • Nucleic acid molecule(s) also embraces short nucleic acid molecules often referred to as oligonucleotide(s).
  • Preferred MarR family nucleic acid molecules are isolated.
  • An "isolated" nucleic acid molecule is one that is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • genomic DNA e.g. whether chromosomal or episomal
  • isolated includes nucleic acid molecules which are separated from flanking DNA sequences with which the DNA is naturally associated.
  • an "isolated" nucleic acid molecule is free of sequences which naturally flank the nucleic acid molecule (i.e., sequences located at the 5' and 3' ends of the nucleic acid molecule) in the DNA (e.g., chromosomal or episomal) of the organism from which the nucleic acid molecule is derived.
  • isolated DNA is not in its naturally occurring state (although, as described in more detail below, its sequence may be naturally occurring in the sense that has not been altered (e.g., mutated) from its naturally occurring form).
  • an isolated MarR family nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb, 0.1 kb, or 0.05kb of nucleotide sequences which naturally flank the nucleic acid molecule in DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • an “isolated” MarR family nucleic acid molecule may, however, be linked to other nucleotide sequences that do not normally flank the MarR family sequences in genomic DNA (e.g., the MarR family nucleotide sequences may be linked to vector sequences).
  • an "isolated" nucleic acid molecule such as a cDNA molecule, also may be free of other cellular material.
  • polypeptide(s) refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds.
  • Polypeptide(s) refers to both short chains, commonly referred to as peptides, oligopeptides and oligomers and to longer chains generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene encoded amino acids.
  • Polypeptide(s) include those modified either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature, and they are well known to those of skill in the art.
  • Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains, and the amino or carboxyl termini.
  • Modifications include, for example, acetylation, acylation, ADP- ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine.
  • Polypeptides may be branched or cyclic, with or without branching. Cyclic, branched and branched circular polypeptides may result from post-translational natural processes and may be made by entirely synthetic methods, as well.
  • an "isolated polypeptide” or “isolated protein” refers to a polypeptide or protein that is substantially free of other polypeptides, proteins, cellular material and culture medium when isolated from cells or produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • An “isolated” or “purified” polypeptide or biologically active portion thereof is substantially free of cellular material or other contaminating polypeptides from the cell or tissue source from which the MarR family polypeptide is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of MarR family polypeptide in which the polypeptide is separated from cellular components of the cells from which it is isolated or recombinantly produced.
  • the language “substantially free of cellular material” includes preparations of MarR family polypeptide having less than about 30% (by dry weight) of non- MarR family polypeptide (also referred to herein as a "contaminating polypeptide”), more preferably less than about 20% of non- MarR family polypeptide, still more preferably less than about 10% of non- MarR family polypeptide, and most preferably less than about 5% non- MarR family polypeptide.
  • the MarR family polypeptide or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the polypeptide preparation.
  • culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the polypeptide preparation.
  • substantially free of chemical precursors or other chemicals includes preparations of MarR family polypeptide in which the polypeptide is separated from chemical precursors or other chemicals that are involved in the synthesis of the polypeptide.
  • the language "substantially free of chemical precursors or other chemicals” includes preparations of MarR family polypeptide having less than about 30% (by dry weight) of chemical precursors or non- MarR family chemicals, more preferably less than about 20% chemical precursors or non- MarR family chemicals, still more preferably less than about 10% chemical precursors or non- MarR family chemicals, and most preferably less than about 5% chemical precursors or non- MarR family chemicals.
  • MarR family nucleic acid molecules and polypeptides are "naturally occurring.”
  • a "naturally-occurring" molecule refers to an MarR family molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural MarR family polypeptide).
  • naturally or non-naturally occurring variants of these polypeptides and nucleic acid molecules which retain the same functional activity, e.g., the ability to modulate adaptation to stress and/or virulence in a microbe.
  • Such variants can be made, e.g., by mutation using techniques that are known in the art. Alternatively, variants can be chemically synthesized.
  • variant(s) includes nucleic acid molecules or polypeptides that differ in sequence from a reference nucleic acid molecule or polypeptide, but retains its essential properties. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference nucleic acid molecule. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below. A typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, and/or deletions in any combination.
  • a variant of a nucleic acid molecule or polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant that is not known to occur naturally.
  • Non-naturally occurring variants of nucleic acid molecules and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.
  • MarR family polypeptides described herein are also meant to include equivalents thereof.
  • variants can be made, e.g., by mutation using techniques that are known in the art.
  • variants can be chemically synthesized.
  • mutant forms of MarR family polypeptides which are functionally equivalent, e.g., have the ability to bind to DNA and to regulate transcription from an operon
  • Mutations can include, e.g., at least one of a discrete point mutation which can give rise to a substitution, or by at least one deletion or insertion.
  • random mutagenesis can be used. Mutations can also be made by random mutagenesis or using cassette mutagenesis.
  • telomere mutagenesis can be used.
  • Megaprimer PCR can be used (O.H. Landt, 1990. Gene 96:125-128).
  • such variants have at least about 25, 30, 35, 40, 45, 50, or 60% or more amino acid identity with a naturally occurring MarR family polypeptide. In preferred embodiments, such variants have at least about 70% amino acid identity with a naturally occurring MarR family polypeptide. In more preferred embodiments, such variants have at least about 80% amino acid identity with a naturally occurring MarR family polypeptide. In particularly preferred embodiments, such variants have at least about 90% amino acid identity and preferably at least about 95% amino acid identity with a naturally occurring MarR family polypeptide.
  • a nucleic acid molecule encoding a variant of an MarR family polypeptide is capable of hybridizing under stringent conditions to a nucleic molecule encoding a naturally occurring MarR family polypeptide.
  • Preferred MarR family nucleic acid molecules and MarR family polypeptides are "naturally occurring.”
  • a "naturally-occurring" molecule refers to an MarR family polypeptide encoded by a nucleotide sequence that occurs in nature (e.g., encodes a natural MarR family polypeptide).
  • Such molecules can be obtained from other microbes, e.g., based on their sequence similarity to the MarR family molecules described herein.
  • variants of these polypeptides and nucleic acid molecules which retain the same functional activity, e.g., the ability to modulate microbial responses to environmental stress and. thereby, modulate microbial adaptation to stress and/or microbial virulence are also within the scope of the invention.
  • Such variants can be made, e.g., by mutation using techniques which are known in the art.
  • variants can be chemically synthesized.
  • MarR family polypeptide activity includes the ability to bind to, and control gene expression. Other MarR family polypeptide activities are described in more detail below.
  • the language “marR family promoter” includes a promoter which is positively or negatively regulated by a MarR family polypeptide.
  • the promoter is a marRAB family promoter.
  • a marRAB family promoter initiates transcription of an operon in a microbe and is structurally or functionally related to the marRAB promoter, e.g., is bound by MarA or a protein related to MarA.
  • the marRAB family promoter is a marRAB promoter.
  • marRAB family promoters as defined herein, e.g., the 405-bp Thai fragment from the marO region is a marRAB family promoter (Cohen et al. 1993. J. Bact. 175:7856).
  • MarA has been shown to bind to a 16 bp MarA binding site (referred to as the "marbox" within marO (Martin et al. 1996. J. Bacteriol. 178:2216).
  • MarA also initiates transcription from the acrAB; micF; mlr 1,2,3; sip; nfo; inaA;fpr; sodA; soi-17, 19; zwffumC; or rpsF promoters (Alekshun and Levy. 1997. Antimicrobial Agents and Chemother. 41 :2067).
  • marRAB family promoters are known in the art and include: araBAD, araE, araFGH and araC, which are activated by AraC; Pm, which is activated by XylS; melAB which is activated by MelR; and oriC which is bound by Rob.
  • a first molecule can be said to interact with a second when it inhibits the binding of the second molecule to a target binding partner (a molecule such as a nucleic acid or polypeptide molecule to which that second molecule normally binds, e.g., in a cell), or when it alters the activity of the second molecule, e.g., by steric interaction with a domain of the second molecule that mediates its activity.
  • a target binding partner a molecule such as a nucleic acid or polypeptide molecule to which that second molecule normally binds, e.g., in a cell
  • a DNA binding domain of a MarR family polypeptide can interact with DNA and alter the level of transcription of DNA or with a polypeptide molecule.
  • compounds can interact with (e.g., by binding) to an MarR family polypeptide and alter the activity of the MarR family polypeptide or can interact with (e.g., by binding) to an MarR family nucleic acid molecule and alter transcription of an MarR family polypeptide from that nucleic acid molecule.
  • MDR multiple drug resistance
  • a chromosomal or plasmid encoded genetic locus in an organism, e.g., a marRAB locus, that results in the ability of the organism to minimize the toxic effects of a compound to which it has been exposed, as well as to other non-related compounds, e.g., by stimulating an efflux pump(s) or microbiological catabolic or metabolic processes.
  • reporter gene includes any gene which encodes an easily detectable product which is operably linked to a promoter, e.g., a marRAB family promoter.
  • RNA polymerase may bind to the promoter of the regulatory region and proceed to transcribe the nucleotide sequence of the reporter gene.
  • a reporter gene construct consists o ⁇ marRAB family promoter linked to a reporter gene.
  • sequences which are herein collectively referred to as transcriptional regulatory elements or sequences may also be included in the reporter gene construct.
  • the construct may include sequences of nucleotides that alter translation of the resulting mRNA, thereby altering the amount of reporter gene product.
  • the term "compound” includes any reagent which is tested using the assays of the invention to determine whether it modulates a MarR family polypeptide activity. More than one compound, e.g.. a plurality of compounds, can be tested at the same time for their ability to modulate a MarR family polypeptide activity in a screening assay.
  • Non-peptidic compounds include antibiotic and non- antibiotic compounds.
  • compounds include candidate detergent or disinfectant compounds.
  • Exemplary compounds which can be screened for activity include, but are not limited to, peptides, non-peptidic compounds, nucleic acids, carbohydrates, small organic molecules (e.g.. polyketides), and natural product extract libraries.
  • non-peptidic compound is intended to encompass compounds that are comprised, at least in part, of molecular structures different from naturally-occurring L-amino acid residues linked by natural peptide bonds.
  • non-peptidic compounds are intended to include compounds composed, in whole or in part, of peptidomimetic structures, such as D-amino acids, non-naturally-occurring L-amino acids, modified peptide backbones and the like, as well as compounds that are composed, in whole or in part, of molecular structures unrelated to naturally-occurring L-amino acid residues linked by natural peptide bonds.
  • Non-peptidic compounds also are intended to include natural products.
  • MarR family member polypeptide sequences are "structurally related" to one or more known MarR family members, preferably to MarR. This structural relatedness is shown by sequence similarity between two MarR family polypeptide sequences or between two MarR family nucleotide sequences. Sequence similarity can be shown, e.g., by optimally aligning MarR family member sequences using an alignment program for purposes of comparison and comparing corresponding positions. To determine the degree of similarity between sequences, they will be aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of one protein for nucleic acid molecule for optimal alignment with the other protein or nucleic acid molecules). The amino acid residues or bases and corresponding amino acid positions or bases are then compared.
