WO2011113999A1

WO2011113999A1 - New polypeptides from endophytes of empetrum nigrum and their fragments, encoding them, their production and uses

Info

Publication number: WO2011113999A1
Application number: PCT/FI2011/050220
Authority: WO
Inventors: Mysore V. Tejesvi; Pedro Picart Faiget; Anna Maria PIRTTILÄ
Original assignee: University Of Oulu
Priority date: 2010-03-15
Filing date: 2011-03-15
Publication date: 2011-09-22

Abstract

The present invention relates to novel polypeptides and fragments thereof, and novel polypeptides and fragments for use in therapy, particularly as an antimicrobial agent. The invention also relates to a new method for isolating genes encoding microbial metabolites and nucleic acids encoding the polypeptides or fragments of this invention. Invention also relates to use of a polypeptide or fragment thereof as a medicament, feed additive, preservative or surfactant and a method of killing or inhibiting growth of microbes using the novel polypeptides or fragments. Also antimicrobial compositions comprising them are within scope of this invention.

Description

NEW POLYPEPTIDES FROM ENDOPHYTES OF EMPETRUM NIGRUM AND THEIR FRAGMENTS, GENES ENCODING THEM, THEIR PRODUCTION AND USES

FIELD OF INVENTION

The present invention concerns novel polypeptides and fragments thereof. The present invention also concerns isolation of a polypeptide with antimicrobial activity from plant endophytes by metagenomic approach. Further, the invention concerns polynucleotides encoding the polypeptides or their fragments, an antimicrobial composition containing said polypeptide or its fragment(s), a polypeptide or a fragment thereof for use in therapy and the use of said polypeptide or its fragment(s), as a medicament, feed additive, preservative or surfactant. Still, the present invention concerns a method of killing or inhibiting growth of microbes

DESCRIPTION OF RELATED ART

The presence of microbes in a wide variety of habitats including hot and cold springs, chemically contaminated areas (Whitman et al., 1998) and inside plant or animal tissue as endophytes or endosymbionts (Schulz and Boyle, 2005, Baumann, 2005), provides an insight into the vast microbial and metabolic diversity that exists in our biosphere (Turnbaugh and Gordon, 2008). The adaptability of microbes to different environmental conditions provides an opportunity to study and screen for novel enzymes and metabolites for industrial applications. The limitation in the study of microbes is their cultivability on laboratory media as only less than 1% of the strains can be cultured in vitro (Amann et al., 1995). Therefore, screening microbes by traditional methods has limitations with respect to functional biodiversity (Park et al, 2007). The use of metagenomics allows the study of total or partial genomes in a particular environment or habitat. Metagenomic tools have been at the forefront of biotechnology for obtaining new insight on the genomes of unculturable microorganisms and provide an opportunity to investigate the metabolic features of microbial communities without culturing them, the results yielding potential to impact a wide range of disciplines from biomedicine to bioenergy, bioremediation, and biodefense. Metagenomic libraries are constructed from DNA isolated from different sources that is size-restricted, cloned, and expressed in various expression vectors (Schloss and Handelsman, 2003). The screening of metagenome libraries has already resulted in identification of useful enzymes and metabolites (Lorenz et al, 2002, Galvao et al, 2005, Cowan et al, 2005).

Metagenomics techniques have not been used for studying the functional diversity of endophytes, which live inside the plant tissue without causing any visible symptoms of disease (Schulz and Boyle, 2005). Endophytes are promising producers of a wide array of secondary metabolites with potential of application in bio medicine, pharmaceutical and healthcare industries (Raghukumar, 2008, Mitchell et al., 2008). The methodology typically used for studying functional diversity of endophytes is based on isolation with an emphasis towards fast-growing strains, thus not representing the full biodiversity (Hyde and Soytong, 2008, Tejesvi et al. 2010). Methods such as denaturing gradient gel electrophoresis (DGGE) (Duong et al., 2006), restriction fragment length polymorphism (RFLP) (Nikolcheva and Barlocher, 2005) or cloning and direct sequencing (Seena et al., 2008, Tejesvi et al. 2010) are used for analyzing the diversity of unculturable fungal communities in plants, but such methods are not suitable for the functional studies. A frequent problem with endophytes is that they produce metabolites only for a certain period of time in vitro and then become inactive, lose the viability or lose the ability to produce the bioactive secondary metabolite. Such culturing problems on other microbes have earlier been one of the driving forces for development of new methods to access the vast microbial wealth (Handelsman, 2004; Green and Keller, 2006).