  • amino acid residues are not identical, they may be similar.
  • an amino acid residue is "similar" to another amino acid residue if the two amino acid residues are members of the same family of residues having similar side chains. Families of amino acid residues having similar side chains have been defined in the art (see, for example. Altschul et al. 1990. J. Mol. Biol. 215:403) including basic side chains (e.g..
  • lysine, arginine, histidine acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta- branched side chains (e.g., threonine, valine. isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan).
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • Alignment strategies are well known in the art; see, for example, Altschul et al. supra for optimal sequence alignment.
  • MarR family polypeptides share some amino acid sequence similarity with MarR.
  • the nucleic acid and amino acid sequences of MarR as well as other MarR family polypeptides are available in the art.
  • the nucleic acid and amino acid sequence of MarR can be found, e.g.. on GeneBank (accession number M96235 or in Cohen et al. 1993. j. Bacteriol. 175:1484. or in SEQ ID NO:l).
  • the nucleic acid and protein sequences of MarR can be used as "query sequences" to perform a search against databases (e.g., either public or private) to, for example, identify other MarR family members having related sequences.
  • databases e.g., either public or private
  • Such searches can be performed, e.g., using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and NBLAST
  • XBLAST and NBLAST can be used. See http://www.ncbi.nlm.nih.gov.
  • MarR family members can also be identified as being structurally similiar based on their ability to specifically hybridize to nucleic acid sequences specifying MarR.
  • Such stringent conditions are known to those skilled in the art and can be found e.g., in Current Protocols in Molecular Biology. John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • a preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C.
  • SSC sodium chloride/sodium citrate
  • Tm melting temperature
  • Tm is the temperature in °C at which half the molecules of a given sequence are melted or single-stranded.
  • the Tm can be estimated in degrees C as 2(number of A+T residues) + 4(number of C+G residues).
  • Hybridization or annealing of nucleic acid molecules should be conducted at a temperature lower than the Tm, e.g., 15 °C, 20°C, 25°C or 30°C lower than the Tm.
  • the nucleic acid sequence of a MarR family member identified in this way is at least about 10%, 20%, more preferably at least about 30%, more preferably at least about 40% identical and most preferably at least about 50%, or 60%, 70%, 80%, 90% or more identical or more with a MarR nucleotide sequence, e.g., with the entire length of the nucleotide sequence or a portion thereof.
  • a MarR family member nucleic acid molecule has at least about 10%, 20%, 30%, 40% identity and most preferably at least about 50%, 60%, 70%, 80%, 90% or more identity with a nucleic acid molecule comprising at least about 100, 200, 300, 400, 500, or more contiguous nucleotides of a MarR family member, e.g., as listed in the Table above.
  • MarR family members have an amino acid sequence at least about 20%, more preferably at least about 30%. more preferably at least about 40% identical and most preferably at least about 50%, or 60%, 70%, 80%, 90% or more identical with a MarR amino acid sequence.
  • the level of sequence similarity among microbial regulators of gene transcription is not necessarily high. This is particularly true in the case of divergent genomes where the level of sequence identity may be low, e.g., less than 20% (e.g., B. burgdorferi as compared e.g., to B. subtilis).
  • the level of amino acid sequence homology between MarR and Pecs is about 31% and the level of amino acid sequence homology between MarR and PapX is about 28% when determined as described above. Accordingly, structural similarity among MarR family members can also be determined based on "three-dimensional correspondence" of amino acid residues.
  • three-dimensional correspondence is meant to includes residues which spatially correspond, e.g., are in the same functional position of a MarR family protein member as determined, e.g., by x-ray crystallography, but which may not correspond when aligned using a linear alignment program.
  • the language "three-dimensional correspondence” also includes residues which perform the same function, e.g., bind to DNA or bind the same cofactor, as determined, e.g., by mutational analysis.
  • Preferred MarR family polypeptides include: MarR, EmrR, Ecl7kD, MexR,
  • a MarR family polypeptide is selected from the group consisting of: MarR, EmrR, Ecl7kD, and MexR .
  • a MarR family polypeptide is MarR.
  • MarR family members have a MarR family polypeptide activity, i.e., they bind to DNA and regulate transcription. Some MarR family members positively regulate transcription (e.g., SlyA, BadR, NhhD, or MexR), while others negatively regulate transcription (e.g., MarR).
  • MarR family polypeptides can control the expression of microbial loci involved in: regulation of antibiotic resistance [e.g., MarR (Cohen et al. 1993. J. Bacteriol. 175:1484), EmrR (Lomovskaya and Lewis. 1992. Proc. Natl. Acad. Sci. 89:8938), and Ecl7kD (Sulavik et al. 1995. Mol. Med. 1 :436), and MexR (Poole et al. 1996. Antimicrob. Agents. Chemother.
  • tissue-specific adhesive properties e.g., PapX (Marklund et al., 1992. Mol. Microbiol. 6:2225)]
  • regulation of expression of a cryptic hemolysin e.g., SlyA (Ludwig et al. 1995 249:4740)
  • regulation of protease production e.g., Hpr from B. subtilis (Perago and Hoch. 1988. J. Bacteriol. 170:2560) and PecS from Erwinia chrysanthemi (Reverchon et al., 1994. Mol. Microbiol. 11 :1127)
  • regulation of sporulation e.g., Hpr (Perego and Hoch. 1988. J.
  • MarR family polypeptides The activity of MarR family polypeptides is antagonized by salicylate (Lomovskaya et al., 1995. J. Bacteriol. 177:2328; Sulavik et al. 1995. Mol. Med. 1 :436).
  • Preferred MarR family polypeptide activities include regulation of multiple drug resistance and/or regulation of virulence.
  • a fragment of a MarR family polypeptide refers to a portion of a full-length MarR family polypeptide which is useful in a screening assay to identify compounds which modulate a biological activity (e.g., DNA binding) of a MarR family polypeptide.
  • MarR family polypeptides for use in the instant screening assays can be full length MarR family member proteins or fragments thereof.
  • a MarR family polypeptide can comprise, consist essentially of, or consist of an amino acid sequence derived from the full length amino acid sequence of a MarR family member.
  • a polypeptide comprising a MarR family polypeptide DNA interacting domain can be used in a screening assay .
  • compounds can be tested for their ability to modulate, e.g., interfere with and reduce the ability of a MarR family polypeptide to directly bind to DNA.
  • compounds can be tested for their ability to alter downstream effects of this DNA binding, e.g., to alter the ability of a MarR family polypeptide to regulate transcription of an operon.
  • a polypeptide comprising a MarR family member protein interacting domain can be used in a screening assay .
  • compounds can be tested for their ability to bind to a protein interactive domain of a MarR family polypeptide. Such binding can lead to allosteric changes in the polypeptide that interfere with or enhance the ability of the a MarR family polypeptide to interact with DNA or can block or alter the ability of other proteins (which are necessary for the MarR family polypeptide to regulate transcription) to interact with MarR.
  • compounds can be tested for their ability to alter events downstream of such an interaction, e.g., the ability of a compound to bind to a polypeptide and alter the ability of the polypeptide to regulate transcription of an operon can be tested.
  • Full length MarR family polypeptides also comprise MarR family polypeptide helix-turn-helix domains.
  • helix-turn-helix domain includes the art recognized definition of the term.
  • Helix-turn helix (HTH) domains have been implicated in DNA binding (A nn Rev. of Biochem. 1984. 53:293).
  • An example of a consensus sequence of a helix-turn-helix domain can be found in Brunelle and Schleif (1989. J. Mol. Biol. 209:607). The domain has been illustrated by the sequence
  • JJJBbJJXOXJJJJBJJXX (adapted from Kelley and Yanofsky, 1985). This sequence indicates if an amino acid is exposed to solvent (J), partially exposed (X), completely buried (B), non-branched chain (b), glycine or alanine (O), or makes contacts within DNA (bold) (Branden, C. and Tooze, J. (1991) In Introduction to Protein Structure. New York, NY USA; London, England: Garland Publishing, Inc., pp. 87-111.; Pabo, CO. and Sauer, R.T. (1984) Ann. Rev. Biochem. 53: 293-321.; Pabo, CO. and Sauer, R.T. (1992) Annu. Rev. Biochem. 61 : 1053-1095).
  • the two helix-turn-helix domains appear to be in the middle and C-terminal DNA binding domain of the polypeptide and comprise from about amino acids 61-80 and from about amino acids 97-116 of MarR.
  • the locations of HTH domains of other MarR family members which correspond to those identified in MarR and which can be used in the subject screening assays can be identified by one of skill in the art.
  • MarR polypeptide sequence and an alignment program e.g., ALIGN, BLAST, or MultAlin
  • an alignment program e.g., ALIGN, BLAST, or MultAlin
  • helix-turn-helix domains in the MarR family members and use them in screening assays.
  • the first and second helix-turn-helix domains of MarR are shown in Figure 5.
  • a polypeptide for use in a screening assay comprises a DNA interacting domain of a MarR family polypeptide.
  • such aa polypeptide for use in a screening assay comprises a sequence that corresponds to the amino acid sequence shown from about amino acid 41 to about amino acid 144 of MarR.
  • a MarR family polypeptide comprises from between about amino acid 41 to about amino acid 144 of MarR.
  • a polypeptide for use in a screening assay comprises a protein interacting domain of a MarR family polypeptide.
  • such a polypeptide comprises a protein interacting domain comprising an amino acid sequence that corresponds to the amino acid sequence shown from about amino acid 1 to about amino acid 41 of MarR.
  • a MarR family polypeptide comprises a protein interacting sequence shown from about amino acid 1 to about amino acid 41 of MarR.
  • a polypeptide for use in a screening assay comprises a helix- turn-helix domain of a MarR family polypeptide.
  • a polypeptide comprises a helix-turn-helix domain comprising a sequence that corresponds to the amino acid sequence shown from about amino acid 61 to about amino acid 80 of MarR and/or from about amino acid 97 to about amino acid 116 of MarR.
  • a polypeptide comprises an amino acid sequence from about amino acid 61 to about amino acid 80 of MarR and/or from about amino acid 97 to about amino acid 116 of MarR.
  • polypeptide sequences may also be present, e.g., portions of a MarR family polypeptide that are not absolutely required for MarR function.
  • non-MarR family polypeptide sequences i.e., derived from a different protein
  • a polypeptide for use in screening compounds consists essentially of a protein binding domain of a MarR family polypeptide, e.g., a polypeptide comprising an amino acid sequence that corresponds to the amino acid sequence shown from between about amino acid 1 to about amino acid 41 of MarR.