There is a need for improved methods of isolating genes encoding metabolites of endophytes. There is also a permanent need of new agents having activity against various microbes. This invention meets these needs as explained in the following.

SUMMARY OF THE INVENTION

The present novel technology is aimed at generating metagenomic libraries from the DNA of endophytes in crowberry (Empetrum nigrum L.). The new technology also aims at screening and characterizing antibacterial activity in these libraries. Crowberry is a perennial shrub growing in the northern hemisphere that has traditionally been used for treating infectious diseases and was considered a good candidate for such studies (McCutcheon et al. 1997). The first object of the invention is a polypeptide or a fragment thereof. Characteristic to the said polypeptide or a fragment thereof is that it comprises an amino acid sequence which has at least 31 % identity with SEQ ID NO: 4 or a fragment or fragments thereof.

One embodiment of the invention is to provide a polypeptide or a fragment thereof having an antimicrobial activity, said polypeptide or fragment thereof being isolated from an endophyte of Empetrum nigrum.

The second object of the invention is a polynucleotide. Characteristic to the said polynucleotide is that it has at least part of the sequence shown as SEQ ID NO: 44, encoding a polypeptide or a fragment according to this invention.

Considerable advantages are obtained by the invention. Thus, the polypeptide or fragments thereof have activity against microorganisms of the family Staphylococcusa in particular against microorganisms of the genus Staphylococcus, more particularly against

microorganisms of the species S. aureus.

The third object of the invention is a method for isolating genes encoding microbial metabolites. Said method comprises the steps of

- grinding plant material containing the microbe(s);

- isolating total DNA of the ground plant material;

- separating DNA from the plant DNA using pulse-field gel electrophoresis;

- digesting DNA and constructing a genomic library based on the digested DNA;

- screening the library for a preselected activity; and

- isolating clones carrying the preselected activity.

The fourth object of the invention is a polypeptide obtained by the process described here. The fifth object of the invention is an antimicrobial composition comprising an antimicrobial polypeptide or a fragment thereof as described here or an antimicrobial polypeptide or a fragment thereof or obtained by a method described here. The sixth object of the invention is a polypeptide or a fragment thereof, as defined here, for use in therapy, particularly for use as an antimicrobial agent.

The seventh object of this invention is a method of killing or inhibiting growth of microbes. Said method comprises the step of contacting said microbes with a polypeptide or fragment thereof, or a polypeptide of fragment obtained by a method as described here.

The eighth object of the invention is a use of a polypeptide or fragment thereof, or a polypeptide of fragment obtained by a method as described here, as a medicament, feed additive, preservative or surfactant.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Antibacterially non active subclones (A) and active subclones (B) that were found in E. co/z^'-based endophytic metagenomic library using S. aureus as an assay strain.

Figure 2. Restriction digest analysis of antibacterial clones digested with BamHl. The arrow indicates the empty pCClFos fosmid. Lane 1, size markers; lane 2, antibacterial subclone pFosSIA and lane 3, antibacterial fosmid clone.

Figure 3. Predicted amino acid sequence of the antibacterial ORF. The predicted protein has 549 residues. Highly conserved PPR-sequences are underlined.

Figure 4. Antibacterial activity of trypsisn digested protein against S. aureus at different time intervals. 15 mins; 2. 30 mins; 3. 1 hour; 4. 2 hours; 5. Centre - Streptomycin (positive control). Figure 5. Antibacterial activity of trypsin digested protein against S. aureus with different concentration. PC - Streptomycin, Positive control; NC - Negative control, 0.01% Acetic acid; 15 - 15 μΐ of trypsin digested protein; 30 - 30 μΐ of trypsin digested protein.