  • a polypeptide for use in screening compounds consists essentially of a DNA interacting domain, e.g., a polypeptide comprising an amino acid sequence corresponding to the amino acid sequence shown from about amino acid 41 to about amino acid 144 of MarR.
  • a polypeptide for use in screening consists essentially of an HTH domain from a MarR family polypeptide, e.g., having an amino acid sequence that corresponds to that shown from about amino acids 61-80 or from about amino acids 97- 116 of MarR.
  • a polypeptide for use in the subject screening assays consists essentially of a portion of a MarR polypeptide.
  • MarR family polypeptides are "naturally occurring.”
  • a "naturally-occurring" molecule refers to an MarR family molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural MarR family protein).
  • variants of these polypeptides and nucleic acid molecules which retain the same functional activity, e.g., the ability to bind to DNA and regulate transcription.
  • Such variants can be made, e.g., by mutation using techniques which are known in the art.
  • variants can be chemically synthesized.
  • MarR family polypeptides described herein are also meant to include equivalents thereof.
  • mutant forms of MarR family polypeptides which are functionally equivalent, can be made using techniques which are well known in the art. Mutations can include, e.g., at least one of a discrete point mutation which can give rise to a substitution, or by at least one deletion or insertion. For example, random mutagenesis can be used. Mutations can be made, e.g., by random mutagenesis or using cassette mutagenesis.
  • telomere mutagenesis can be used.
  • Megaprimer PCR can be used (O.H. Landt, Gene 96:125-128).
  • polypeptides suitable for use in the claimed assays such as those which retain their function (e.g., the ability to bind to DNA, to regulate transcription from an operon) or those which are critical for binding to regulatory molecules (such as test compounds) can be easily determined by one of ordinary skill in the art, e.g, using standard truncation or mutagenesis techniques and used in the instant assays. Exemplary techniques are described by Gallegos et al. (1996. J. Bacteriol. 178:6427).
  • MarR family polypeptides comprising only naturally-occurring amino acids
  • MarR family peptidomimetics are also provided.
  • Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the template peptide. These types of non-peptide compound are termed "peptide mimetics” or “peptidomimetics” (Fauchere, J. (1986) Adv. Drug Res. 15: 29; Veber and Freidinger (1985) TINS p.392; and Evans et al. (1987) J. Med. Chem 30: 1229, which are incorporated herein by reference) and are usually developed with the aid of computerized molecular modeling.
  • Peptide mimetics that are structurally similar to therapeutically useful peptides may be used to produce an equivalent therapeutic or prophylactic effect.
  • Such peptide mimetics may have significant advantages over polypeptide embodiments, including, for example: more economical production, greater chemical stability, enhanced pharmacological properties (half-life, abso ⁇ tion, potency, efficacy, etc.), altered specificity (e.g., a broad-spectrum of biological activities), reduced antigenicity, and others.
  • Labeling of peptidomimetics usually involves covalent attachment of one or more labels, directly or through a spacer (e.g., an amide group), to non-interfering position(s) on the peptidomimetic that are predicted by quantitative structure-activity data and/or molecular modeling.
  • Such non-interfering positions generally are positions that do not form direct contacts with the macromolecules(s) to which the peptidomimetic binds to produce the therapeutic effect.
  • Derivitization (e.g., labelling) of peptidomimetics should not substantially interfere with the desired biological or pharmacological activity of the peptidomimetic.
  • Systematic substitution of one or more amino acids of an MarR family amino acid sequence with a D-amino acid of the same type may be used to generate more stable peptides.
  • constrained peptides comprising an MarR family amino acid sequence or a substantially identical sequence variation may be generated by methods known in the art (Rizo and Gierasch (1992) Ann. Rev. Biochem. 61 : 387, inco ⁇ orated herein by reference); for example, by adding internal cysteine residues capable of forming intramolecular disulfide bridges which cyclize the peptide.
  • MarR family polypeptides identified herein will enable those of skill in the art to produce polypeptides corresponding to MarR family peptide sequences and sequence variants thereof.
  • Such polypeptides may be produced in prokaryotic or eukaryotic host cells by expression of polynucleotides encoding an MarR family peptide sequence, frequently as part of a larger polypeptide. Alternatively, such peptides may be synthesized by chemical methods.
  • Peptides can be produced, typically by direct chemical synthesis, and used e.g., as agonists or antagonists of an MarR familymolecule, e.g., to modulate binding of an MarR family polypeptide and a molecule with which it normally interacts.
  • Peptides can be produced as modified peptides, with nonpeptide moieties attached bycovalent linkage to the N-terminus and/or C-terminus.
  • either the carboxy-terminus or the amino-terminus, or both are chemically modified. The most common modifications of the terminal amino and carboxyl groups are acetylation and amidation, respectively.
  • Amino-terminal modifications such as acylation (e.g., acetylation) or alkylation (e.g., methylation) and carboxy-terminal-modifications such as amidation, as well as other terminal modifications, including cyclization, may be inco ⁇ orated into various embodiments of the invention.
  • Certain amino-terminal and/or carboxy-terminal modifications and/or peptide extensions to the core sequence can provide advantageous physical, chemical, biochemical, and pharmacological properties, such as: enhanced stability, increased potency and/or efficacy, resistance to serum proteases, desirable pharmacokinetic properties, and others.
  • Peptides may be used to control bacterial growth, e.g, to treat infection or to clean surfaces.
  • the instant invention also pertains to isolated MarR family member polypeptides, portions thereof, and the nucleic acid molecules encoding them, including naturally occurring and mutant forms.
  • MarR family polypeptides for use in screening assays are "isolated or recombinant" polypeptides.
  • MarR family polypeptides can be made from nucleic acid molecules.
  • Nucleic acid molecules encoding MarR family polypeptides can be used to produce MarR family polypeptides for use in the instant assays.
  • nucleic acid molecules encoding a MarR family polypeptide can be isolated (e.g., isolated from the sequences which naturally flank it in the genome and from cellular components) and can be used to produce a MarR family polypeptide.
  • nucleic acid molecule which has been (1) amplified in vitro by, for example, polymerase chain reaction (PCR); (2) recombinantly produced by cloning, or (3) purified, as by cleavage and gel separation; or (4) synthesized by, for example, chemical synthesis can be used to produce MarR family polypeptides.
  • PCR polymerase chain reaction
  • nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • Nucleic acid molecules specifying MarR family polypeptides can be placed in a vector.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked.
  • expression vector includes any vector, (e.g., a plasmid, cosmid or phage chromosome) containing a gene construct in a form suitable for expression by a cell (e.g., linked to a promoter).
  • plasmid and vector are used interchangeably, as a plasmid is a commonly used form of vector.
  • the invention is intended to include other vectors which serve equivalent functions.
  • Exemplary expression vectors for expression of a gene encoding a MarR family polypeptide and capable of replication in a bacterium such as a bacterium from a genus selected from the group consisting of: Escherichia, Bacillus. Streptomyces, Streptococcus, or in a cell of a simple eukaryotic fungus such as a Saccharomyces or, Pichia, or in a cell of a eukaryotic organism such as an insect, a bird, a mammal, or a plant, are known in the art.
  • Such vectors may carry functional replication-specifying sequences (replicons) both for a host for expression, for example a Streptomyces, and for a host, for example, E. coli, for genetic manipulations and vector construction. See e.g. U.S.P.N 4,745,056. Suitable vectors for a variety of organisms are described in Ausubel, F. et al , Short Protocols in Molecular Biology, Wiley, New York ( 1995), and for example, for Pichia, can be obtained from Invitrogen (Carlsbad, CA).
  • replicons replication-specifying sequences
  • Useful expression control sequences include, for example, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is ' directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda , the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast ⁇ -mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • a useful translational enhancer sequence is described in U.S.P.N. 4,820,639.
  • Transcriptional regulatory sequence is a generic term to refer to DNA sequences, such as initiation signals, enhancers, operators, and promoters, which induce or control transcription of protein coding sequences with which they are operably linked. It will also be understood that a recombinant gene encoding a MarR family polypeptide can be under the control of transcriptional regulatory sequences which are the same or which are different from those sequences which control transcription of the naturally-occurring MarR family gene.
  • Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in En ⁇ ymology 185, Academic Press, San Diego, CA (1990).
  • any of a wide variety of expression control sequences that control the expression of a DNA sequence when operatively linked to it, may be used in these vectors to express DNA sequences encoding the MarR family proteins of this invention.
  • Appropriate vectors are widely available commercially and it is within the knowledge and discretion of one of ordinary skill in the art to choose a vector which is appropriate for use with a given microbial cell.
  • sequences encoding MarR family polypeptides can be introduced into a cell on a self-replicating vector or may be introduced into the chromosome of a microbe using homologous recombination or by an insertion element such as a transposon.
  • Such vectors can be introduced into cells using standard techniques, e.g., transformation or transfection.
  • transformation and “transfection” mean the introduction of a nucleic acid, e.g., an expression vector, into a recipient or "host” cell.
  • transduction means transfer of a nucleic acid sequence, preferably DNA, from a donor to a recipient cell, by means of infection with a virus previously grown in the donor, preferably a bacteriophage.
  • Nucleic acids can also be introduced into microbial cells by transformation using calcium chloride or electroporation.
  • Cells are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny or potential progeny of such a cell.
  • cells used to express MarR family polypeptides for purification e.g., host cells, comprise a mutation which renders any endogenous MarR family polypeptide nonfunctional or causes the endogenous polypeptide to not be expressed.
  • mutations may also be made in other related genes of the host cell, such that there will be no interference from the endogenous host loci.
  • a MarR family polypeptides e.g., recombinantly expressed polypeptides
  • the medium can be collected.
  • the host cells can be lysed to release the MarR family polypeptide.
  • Such spent medium or cell lysate can be used to concentrate and purify the MarR family polypeptide.
  • the medium or lysate can be passed over a column, e.g., a column to which antibodies specific for the MarR family member polypeptide have been bound.
  • a column e.g., a column to which antibodies specific for the MarR family member polypeptide have been bound.
  • Such antibodies can be specific for a non- MarR family member polypeptide which has been fused to the MarR family polypeptide (e.g., as a tag) to facilitate purification of the MarR family member polypeptide.
  • Other means of purifying MarR family member polypeptides are known in the art.
  • the invention provides a method (also referred to herein as a "screening assay") to identify those compounds which modulate (enhance (agonists) or block (antagonists)) the action of MarR family polypeptides or nucleic acid molecules, particularly those compounds that are bacteriostatic and/or bactericidal or prevent the infectious process.
  • the subject screening assays can be used to identify modulators, i.e.. candidate or test compounds or agents (e.g., polypeptides, peptides, peptidomimetics. small molecules or other drugs) which modulate MarR family polypeptides, i.e., have a stimulatory or inhibitory effect on, for example, MarR family polypeptide expression or MarR family polypeptide activity.