DESCRIPTION OF PREFERRED EMBODIMENTS

Generally, endophytes, organisms that live inside plant tissues, are promising producers of natural products and small molecules to be used as pharmaceutical compounds. To access the genetic resources of unculturable endophytes, a metagenomic fosmid library containing 8,000 clones from the plant Empetrum nigrum L. was constructed. The library was screened to select antibacterial clones using Staphylococcus aureus as a target organism by the double-agar-layer method. One unique clone exhibiting antibacterial activity was selected from the metagenomic library. Secondary libraries were generated to obtain antibacterial subclones with reduced insert size for characterization of the gene responsible for the antibacterial activity. DNA sequence analysis of the subclone revealed that the protein encoded by the gene responsible for antibacterial activity was novel, with no similarities to known sequences. Based on the deduced amino acid similarity, the metagenomic gene encoded for a protein without any known signal peptide, having three conserved pentatricopeptide-motifs and displaying 26-31% homology to other hypothetical proteins. The protein homology search was done using a program BLASTp at GenBank (http://l3last.ncbi.nlm.n1h.gov/Blast.cgi) and the closest matches were hypothetical proteins of Malassezia globosa, Ustilago maydis, Laccaria bicolor, Coprinopsis cinerea and Cryptococcus neoformans. The fragments obtained as tryptic digests do not have any homology to known peptides, this is thus believed to be a new class of antimicrobial peptides.

Our work suggests that metagenomics is a potential tool to search for novel compounds and proteins from unculturable endophytes of plants. In fact, this is the first report of a gene obtained from an endophytic metagenome.

The antimicrobial properties of the fragments have been estimated as explained in Examples. In one embodiment of the invention the polypeptide, a fragment or fragments thereof has(have) an amino acid sequence which is shown as SEQ ID NO: 4, or alternatively, it has a 30 %, or 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 % or 98 % identity with amino acids or amino acid sequence shown as SEQ ID NO: 4.

In this connection phrase "a fragment or fragments thereof includes a single fragment or a mixture of at least two fragments of the polypeptide. It is also possible that the fragments are linked to each other. Phrase "fragment" means a part of polypeptide having SEQ ID NO: 4 or showing identity to it. Tryptic peptides of SEQ ID NO: 4 are examples of fragments.

In this connection the term "identity" refers to the global identity between two amino acid sequences compared to each other from the first amino acid to the last corresponding amino acid. Thus a fragment can only be compared to respective amino acids (essentially equal number of amino acids) of the mature polypeptide.

One embodiment of the invention is a fragment of the polypeptide having a sequence shown as SEQ ID NO: 4. In one embodiment said fragment is obtainable by digesting the polypeptide having SEQ ID NO: 4. In another embodiment said fragment is obtainable by trypsinating the polypeptide having SEQ ID NO: 4. In a further embodiment the fragment or polypeptide has been recombinantly produced. In a still further embodiment the fragment is chemically synthesized (synthetic peptide). In one embodiment the polypeptide and/or fragments thereof are isolated from the endophyte, recombinant growth medium or synthesis medium.

In one embodiment said fragment has a length of 5 to 41 amino acids, preferably 5 to 26 amino acids, more preferably 5 to 19 amino acids and most preferably 5 to 10 amino acids. It has surprisingly been found that relatively small fragments may have desired activity. The fragments according to this invention may originate from any region of the polypeptide according to SEQ ID NO:. 4 or its homologue having at least 30 % identity with said sequence. However, preferred regions of origin are located in regions ranging from amino acid 20 - amino acid 320, particularly amino acid 20 to 220 and amino acid 260 to 320; amino acid 380 to 420; and amino acid 485 to 546 of SEQ ID NO: 4. Particularly preferred are the regions amino acid 145 to 165 and amino acid 480 to 546.

In one embodiment the fragment or the fragments comprise at least one fragment selected from fragment having essentially SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14; SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 42 or a fragment having at least 80% identity to said sequence. In a further embodiment the fragment or fragments consist of one or more fragment(s) listed above.