  • Test compounds may be natural substrates and ligands or may be structural or functional mimetics. See, e.g.. Coligan et al., Current Protocols in Immunology 1(2): Chapter 5 (1991).
  • MarR family polypeptide agonists and antagonists can be assayed in a variety of ways.
  • the invention provides for methods for identifying a compound which modulates an MarR family molecule, e.g., by detecting or measuring the ability of the compound to interact with an MarR family nucleic acid molecule or an MarR family polypeptide or the ability of a compound to modulate the activity or expression of an MarR family polypeptide.
  • the ability of a compound to modulate the binding of an MarR family polypeptide or MarR family nucleic acid molecule to a molecule to which they normally bind e.g., an MarR family binding polypeptide can be tested.
  • Compounds for testing in the instant methods can be derived from a variety of different sources and can be known or can be novel. Each of the DNA sequences provided herein may be used in the discovery and development of antibacterial compounds. The encoded proteins, upon expression, can be used as a target for the screening of antibacterial drugs. In another embodiment, antisense nucleic acid molecules or nucleic acid molecules that encode for dominant negative MarR family mutants can also be tested in the subject assays. In one embodiment, libraries of compounds are tested in the instant methods. In another embodiment, known compounds are tested in the instant methods. In another embodiment, compounds among the list of compounds generally regarded as safe (GRAS) by the Environmental Protection Agency are tested in the instant methods. In one embodiment, a library of compounds can be screened in the subject assays.
  • GRAS chemical regarded as safe
  • a recent trend in medicinal chemistry includes the production of mixtures of compounds, referred to as libraries. While the use of libraries of peptides is well established in the art, new techniques have been developed which have allowed the production of mixtures of other compounds, such as benzodiazepines (Bunin et al. 1992. J. Am. Chem. Soc. 114:10987; DeWitt et al. 1993. Proc. Natl Acad. Sci. USA 90:6909) peptoids (Zuckermann. 1994. J. Med. Chem. 37:2678) oligocarbamates (Cho et al. 1993. Science. 261 : 1303), and hydantoins (DeWitt et al. supra).
  • Exemplary compounds which can be screened for activity include, but are not limited to, peptides, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. 1998. Science 282:63), and natural product extract libraries.
  • the test compound is a peptide or peptidomimetic.
  • the compounds are small, organic non-peptidic compounds.
  • combinatorial polypeptides can be produced from a cDNA library.
  • the efficacy of the agonist or antagonist can be assessed by generating dose response curves from data obtained using various concentrations of the test modulating agent.
  • a control assay can also be performed to provide a baseline for comparison.
  • a variety of different techniques can be used to perform such an assay, e.g., by determining whether a compound modulates binding of a MarR family protein to a molecule with which it normally interacts, (a binding partner such as DNA, e.g., a marRAB promoter, or a polypeptide). For example, the ability of a compound to decrease binding of a MarR family polypeptide to DNA, e.g., to decrease the binding of MarR to marO, or the ability of the compound to reduce MarR family polypeptide- mediated regulation of transcription from such a promoter can be measured. As described in more detail below, either whole cell or cell free assay systems can be employed.
  • assays are useful in the identification of compounds which will lead to an alteration in the expression of genetic loci in microbes that are controlled by MarR family members, e.g., loci which mediate MDR or virulence. If compounds are identified as interfering with transcription of a desirable gene or gene locus, the use of such compounds can be minimized, whereas if compounds are identified as enhancing transcription of an undesirable gene or gene locus, the use of such compounds can be promoted. For example, compounds that interfere with the activity of MarR and promote MDR can be identified and their use as antimicrobials or disinfectants curtailed. As described in more detail below, either whole cell or cell free assay systems can be employed.
  • the subject screening assays can be performed using whole cells.
  • the step of determining whether a compound modulates, e.g., reduces the activity or expression of an MarR family polypeptide comprises contacting a cell expressing an MarR family polypeptide with a compound and measuring the ability of the compound to modulate the activity or expression of an MarR family polypeptide.
  • modulators of MarR family polypeptide expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of MarR family polypeptide mRNA or protein in the cell is determined.
  • the level of expression of MarR family polypeptide mRNA or protein in the presence of the candidate compound is compared to the level of expression of MarR family polypeptide mRNA or polypeptide in the absence of the candidate compound.
  • the candidate compound can then be identified as a modulator of MarR family polypeptide expression based on this comparison. For example, when expression of MarR family polypeptide mRNA or protein is greater (e.g., statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of MarR family polypeptide mRNA or protein expression.
  • the candidate compound when expression of MarR family polypeptide mRNA or protein is less (e.g., statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of MarR family mRNA or protein expression.
  • the level of MarR family mRNA or protein expression in the cells can be determined by methods described herein for detecting MarR family mRNA or protein.
  • transcription of an MarR family gene can be measured in control cells which have not been treated with the compound and compared with that of test cells which have been treated with the compound.
  • cells which express endogenous MarR family polypeptides or which are engineered to express or overexpress recombinant MarR family polypeptides can be caused to express or overexpress a recombinant MarR family polypeptide in the presence and absence of a test modulating agent of interest, with the assay scoring for modulation in MarR family polypeptide responses by the target cell mediated by the test agent.
  • modulating agents which produce a change e.g., a statistically significant change in MarR family polypeptide -dependent responses (either an increase or decrease) can be identified.
  • Recombinant expression vectors that can be used for expression of MarR family polypeptide are known in the art (see discussions above).
  • the MarR family polypeptide -coding sequences are operatively linked to regulatory sequences that allow for constitutive or inducible expression of MarR family polypeptide in the indicator cell(s).
  • Use of a recombinant expression vector that allows for constitutive or inducible expression of MarR family polypeptide in a cell can be used for identification of compounds that enhance or inhibit the activity or expression of MarR family polypeptide.
  • the MarR family polypeptide coding sequences are operatively linked to regulatory sequences of the endogenous MarR family polypeptide gene (i.e., the promoter regulatory region derived from the endogenous gene).
  • regulatory sequences of the endogenous MarR family polypeptide gene i.e., the promoter regulatory region derived from the endogenous gene.
  • the step of determining whether a compound reduces the activity of a MarR family polypeptide comprises contacting the cell with a compound and detecting the ability of the compound to increase transcription from a marRAB promoter.
  • the MarR family polypeptide regulates transcription (either positively, e.g., SlyA, BadR, NhhD, or MexR, or negatively, e.g., MarR)
  • a compound would be identified based on its ability to modulate, e.g. increase or decrease, the control level of transcription as compared to the level of transcription in a cell which has not been treated with the compound.
  • the level of transcription can be determined by measuring the amount of RNA produced by the cell.
  • RNA can be isolated from cells which express an MarR family polypeptide and that have been incubated in the presence or absence of compound.
  • Northern blots using probes specific for the sequences to be detected can then be performed using techniques known in the art. Numerous other, art- recognized techniques can be used.
  • western blot analysis can be used to test for MarR family.
  • transcription of specific RNA molecules can be detected using the polymerase chain reaction, for example by making cDNA copies of the RNA transcript to be measured and amplifying and measuring them.
  • RNAse protection assays such as SI nuclease mapping or RNase mapping can be used to detect the level of transcription of a gene.
  • sequences not normally regulated by a MarR family member can be regulated by a MarR family polypeptide by linking them to a promoter that is regulated by a MarR family member polypeptide.
  • sequences can be linked to a marRAB family promoter, including, for example, endogenous sequences or reporter gene sequences.
  • Exemplary endogenous sequences which can be detected include: acrAB; micF; mlr 1,2,3; sip; inaA; fpr; sodA; soi-17,19; zwffumC; or rpsF; another example, would be araBAD, araE, araFGH and araC, which are activated by AraC; Pm, which is activated by XylS; melAB which is activated by MelR; and oriC which is bound by Rob, as well as sequences from genetic loci that are identified using the assays described infra.
  • the ability of a compound to induce a change in transcription from a marRAB promoter can be accomplished by detecting the amount of a polypeptide produced by the cell.
  • Polypeptides which can be detected include any polypeptides which are produced upon the activation of a MarR family responsive promoter, including, for example, both endogenous sequences and reporter gene sequences.
  • Exemplary endogenous polypeptides which can be detected include: AcrAB; Mlr 1,2,3; Sip; InaA; Fpr; SodA; Soi-17,19: Zwf; FumC; or RpsF (Alekshun and Levy. 1997. Antimicrobial Agents and Chemother. 41 :2067).
  • the ability of a compound to modulate a MarR family polypeptide activity can be tested by detecting the ability of the compound to affect the a virulence or drug resistance phenotype in a microbe, e.g. by testing the ability of the microbe to resist antibiotics or to cause infection.
  • the ability of a compound to induce a change in transcription or translation of an MarR family polypeptide can be accomplished by measuring the amount of MarR family polypeptide produced by the cell.
  • Polypeptides which can be detected include any polypeptides which are produced upon the activation of an MarR family responsive promoter, including, for example, both endogenous sequences and reporter gene sequences.
  • the amount of polypeptide made by a cell can be detected using an antibody against that polypeptide. In other embodiments, the activity of such a polypeptide can be measured.
  • sequences which are regulated by an MarR family promoter can be detected.
  • sequences not normally regulated by an MarR family promoter can be assayed by linking them to a promoter that regulates transcription of an MarR family polypeptide.
  • such a promoter is linked to a reporter gene, the transcription of which is readily detectable.
  • a bacterial cell e.g., an E. coli cell
  • reporter genes include, but are not limited to, CAT (chloramphenicol acetyl transferase) (Alton and Vapnek (1979), Nature 282: 864-869) luciferase, and other enzyme detection systems, such as beta-galactosidase; firefly luciferase (deWet et al.
  • the ability of a compound to modulate the binding of an MarR family polypeptide to an MarR family binding partner can be determined.
  • exemplary MarR family binding partners include nucleic acid molecules, such as DNA, as well as polypeptides that are downstream of MppA, a periplasmic binding protein in E. coli which functions upstream of MarA in a signal transduction pathway (Li and Park. 1999. J. of Bacteriology. 181 :4842) and related molecules.
  • MarR family binding polypeptides can be identified using techniques which are known in the art. For example, in the case of binding polypeptides that interact with MarR family polypeptides, interaction trap assays or two hybrid screening assays can be used.
  • MarR family binding partners can be identified e.g., e.g., by using an MarR family polypeptides or portions thereof of the invention as a "bait proteins" in a two- hybrid assay or three-hybrid assay (see, e.g.. U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; Iwabuchi et al.
  • MarR family -binding polypeptides bind to or interact with MarR family polypeptides
  • MarR family -binding polypeptides bind to or interact with MarR family polypeptides
  • Such MarR family-binding polypeptides are also likely to be involved in the propagation of signals by the MarR family polypeptides or to associate with MarR family polypeptides and enhance or inhibit their activity.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • the gene that codes for an MarR family polypeptide is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence, from a library of DNA sequences, that encodes an unidentified protein (“prey" or "sample”) is fused to a gene that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the polypeptide which interacts with the MarR family polypeptide.
  • a reporter gene e.g., LacZ
  • MarR family binding partners may also be identified in other ways.
  • a library of molecules can be tested for the presence of MarR family binding polypeptides.
  • the library of molecules can be tested by expressing them in an expression vector, e.g., a bacteriophage.
  • Bacteriophage can be made to display on their surface a plurality of polypeptide sequences, each polypeptide sequence being encoded by a nucleic acid contained within the bacteriophage.
  • the phage expressing these candidate MarR family binding polypeptides can be tested for the ability to bind an immobilized MarR family polypeptide, to obtain those polypeptides having affinity for the MarR family polypeptide.
  • the method can comprise: contacting the immobilized MarR family polypeptide with a sample of the library of bacteriophage so that the MarR family polypeptide can interact with the different polypeptide sequences and bind those having affinity for the MarR family polypeptide to form a set of complexes consisting of immobilized MarR family polypeptide and bound bacteriophage.
  • the complexes which have not formed a complex can be separated.
  • the complexes of MarR family polypeptide and bound bacteriophage can be contacted with an agent that dissociates the bound bacteriophage from the complexes; and the dissociated bacteriophage can be isolated and the sequence of the nucleic acid moleculeencoding the displayed polypeptide obtained, so that amino acid sequences of displayed polypeptides with affinity for MarR family polypeptides are obtained.
  • MarR family binding polypeptides can be identified, e.g., by contacting an MarR family nucleotide sequence with candidate MarR family binding polypeptides (e.g., in the form of microbial extract) under conditions which allow interaction of components of the extract with the MarR family nucleotide sequence. The ability of the MarR family nucleotide sequence to interact with the components can then be measured to thereby identify a polypeptide that binds to an MarR family nucleotide sequence.
  • candidate MarR family binding polypeptides e.g., in the form of microbial extract
  • the subject screening methods can involve cell-free assays, e.g., using high- throughput techniques.
  • a synthetic reaction mix comprising an MarR family molecule and a labeled substrate or ligand of such polypeptide is incubated in the absence or the presence of a candidate molecule that may be an agonist or antagonist.
  • the reaction mix can further comprise a cellular compartment, such as a membrane, cell envelope or cell wall, or a combination thereof.
  • the ability of the test compound to agonize or antagonize the MarR family polypeptide is reflected in decreased binding of the MarR family polypeptide to an MarR family binding partner or in a decrease in MarR family polypeptide activity.
  • test modulating agents In many drug screening programs which test libraries of modulating agents and natural extracts, high throughput assays are desirable in order to maximize the number of modulating agents surveyed in a given period of time.
  • Assays which are performed in cell-free systems such as may be derived with purified or semi-purified proteins, are often preferred as "primary" screens in that they can be generated to permit rapid development and relatively easy detection of an alteration in a molecular target which is mediated by a test modulating agent.
  • the effects of cellular toxicity and/or bioavailability of the test modulating agent can be generally ignored in the in vitro system.
  • the ability of a compound to modulate the activity or expression of an MarR family polypeptide is accomplished using isolated MarR family polypeptides or MarR family nucleic acid molecule in a cell-free system.
  • the step of measuring the ability of a compound to modulate the activity or expression of the MarR family polypeptide is accomplished, for example, by measuring direct binding of the compound to an MarR family polypeptide or MarR family nucleic acid molecule or the ability of the compound to alter the ability of the MarR family polypeptide to bind to a binding partner to which the MarR family polypeptide normally binds (e.g., protein or DNA).
  • an assay of the present invention is a cell-free assay in which an MarR family polypeptide or portion thereof is contacted with a test compound and the ability of the test compound to bind to the MarR family polypeptide or biologically active portion thereof is determined.
  • Determining the ability of the test compound to modulate the activity of an MarR family polypeptide can be accomplished, for example, by determining the ability of the MarR family polypeptide to bind to an MarR family target molecule by one of the methods described above for determining direct binding. Determining the ability of the MarR family polypeptide to bind to an MarR family target molecule can also be accomplished using a technology such as realtime Biomolecular Interaction Analysis (BIA). Sjolander, S.
  • BIOA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g.. BIAcore). Changes in the optical phenomenon of surface plasmon resonance (SPR) can be used as an indication of realtime reactions between biological molecules.
  • SPR surface plasmon resonance
  • the cell-free assay involves contacting an MarR family polypeptide or biologically active portion thereof with a known compound which binds the MarR family polypeptide to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the MarR family polypeptide, wherein determining the ability of the test compound to interact with the MarR family polypeptide comprises determining the ability of the MarR family polypeptide to preferentially bind to or modulate the activity of an MarR family binding partner.
  • Exemplary MarR family binding partners include nucleic acid molecules, such as DNA, as well as polypeptides that are downstream of MppA, a periplasmic binding protein in E. coli which functions upstream of MarA in a signal transduction pathway (Li and Park. 1999. J. of Bacteriology. 181 :4842) and related molecules.
  • the cell-free assays of the present invention are amenable to use of both soluble and/or membrane-bound forms of proteins (e.g., MarR family polypeptides or MarR family binding polypeptides).
  • a membrane-bound form of a polypeptide it may be desirable to utilize a solubilizing agent such that the membrane-bound form of the polypeptide is maintained in solution.
  • non-ionic detergents such as n- octylgluco
  • compounds can be tested for their ability to directly bind to an MarR family nucleic acid molecule or an MarR family polypeptide or portion thereof, e.g., by using labeled compounds, e.g., radioactively labeled compounds.
  • an MarR family polypeptide sequence can be expressed by a bacteriophage.
  • phage which display the MarR family polypeptide would then be contacted with a compound so that the polypeptide can interact with and potentially form a complex with the compound.
  • Phage which have formed complexes with compounds can then be separated from those which have not.
  • the complex of the polypeptide and compound can then be contacted with an agent that dissociates the bacteriophage from the compound.
  • any compounds that bound to the polypeptide can then be isolated and identified.
  • the ability of a compound to bind to an MarR family nucleic acid molecule can be measured (e.g.. MarR binding to marO).
  • gel shift assays or restriction enzyme protection assays can be used. Gel shift assays separate polypeptide-DNA complexes from free DNA by non-denaturing polyacrylamide gel electrophoresis. In such an experiment, the level of binding of a compound to DNA can be determined and compared to that in the absence of compound. Compounds which change the level of this binding are selected in the screen as modulating the activity of an MarR family polypeptide.
  • assays will be performed in which direct binding is measured, e.g., protection o ⁇ marO restriction enzyme digestion as described in the appended examples, in order to rule out indirect effects of compounds, e.g., on mRNA stability.
  • the invention provides a method for identifying compounds that modulate antibiotic resistance by assaying for test compounds that bind to MarR family nucleic acid molecules and interfere, e.g., with gene transcription.
  • an MarR family nucleic acid molecule and an MarR family binding polypeptide that normally binds to that nucleotide sequence are contacted with a test compound to identify compounds that block the interaction of an MarR family nucleic acid molecule and an MarR family binding polypeptide.
  • the MarR family nucleotide sequence and/or the MarR family binding polypeptide are contacted under conditions which allow interaction of the compound with at least one of the MarR family nucleic acid molecule and the MarR family binding polypeptide.
  • the ability of the compound to modulate the interaction of the MarR family nucleotide sequence with the MarR family binding polypeptide is indicative of its ability to modulate an MarR family polypeptide activity.
  • Determining the ability of the MarR family polypeptide to bind to or interact with an MarR family binding polypeptide can be accomplished, e.g., by direct binding.
  • the MarR family polypeptide could be coupled with a radioisotope or enzymatic label such that binding of the MarR family polypeptide to an MarR family polypeptide target molecule can be determined by detecting the labeled MarR family polypeptide in a complex.
  • MarR family polypeptides can be labeled with 125 ⁇ ⁇ 35 ⁇ 7 14 O ⁇ 3]T 5 either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • MarR family polypeptide molecules can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • horseradish peroxidase alkaline phosphatase
  • luciferase a protein phosphatase
  • a fusion protein can be provided which adds a domain that allows the polypeptide to be bound to a matrix.
  • glutathione-S- transferase/ MarR family polypeptide (GST/ MarR family polypeptide) fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St.
  • the cell lysates e.g. an 35s-labeled
  • the test modulating agent glutathione derivatized microtitre plates
  • the mixture incubated under conditions conducive to complex formation, e.g., at physiological conditions for salt and pH, though slightly more stringent conditions may be desired.
  • the beads are washed to remove any unbound label, and the matrix immobilized and radiolabel determined directly (e.g. beads placed in scintilant), or in the supernatant after the complexes are subsequently dissociated.
  • the complexes can be dissociated from the matrix, separated by SDS-PAGE, and the level of MarR family polypeptide -binding polypeptide found in the bead fraction quantitated from the gel using standard electrophoretic techniques.
  • Other techniques for immobilizing proteins on matrices are also available for use in the subject assay.
  • either an MarR family polypeptide or polypeptide to which it binds can be immobilized utilizing conjugation of biotin and streptavidin.
  • biotinylated MarR family polypeptide molecules can be prepared from biotin- NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford. IL).
  • Exemplary methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the MarR family binding polypeptide, or which are reactive with MarR family polypeptide and compete with the binding polypeptide; as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the binding partner, either intrinsic or extrinsic activity.
  • the enzyme can be chemically conjugated or provided as a fusion protein with the MarR family binding polypeptide.
  • the MarR family polypeptide can be chemically cross-linked or genetically fused with horseradish peroxidase, and the amount of protein trapped in the complex can be assessed with a chromogenic substrate of the enzyme, e.g. 3,3'-diamino-benzadine terahydrochloride or 4-chloro-l-napthol.
  • a fusion protein comprising the protein and glutathione- S-transferase can be provided, and complex formation quantitated by detecting the GST activity using 1 -chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).
  • the polypeptide to be detected in the complex can be "epitope tagged" in the form of a fusion protein which includes, in addition to the MarR family polypeptide sequence, a second polypeptide for which antibodies are readily available (e.g. from commercial sources).
  • the GST fusion proteins described above can also be used for quantification of binding using antibodies against the GST moiety.
  • Other useful epitope tags include myc-epitopes (e.g., see Ellison et al.
  • a microphysiometer can be used to detect the interaction of MarR family polypeptide with its target molecule without the labeling of either MarR family polypeptide or the target molecule. McConnell, H. M. et al. (1992) Science 257:1906-1912.
  • a "microphysiometer” e.g., Cytosensor
  • LAPS light-addressable potentiometric sensor
  • This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in methods of reducing drug resistance in microbes, e.g., in vivo or ex vivo. Such agents can be used in methods of treatment (in vivo or ex vivo) or in methods of reducing resistance to drugs in the environment.