In this connection the phrase "having essentially SEQ ID NO:" means that the amino acid sequence has at least 80% identity to said sequence and/or that only conservative amino acid substitutions have been made to the fragment.

In a preferred embodiment the fragment or the fragments comprise at least one fragment selected from fragment having essentially SEQ ID NO: 6 (encoding Trypsin6), SEQ ID NO: 14 (encoding Trypsinl4), SEQ ID NO: 15 encoding (Trypsinl5), SEQ ID NO: 42 (encoding Trypsin42) and SEQ ID NO: 43 (encoding Trypsin43) 42 or a fragment having at least 80% identity to said sequence. In a further embodiment the fragment or fragments consist of one or more fragment(s) listed above. In one embodiment the polypeptide is isolated or genetically originating from an endophyte of Empetrum nigrum. With phrase "genetically originating" it is meant that the gene has been isolated from an endophyte of Empetrum nigrum or a sequence information obtained from said endophyte has been used for producing a production construct. In one embodiment the polypeptide, a fragment thereof or fragments thereof has an activity against microorganisms of the family Staphylococcus in particular against microorganisms of the genus Staphylococcus, more particularly against microorganisms of the species S. aureus. S. aureus is one of the most important human pathogens and develops easily resistance towards antibiotics. The strain has already developed resistance to most of the commercially available antibiotics. Antimicrobial peptides, such as we describe here, are a promising class of antibiotics as they often do not induce resistance in microorganisms.

In one embodiment of the invention a polynucleotide encoding SEQ ID NO: 44 or a fragment thereof has a sequence of a sufficient length of base pairs for being able to encode said polypeptide or an active fragment thereof. Polynucleotide of SEQ ID NO: 44 encodes the polypeptide of SEQ ID NO: 4. Recombinant production of the polypeptide has been demonstrated in the experimental part of the description. The polypeptide obtained can be used as such or processed further e.g. by digestion to fragments. It is also possible to directly produce fragments of polypeptide of SEQ ID NO: 4.

In another embodiment the fragment encodes one or more of the sequence(s) selected from SEQ ID NO: 2; SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14; SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 SEQ ID NO: 42 and SEQ ID NO: 42 or a fragment having at least 80% identity to said sequence. In a further embodiment the fragment encodes one or more of the sequence selected from SEQ ID NO: 6 encoding Trypsin6, SEQ ID NO: 14 encoding Trypsinl4, SEQ ID NO: 15 encoding Trypsinl5, SEQ ID NO: 42 encoding Trypsin42 and SEQ ID NO: 43 encoding Trypsin43 or a fragment having at least 80% identity to said sequence. Polynucleotides can be used for constructing a production vector for the metabolite or active agent. Active agent can be a polypeptide or at least one fragment thereof. Also a mixture of fragments can be recombinantly produced.

In one embodiment the pulse-field gel electrophoresis includes the following steps:

- N/S - 60 s and E/W - 60 s for 6 h;

- N/S - 90 s and E/W - 90 s for 6 h;

- N/S - 99 s and E/W - 99 s for 6 h, respectively,

- a voltage of 6 V/cm,

- a 120° fixed angle and - using a 0.15xTris-borate-EDTA (TBE) buffer.

These conditions allow an efficient separation of plant and endophyte DNA. In a preferred embodiment the gene encoding a metabolite or the antimicrobial activity is further isolated and cloned for production. Recombinant technologies allow also a large scale production of polypeptides and/ or their fragment(s) having desired activity.

In one embodiment the polypeptide, a fragment or fragments thereof exhibits an activity against microorganisms of the family Staphylococcusa in particular against microorganisms of the species S. aureus. S. aureus has developed resistance to most of the commercially available antibiotics and antimicrobial peptides, such as we describe here, are a promising class of new antibiotics, as they often do not induce resistance in microorganisms.