  • microbes suitable for testing Numerous different microbes are suitable for use in testing for compounds that affect MarR or as sources of materials for use in the instant assays.
  • the term "microbe” includes any microorganism having a MarR family member polypeptide. Preferably unicellular microbes including bacteria, fungi, or protozoa.
  • microbes suitable for use in the invention are multicellular, e.g., parasites or fungi.
  • microbes are pathogenic for humans, animals, or plants.
  • microbes causing environmental problems, e.g., fouling or spoilage or that perform useful functions such as breakdown of plant matter are preferred. As such, any of these disclosed microbes may be used as intact cells or as sources of materials for cell-free assays as described herein.
  • microbes for use in the claimed methods are bacteria, either Gram-negative or Gram-positive bacteria.
  • any bacteria that are shown to become resistant to antibiotics, e.g., to display MDR are appropriate for use in the claimed methods.
  • microbes suitable for testing are bacteria from the family Enterobacteriaceae.
  • bacteria of a genus selected from the group consisting of: Escherichia. Proteus, Salmonella, Klebsiella, Providencia, Enter obacter, Burkholderia, Pseudomonas. .
  • the microbes to be tested are Gram-positive bacteria and are from a genus selected from the group consisting of: Lactobacillus,
  • the microbes to be tested are acid fast bacilli, e.g., from the genus Mycobacterium.
  • the microbes to be tested are, e.g., selected from a genus selected from the group consisting of: Methanobacterium, Sulfolobus, Archaeoglobu, Rhodobacter, or Sinorhizobium.
  • the microbes to be tested are fungi.
  • the fungus is from the genus Mucor or Candida, e.g., Mucor racemosus or Candida albicans.
  • the microbes to be tested are protozoa.
  • the microbe is a malaria or cryptosporidium parasite.
  • antiinfective compound includes a compound which reduces the ability of a microbe to produce infection in a host or which reduces the ability of a microbe to multiply or remain infective in the environment.
  • exemplary antiinfective compounds include e.g., disinfectants or antibiotics.
  • Antiinfective compounds include those compounds which are static or cidal for microbes, e.g., an antimicrobial compound which inhibits the proliferation and/or viability of a microbe.
  • Preferred antiinfective compounds increase the susceptibility of microbes to antibiotics or decrease the infectivity or virulence of a microbe.
  • antibiotics is art recognized and includes antimicrobial agents synthesized by an organism in nature and isolated from this natural source, and chemically synthesized antibiotics.
  • the term includes but is not limited to: polyether ionophore such as monensin and nigericin; macrolide antibiotics such as erythromycin and tylosin; aminoglycoside antibiotics such as streptomycin and kanamycin; ⁇ -lactam antibiotics such as penicillin and cephalosporin; and polypeptide antibiotics such as subtilisin and neosporin.
  • Semi-synthetic derivatives of antibiotics, and antibiotics produced by chemical methods are also encompassed by this term.
  • Antimicrobial agents such as isoniazid, trimethoprim, quinolones, fluoroquinolones and sulfa drugs are considered antibacterial drugs, although the term antibiotic has been applied to these. It is within the scope of the screens of the present invention to include compounds derived from natural products and compounds that are chemically synthesized.
  • the term "antibiotic” includes the antimicrobial agents to which the Mar phenotype has been shown to mediate resistance and, as such, includes disinfectants, antiseptics, and surface delivered compounds.
  • antibiotics, biocides, or other type of antibacterial compounds, including agents which induce oxidative stress agents, and organic solvents are included in this term.
  • biocide is art recognized and includes an agent that those ordinarily skilled in the art prior to the present invention believed would kill a cell "non- specifically," or a broad spectrum agent whose mechanism of action is unknown, e.g., prior to the present invention, one of ordinary skill in the art would not have expected the agent to be target-specific.
  • biocides include paraben, chlorbutanol, phenol, alkylating agents such as ethylene oxide and formaldehyde, halides, mercurials and other heavy metals, detergents, acids, alkalis, and chlorhexidine.
  • bactericidal refers to an agent that can kill a bacterium; "bacteriostatic” refers to an agent that inhibits the growth of a bacterium.
  • an antibiotic or an "anti-microbial drug approved for human use” is considered to have a specific molecular target in a microbial cell.
  • a microbial target of a therapeutic agent is sufficiently different from its physiological counte ⁇ art in a subject in need of treatment that the antibiotic or drug has minimal adverse effects on the subject.
  • Compounds for testing in the instant methods can be derived from a variety of different sources and can be in solution or immobilized on a surface.
  • libraries of compounds are tested in the instant methods to identify compounds that modulate the ability of a MarR family polypeptide to negatively regulate transcription of an operon, the transcription of which leads to MDR.
  • a recent trend in medicinal chemistry includes the production of mixtures of compounds, referred to as libraries. While the use of libraries of peptides is well established in the art, new techniques have been developed which have allowed the production of mixtures of other compounds, such as benzodiazepines (Bunin et al. 1992. J. Am. Chem. Soc. 1 14:10987; DeWitt et al. 1993. Proc. Natl. Acad. Sci. USA 90:6909) peptoids (Zuckermann. 1994. J. Med. Chem. 37:2678) oligocarbamates (Cho et al. 1993. Science. 261 :1303), and hydantoins (DeWitt et al. supra).
  • the compounds for screening in the assays of the present invention can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the 'one- bead one-compound' library method, and synthetic library methods using affinity chromatography selection.
  • biological libraries include biological libraries; spatially addressable parallel solid phase or solution phase libraries, synthetic library methods requiring deconvolution, the 'one- bead one-compound' library method, and synthetic library methods using affinity chromatography selection.
  • the biological library approach is limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. Anticancer Drug Des. 1997. 12:145).
  • Exemplary compounds which can be screened for activity include, but are not limited to, peptides, nucleic acids, carbohydrates, small organic molecules (e.g., polyketides) (Cane et al. 1998. Science 282:63). and natural product extract libraries.
  • the test compound is a peptide or peptidomimetic.
  • the compounds are small, organic non-peptidic compounds.
  • combinatorial polypeptides can be produced from a cD ⁇ A library.
  • compounds to be tested do not include salicylate or related compounds. Examples of such related compounds include sodium benzoate and acetyl salicylate (Rosner, J.L. 1985. Proc. ⁇ atl. Acad. Sci. USA 82:8771).
  • Example 1 Structurally diverse compounds bind to MarR
  • the multiple antibiotic resistance (mar) operon o ⁇ Escherichia coli is a chromosomally encoded locus that controls an adaptational response to antibiotics and other environmental hazards (Alekshun. M. N. and Levy 1997, Antimicrob. Agents Chemother). This control is accomplished on the genomic level whereby MarA, a transcriptional activator encoded within the marRAB operon, regulates the expression of multiple genes on the E. coli chromosome (Alekshun, M. N. and Levy 1997, Antimicrob. Agents Chemother 10:2067
  • MarR negatively regulates expression of the marRAB operon (Cohen et al, 1993. J. Bacteriol. 175: 7856; Martin and Rosner, 1995, Proc. Natl. Acad. Sci. 92: 5456 and Seoane and Levy, 1995, J. Bacteriol. 177: 3414). DNA footprinting experiments suggest that MarR oligomerizes at two locations, sites I and II, within the mar operator (marO) (Martin and Rosner, 1995, Proc. Natl Acad. Sci. 92: 5456).
  • Site I is positioned within the -35 and -10 hexamers and site II spans the putative MarR ribosome binding site (reviewed in (Alekshun and Levy, 1997, Antimicrob. Agents Chemother. 10: 2067)).
  • the inducer concentration needed to achieve derepression of a Pmarjj-ccdB fusion in the bacterial cell was determined using a "killer" system.
  • Example 1 The following methods were used in Example 1 :
  • a low copy number wild type MarR expression vector was constructed using a modified version of pACT7 (Maneewannakul, K., et al 1992. Mol. Microbiol. 6, 2961-2973).
  • a high-copy number wild type MarR expression vector was constructed in p ⁇ T13a (Studier, F.W., et al 1990. Meth. Enzymol 185, 60-89), a kanamycin resistance version of pET311 a (Novagen, Madison. WI).
  • PCR amplification of marR was performed as described above using forward and reverse primers containing Vspl and BamRl restriction sites, respectively, to facilitate directional cloning into Ndel/BamHl digested pET13a.
  • pMarR-WT expression of MarR is under the control of the T7-RNA polymerase promoter and a strong ribosome binding site. DNA sequence analysis was performed as described above.
  • a Pmarf j -ccdB fusion was created in a modified version of pETl Id (Novagen, Madison, WI).
  • pETl Id was digested with EcoRV to remove the majority o ⁇ lacl, the vector was purified using the Qiagen gel purification kit (Qiagen, Santa Clarita, CA), and religated.
  • Pmar jj containing the mar operator (marO) and promoter sequences, was amplified by PCR from AG100 chromosomal DNA as described above and blunt-end cloned into the Eagl site of pETl Id (lacking lad).
  • lacO-ccdB portion of pKIL 18 (Bernard, P., et al 1994. Gene 148, 71-74) was amplified by PCR so as to exclude the tac promotor sequences.
  • Xhol and Bsml restriction sites were inco ⁇ orated into the forward and reverse primers, respectively, to facilitate directional cloning downstream o ⁇ Pmarjj, into the Aval- Bsml digested vector.
  • the resulting plasmid was designated pSup-Test (FIG. 3).
  • the column was washed with 50 mM Tris-HCl (pH 7.4) and MarR was eluted using a linear gradient of 0-1 m NaCl in 50 mM Tris-HCl (pH 7.4).
  • the purified protein eluted at 0.2-0.3 mM NaCl and was dialyzed against 100 volumes of 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 10% glycerol, and 1 mM phenylmethlysulfonyl fluoride (serine protease inhibitor) overnight at 4°C
  • the purified MarR was judged to be >90% pure on a SDS-PAGE Coomassie stained gel, was stored in aliquots at -70°C until further use.
  • reaction mixtures were terminated by the addition of 1.5 ⁇ l stop buffer (0.25M EDTA (pH 8.0), 1% SDS) and 5 ⁇ l 6X agarose gel loading buffer (0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol) and analyzed on 0.7% agarose (Life Technologies, Gaithersburg, MD) gels.
  • stop buffer 0.25M EDTA (pH 8.0), 1% SDS
  • 5 ⁇ l 6X agarose gel loading buffer 0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol
  • the affinity of MarR for marO was estimated by determining the point of 50% protection, as judged by visual inspection of ethidium bromide stained gels according to established methods (Joachimiak, A.J., et al 1983. Proc. Natl. Acad. Sci. USA 80, 668- 672; Melville, S.B., et al 1996. Proc. Natl. Acad. Sci. USA 93, 1226-1231 and Smith, H.Q., et al 1997. J. Bacteriol. 179, 5914-5921). Consistently, an average value, ⁇ Ky of 1.9 and 0.95 ⁇ M (assuming the monomeric and dimeric forms of MarR, respectively) was obtained.
  • the restriction site protection assays were repeated in the presence of various inducers to confirm the gradient plate data and to test putative inducers that are potent antibiotics, i.e., tetracycline, chloramphenicol, ampicillin, and norfloxacin.
  • the DNA binding activity of MarR in vitro was antagonized by weak acids and oxidative stress agents.
  • vector (pSup-Test, 3.4 nM) alone with and without digestion with Sspl, and pSup-Test in the presence of MarR (2.92-3.6 ⁇ g, 9.1-1 1.2 ⁇ M, assuming the monomeric form of MarR) digested with Sspl were electrophoresed along with the samples.
  • both antibiotics may simply increase mRNA stability (Lopez, F.J., et al 1998. Proc. Natl. Acad. Sci. 95, 6067-6072).
  • both the inducing and non-inducing chemicals were added to the Sspl restriction endonuclease, no altered activity or specificity of the enzyme was seen.
  • Bacillus subtilis, BmrR the negative regulator of the Bmr mutidrug transporter, binds chemicals (rhodamine 6G and tetraphenylphosphonium) that are substrates of the pump (Ahmed, M., et al 1994. J. Biol. Chem. 269, 28506-28513).
  • the gene product o ⁇ fabl in E. coli, encoding enoyl reductase binds natural fatty acid substrates and interacts with triclosan and, presumably, diazaborine, that inhibit function of the protein (McMurry, L.M., et al 1998. N ⁇ twre 394, 531-532; Hearth et al.
  • Salicylate (Sodium salt) 0.53 ⁇ 0.64
  • Menadione (Sodium bisulfite) 2.8 ⁇ 1.41 a As determined by gradient plates containing potential inducers at the concentrations indicated: plumbagin (0.4 mM), 2,4-dinitrophenol (0.4 mM), sodium salicylate (10 mM), sodium benzoate (10 mM), and menadione (5 mM).
  • Negative complementing marR mutants were generated in order to identify the DNA binding domain of this repressor.
  • the functional regions of the multimeric lac (LacR) and trp (T ⁇ R) repressors were characterized by studying mutant proteins (Betz, 1987, J Mol. Biol. 195: 495; Hurlburt and Yanofsky, 1990, J Biol. Chem. 265: 7853; Kelley and Yanofsky, 1985, Proc. Natl. Acad. Sci. USA 82: 482; Klig and Yanofsky. 1988, J Biol. Chem. 263: 243; Miller, 1980, The operon. Cold Spring Harbor. NY.
  • Negative complementing trpR and particular lad alleles encode proteins with impaired DNA binding properties but that are still able to form multimers, resulting in association with, and inactivation of (tram-dominance), wild type subunits (Adler et al, 1971, Nature 237: 322; Gilbert and M ⁇ ller-Hill, 1970, The lactose operon; Kelley and Yanofsky, 1985, Proc. Natl. Acad. Sci. USA 82: 482; Miller, 1980, 77ze operon Cold Spring Harbor, NY.
  • the marR'" mutants isolated in this study are clustered in two regions and lie within domains that show homology to HTH binding motifs of known crystal structure. Particular mutations in each putative HTH yield proteins that are unable to form wild type complexes with operator DNA. Another class of mutants that are able to bind DNA but have limited intracellular functions have been identified and have novel implications.
  • SPC105 (Cohen, et al., 1993, J. Bacteriol 175: 7856) contains a wild type mar locus and SPC107 (MO 100) has a 39-kb deletion that includes the mar locus (Seoane and Levy, 1995, J. Bacteriol. ll: 3414); both strains bear a chromosomal (AmpR) Pmarjj-lacZ fusion on ⁇ at the attachment site (Cohen, et al., 1993, J. Bacteriol. 175: 7856; Seoane and Levy, 1995, J. Bacteriol. 177:3414).
  • E. coli BL21(DE3) (Novagen, Madison, WI) was the strain used for high level MarR expression.
  • Mutant marR alleles were sequenced from plasmids purified using the Qiagen plasmid purification kit (Qiagen) or from PCR products generated using the mutant plasmids as template DNA and Platinum Taq DNA polymerase high fidelity according to the manufacturer's protocols (Life Technologies). DNA sequence analysis was performed using an ABI automated DNA sequencer. Competent cells were prepared as previously described (Tang et al., 1994, Nuc. Acids Res. 22: 2857). Plasmid pAC-MarR (WT) (Kan R ) was constructed from pACT7 (Maneewannakul et al., 1992. Mol. Micorbiol.
  • the wild type and marR-d mutant alleles were amplified by PCR from the mutant low copy number vectors [pAC-MarR (WT) derivatives] and subsequently cloned into pET13a (Studier et al, 1990, Meth. Enzymol 185: 60).
  • expression o ⁇ marR is regulated by the T7 RNA polymerase promoter and a near consensus ribosome binding site.
  • Mutant marR alleles which specify peptides that can interact with and inactivate wild type marR are defined as negative complementing or tr ⁇ r ⁇ -dominant.
  • a mixture of hydroxylamine and nitrosoguanidine mutagenized pAC-MarR (WT) plasmids were transformed into SPC105 and plated on MacConkey lactose agar containing ampicillin (100 ⁇ g/ml), kanamycin (30 ⁇ g/ml), and IPTG (50 ⁇ M). The host alone or containing pACT7 (Maneewannakul, et al., 1992, Mol Microbiol.
  • SPC107 and BL21(D ⁇ 3) bearing marR alleles in trans were grown in LB broth containing the appropriate antibiotics to mid-logarithmic phase and induced with IPTG.
  • Cells were collected by centrifugation, washed, resuspended in 50 mM sodium phosphate (pH 7.4), 2 mM 4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride (AEBSF, serine protease inhibitor) (Sigma), and 5 mM EDTA, and sonicated.
  • Insoluble proteins were removed by centrifugation at 14,900 x g at 4°C and total soluble protein concentration determinations were performed using the Bio-Rad protein assay kit (Bio-Rad).
  • the negative complementing phenotype was presumably attributed to subunit mixing that would occur among the wild type and mutant proteins forming relatively inactive repressors.
  • the mutation at position 73 had the most profound negative complementing tr ⁇ /75-dominant effect on the activity of wild type MarR (Table 3).
  • Mutations identified between amino acids 94 and 116 (Fig. 4 and Table 2) also displayed negative complementing tram-dominant phenotypes (Table 3). It was postulated that the MarR"" mutations identified in these two regions (aa 61-80 and 97-116) might reside in separate HTH DNA binding domains (see below).
  • this allele would encode a 41 residue protein with the following properties: it is negative complementing and tram-dominant in the marR + host and yet it has no repressor activity in the AmarR background (Table 3). This shows that the first 41 amino acids of MarR contain the majority of the contacts necessary to mediate protein-protein interactions.
  • Lanes 2-12 were SPC 107 containing: lane 2: wild type MarR; lane 3, R16H/G69R; lane 4: Q42Amber; lane 5: G69E; lane 6, R73C; lane 7: M74I; lane 8: R77H; lane 9: R94C; lane 10: L100F; lane 11 : T101I; lane 12: Ql lOOchre; and lane 13: G116S.
  • a blot was also done on lysates from cells BL21(DE3) (marR + ) bearing marR alleles on the medium copy number, but high level expression, pET13a (pBR322 derivative) vector.
  • Lane 1 SPC107 alone (negative control).
  • Lanes 2-1 1 are BL21(DE3) containing: lane 2: wild type MarR; lane 3: Q42Amber; lane 4: G69E; lane 5: R73C; lane 6: M74I; lane 7: R77H; lane 8: L100F; lane 9: T101I; lane 10: Ql lOOchre; lane 11 : G116S.
  • the R16H/G69R, G69E, R73C, R77H, and G116S mutants were easily detected and their intracellular levels were comparable to that of the host bearing pAC-MarR (WT).
  • the expression of the M74I and R94C mutants was considerably lower than that of wild type MarR and the Q42 Amber, LI 00F, T101I, Ql lOOchre proteins were undetectable by this method.
  • the mutant alleles were cloned into pET13a and overexpressed in E. coli BL21(DE3). Total cell lysates were prepared from these strains and proteins were again subjected to Western analysis.
  • expression of the M74I and LI 00F mutants was improved and synthesis of the Tl 011 protein, albeit at a much lower level, was now detectable.
  • HTH motifs are usually 20-21 residues in length and the following parameters generally apply: stereochemical requirements exist for residues 4-5, 8-10, and 15; positions 4 and 15 are most often completely buried and, thus, should be nonpolar with Val or He usually being found at position 15; position 5 is generally a small residue, such as alanine or glycine, and should not be a branched chain amino acid (Val, Leu, or He) since these larger residues would interfere with the conformation of the HTH motif; and residue 9 is a small amino acid residue, either glycine (most common) or alanine (Branden and Tooze, 1991, Introduction to protein structure New York, NY USA, London England, Garland Publishing, Inc; Pabo and Sauer, 1984, Ann. Rev. Biochem. 53: 293; Pabo and Sauer, 1992, Annu. Rev. Biochem. 61 : 1053).
  • Gly 69 of MarR would correspond to position 9 and occur in the turn of the MarR-M HTH motif (Fig. 6). Residues within this region of HTH motifs of known crystal structures are critical for the correct orientation of the two helices in the motif (Pabo and Sauer, 1984, Ann. Rev. Biochem. 53: 293; Pabo and Sauer, 1992, Annu. Rev. Biochem. 61: 1053). The size and charge of the glutamic acid or arginine side chains, relative to that of glycine or alanine, in the G69E and R16H/G69R mutants may distort the helices and severely hinder DNA binding. Likewise, the LI OOF and T101I mutants found within MarR-C correspond to positions that are subject to stereochemical constraints (Figs. 5 and 6).
  • Lanes 3-11 2.5 ⁇ g of cell extracts from cells containing MarR mutants; lane 3: Q42Amber; lane 4: G69E; lane 5: R73C; lane 6: M74I; lane 7: R77H; lane 8: L100F; lane 9: T101I; lane 10: Ql lOOchre; and lane 11 : G116S.
  • the mutant may prevent the binding of wild type repressor top marO or affect a point subsequent to this step (see below).
  • Two regions that resemble known HTH motifs (Branden and Tooze, 1991, Introduction to protein structure; Pabo and Sauer, 1984. Ann. Rev. Biochem. 53: 293; Pabo and Sauer, 1992, Annu. Rev. Biochem. 61 : 1053) were identified in MarR (MarR-M, amino acids 61-80, and MarR-C, residues 97-1 16) (Fig. 5 and 6).
  • the two HTH motif proposal is also supported by the phenotype (Table 3) of the nonsense mutation at residue 110 (Ql lOOchre) and its behavior in vitro.
  • the Ql lOOchre allele encodes a 109 residue MarR which would include an intact HTH
  • MarR protects both strands of two regions (sites I and II) within the mar operator fom nuclease cleavage in vitro (Martin and Rosner, 1995, Proc. Natl. Acad. Sci. 92: 5456). Two inverted nucleotide sequence repeats (indicated by arrows in
  • Fig. 7 that are separated by a dyad axis of symmetry (represented as broken lined boxes in Fig. 7) are found in sites I and II (Fig. 7). This type of organization might be expected for a protein with a dual HTH motif and these data suggest that MarR and other members of this family may recognize DNA in a common manner.
  • the chromosomal multiple antibiotic resistance (mar) locus o ⁇ Escherichia coli controls an adaptational response to antibiotics and other environmental hazards (Alekshun, M.N., and Levy, S.B., 199 ', Antimicrob. Agents Chemother. 10: 2067).
  • the expression of multiple genes on the E. coli chromosome are regulated by MarA, a transcriptional activator encoded within the marRAB operon (Alekshun, M.N., and Levy, S.B., 1997 ', Antimicrob. Agents Chemother. 10: 2067).
  • MarR negatively regulates expression of the marRAB operon (Cohen, S.P., et al 1993, J. Bacteriol. 175: 1484; Martin, R.G. and Rosner, J.L., 1995, proc. Natl. Acad. Sci. 92: 5456, Seoane, A.S. and Levy, S.B., 1995, J Bacteriol. 177: 3414). DNA footprinting experiments suggest that MarR dimerizes at two locations, sites I and II, within the mar operator (marO) (Martin, R.G. and Rosner, J.L. 1995, Proc. Natl Acad. Sci.
  • a low-copy number wild type MarR expression vector was constructed using a modified version of pACT7 (Maneewannakul, K. et al. 1992, Mol. Microbiol 6: 2961). marR was amplified by PCR from E. coli AG100 (George, A.M. and Levy, S.B. 1983, J. Bacteriol 155: 531) chromosomal DNA using Taq DNA polymerase according to the manufacturer's protocols (Life Technologies, Gaithersburg, MD). EcoRI and Pstl restriction sites were inco ⁇ orated into the forward and reverse primers to facilitate directional cloning into pACT7 in place of the T7-RNA polymerase gene following digestion with EcoRI and Pstl.
  • pAC-MarR In pAC-MarR (WT), transcription of marR is regulated by the lacPj promoter and protein synthesis is governed by the wild type MarR ribosome binding site (AGGG) and translational initiation (GTG) signals (Cohen, S.P. et al. 1993, J. Bacteriol. 175: 1484).
  • a high-copy number wild type MarR expression vector was constructed in p ⁇ T13a (Studier, F.W. et al. 1990, Meth. Enzymol 185: 60), a kanamycin resistant version of pETl la (Novagen, Madison, WI).
  • PCR amplification o ⁇ marR was performed as described above using forward and reverse primers containing Vspl and Bamrll restriction sites to facilitate directional cloning into Ndel/BamUl digested pET13a.
  • pMarR-WT expression of MarR is under the control of the T7-RNA polymerase promoter and a near consensus ribosome binding site.
  • a Pmarj j /marO-ccdB fusion was created in pETl Id (Novagen, Madison, WI). After digestion with EcoRV to remove the majority o ⁇ lacl, p ⁇ Tl Id was purified using the Qiagen gel purification kit (Qiagen, Santa Clarita, CA), and religated. Pmarjj/marO, containing the marRAB promoter (Pmarj j ) and operator (marO) sequences (Fig.
  • MarR superrepressors In order to identify MarR superrepressors, expression of the lethal ccdB gene product on pSup-Test was exploited. Plasmid pAC-MarR (WT) was mutagenized in vitro and transformed into DH5 ⁇ containing pSup-Test. Transformants were selected in the presence of sodium salicylate, a known marRAB operon inducer (Cohen, S.P. et al. 1993, J. Bacteriol. 175: 7856). Growth of DH5 ⁇ bearing pETl Id or pmarO (pSup-Test lacking ccdB, Table 4) was unaffected by the highest concentration of this and other inducers tested (Table 5).
  • DH5 ⁇ cells containing pSup-Test in the absence or presence of plasmid-encoded wild type marR were non-viable in the presence of sodium salicylate and other inducers (Table 5).
  • cells containing a putative MarR superrepressor survived higher concentrations of known marRAB operon inducers presumably by binding of the mutant protein to marO in front o ⁇ ccdB on pSup-Test and preventing expression of the lethal gene product (Table 5). From a total of 276 transformants, twelve putative MarR superrepressor mutants were independently isolated (Fig. 9).
  • the wild type and marR superrepressor genes were cloned into pET13a, transformed into E. coli BL21(DE3) for overexpression, and the MarR proteins purified.
  • Cells, grown in LB at 37°C to mid-logarithmic phase, were induced for 3 hr with 1 mM IPTG, collected, washed, and frozen at -70°C The frozen cell pellet was resuspended in 10 mL buffer A [50 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 2.5 mM
  • AEBSF 4-(2-aminoethyl)-benzenesulfonylfluoride hydrochloride
  • MarR was eluted using a linear gradient of 0-1 M NaCl in 50 mM Tris-HCl (pH 7.4). Eluting at 0.2-0.3 mM NaCl, MarR was dialyzed against 100 volumes of 50 mM Tris-HCl (pH 7.4), 100 mM NaCl, 10 % glycerol, and 1 mM phenylmethylsulfonyl fluoride (PMSF, serine protease inhibitor) overnight at 4°C Judged to be >90% pure on a SDS-PAGE Coomassie stained gel, MarR was stored in aliquots at -70°C until further use.
  • PMSF phenylmethylsulfonyl fluoride
  • FIG. 8A A unique Sspl recognition sequence within one of two MarR binding sites in marO (Fig. 8A) formed the basis of a restriction enzyme site protection assay (Kelly and Yanofshy. 1985. Proc. Natl. Acad. Sci. USA 82:482; Melville and Gunsalus. 1996. Proc. Natl. Acad. Sci. 92:5456; Smith and Somerville. 1997. J. Bacteriol. 179:5914) to assess MarR binding to marO.
  • the D26N, G95S, and L135F superrepressor mutant proteins displayed at least a 9-fold greater DNA binding activity than the wild type repressor (Table 6). Although the inducer susceptibility profiles of these three mutants were similar in intact E. coli DH5 ⁇ (Fig. 10 and Table 5), their in vitro DNA binding properties were quite different (Table 6). The G95S MarR s mutant showed -2 and 3.5-fold greater DNA binding than did the D26N and L135F mutants (Table 6).
  • the G95S MarR ⁇ mutation occurred within a region that is conserved among all members of the MarR family of proteins and the mutant protein is 30-fold more active than wild type MarR. tram-dominant negative complementing MarR mutants that are in proximity to this residue ( Cohen, S.P. et al. 1993, J. Bacteriol. 175: 7856; Seoane, A.S. and Levy, S.B. 1995, J. Bacteriol. Ill) suggest that it may play a more direct role in DNA binding.
  • the D26N superrepressor mutation results in a charged amino acid being substituted for an uncharged residue.
  • a decrease in electrostatic interactions between the protein and the DNA backbone may be the basis for this superrepressor activity. It is also possible that new hydrogen bonds between the asparagine side chain and the DNA backbone contribute to an increased affinity for DNA. Thus, non-specific DNA binding is expected to form the basis of superrepression.
  • the latter mutation is probably not required since this mutant produced data similar to that of the protein bearing the single D26N change (Fig. 10 and Table 5).
  • the V132M MarR$ mutant showed inducer responses like D26N and G95S mutants in whole cells (Table 5). Since both mutations are expected to lie outside of the putative DNA binding domain of it is speculated that each plays an accessory role in DNA binding. With respect to the L135F mutant, the phenylalanine residue may increase DNA binding through newly acquired interactions with the phosphate backbone (Schildbach, J.F. et al. 1999, Proc. Natl. Acad. Sci. USA 96: 811). The lesion in each mutant may also reside in a region required for proper protein folding, MarR oligomer assembly, or an inducer recognition domain. It is also possible that the superrepressor mutation in these or the other proteins affects transmission of the signal to the DNA binding domain following inducer recognition.

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Abstract

La présente invention concerne des analyses permettant d'identifier des composés qui modulent la capacité des polypeptides de la famille MarR à se lier à l'ADN et à réguler positivement ou négativement la transcription des loci génétiques. Ces analyses instantanées détectent la capacité des composés à se lier aux polypeptides de la famille MarR et/ou à réguler leur activité. L'invention se rapporte, entre autres, à des polypeptides de la famille MarR et à des procédés d'utilisation de ces derniers.
PCT/US2000/010829 1999-04-23 2000-04-21 Identification de modulateurs des proteines de la famille marr WO2000065082A1 (fr)

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WO1996033285A1 (fr) * 1995-04-21 1996-10-24 Microcide Pharmaceuticals, Inc. Inhibiteurs de pompe a ecoulement

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WO1996033285A1 (fr) * 1995-04-21 1996-10-24 Microcide Pharmaceuticals, Inc. Inhibiteurs de pompe a ecoulement

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ALEKSHUN, M. N. (1) ET AL: "Identification and characterization of MarR "superrepressors" of the chromosomal mar locus in Escherichia coli.", ABSTRACTS OF THE INTERSCIENCE CONFERENCE ON ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, (1998) VOL. 38, PP. 106. MEETING INFO.: 38TH INTERSCIENCE CONFERENCE ON ANTIMICROBIAL AGENTS AND CHEMOTHERAPY SAN DIEGO, CALIFORNIA, USA SEPTEMBER 24-27, 1998 AMERICAN, XP000938233 *
ALEKSHUN, MICHAEL N. ET AL: "Alteration of the repressor activity of MarR, the negative regulator of the Escherichia coli marRAB locus, by multiple chemicals in vitro.", JOURNAL OF BACTERIOLOGY, (AUG., 1999) VOL. 181, NO. 15, PP. 4669-4672., XP000938043 *
SEONE, ASUNCION S. ET AL: "Characterization of MaR, the repressor of the multiple antibiotic resistance (mar) operon in Escherichia coli.", JOURNAL OF BACTERIOLOGY, (1995) VOL. 177, NO. 12, PP. 3414-3419., XP000937842 *
SULAVIK, MARK C. ET AL: "The MarR repressor of the multiple antibiotic resistance (mar) operon in Escherichia coli: Prototypic member of a family of bacterial regulatory proteins involved in sensing phenolic compounds.", MOLECULAR MEDICINE (CAMBRIDGE), (1995) VOL. 1, NO. 4, PP. 436-446., XP000937847 *

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WO2004077064A1 (fr) * 2003-02-28 2004-09-10 Martin Fussenegger Detection et decouverte d'un compose anti-infectieux

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