Specifically, a metagenomic library was constructed as follows:

Endophytes, microorganisms which spend their entire life cycle, or parts of it, inside of healthy tissues of a host plant, are found in all plant species (Arnold, 2007). Whereas the majority of endophytes cannot be cultured, it is not surprising that a chemical structure isolated from a plant can actually have a microbial origin. Metagenomics provides a valuable tool to access the resources of bioactive compounds derived from unculturable microorganisms (Handelsman, 2004). In this study, we have constructed a metagenomic library from endophytes and screened for antibacterial activity. To our knowledge, this is the first report of characterizing a gene derived from unculturable endophyte that confers antibacterial activity against S.aureus.

The metagenomic DNA prepared from the total DNA of Empetrum nigrum ranged around 35 kb in size, as confirmed by pulse-field gel electrophoresis, which is suitable for cloning in a fosmid. The DNA prepared from E. nigrum was viscous due to high polysaccharide content which complicated the enzymatic manipulation for library construction. However, we were able to obtain -8,000 metagenome clones from 0.02 mg plant DNA. Regarding the high diversity of endophytic populations in a given plant species, an enormous number of clones should be screened to explore the full endophytic community cloning efficiency being an important factor. The cloning efficiency in this work was not as high as desired, probably due to the problems with DNA quality. However, the metagenomic library that was screened for antibacterial activity ranged one hit in 8,000 clones, which is very high compared to other libraries constructed from soil metagenomes that usually range one hit from 20,000-50,000 clones (Rondon et al, 2000; Brady, 2007; Chung et al, 2008). Our library, constructed from the 0.02 mg of plant DNA and containing -8,000 clones with an average size of 35 kb, represents about 0.28 Gb of genomic DNA and equals to -70 full endophyte genomes.

Screening for biolo ical activity

One antibacterial clone was selected by screening the metagenomic library by the double- agar-layer method. The selected clone exhibited a clear growth inhibition of S. aureus but was not active against E. coli. Restriction fragment analysis revealed that the clone carried an insert of over 30 kb in size, indicating that the fosmid contained a metagenomic DNA insert (Figure 1).

A secondary library was generated from the antibacterial clone to obtain subclones with shorter insert size. Restriction fragment analysis of the antibacterial subclones identified a subclone with an insert of 1.8 kb (Figure 1) that exhibited growth inhibition against S. aureus in the double-agar-layer assay (Figure 2). In this way, the gene responsible for the antibacterial activity was successfully subcloned into pCClFos and the full-length sequence of the insert was obtained. The open reading frame (ORF) responsible for the antibacterial activity was analyzed from the individual subclone named pFosSl A.

Sequence analysis of the antibacterial active clone

The insert of pFosSIA was completely sequenced in both directions. It contained a unique open reading frame of -1650 bp. The nucleotide sequence of this gene was compared with sequences contained in databases (BLAST) but no similarities to known sequences were found, suggesting that the gene encoded a protein of novel structure. The isolated gene coded a predicted protein of 549 amino acids (Figure 3) which displayed 28% and 26% homologies to hypothetical proteins of Laccaria bicolor and Malassezia globosa, respectively, and did not contain any putative amino -terminal signal sequence for secretion or translocation. When the amino acid sequence was analyzed for conserved motifs using the Pfam protein family database (Finn et al., 2008), three pentatricopeptide repeat-motifs were present at positions 77 to 107, 126 to 153 and 392 to 420 aa (Figure 3). The isolated endophytic antimicrobial protein can be produced as recombinant protein in a host which can be bacteria, yeast, fungi, baculovirus, mammalian cell, plant cell or plant. Protein can be expressed with or without fusion tag e.g. his-, GST-, FLAG- or T7-tag so that full length protein or fragments of it is obtained. The nucleotides sequence can be changed in order to optimize codon usage. The antibacterial protein is isolated from a host by separation it from host cell proteins with e.g. chromatographic methods. Antimicrobial activity is measured either from extracts of host expressing the recombinant protein or purified recombinant protein. Antimicrobial activity is tested against variable microbial strains. Thus, in summary, we have developed a new approach to screen for novel protein from metagenomes of endophytes. Our efforts were focused to isolate and separate microbial (endophytic) DNA from plant DNA by pulse-field gel electrophoresis, cloning the derived DNA into fosmid libraries and expressing in E. coli to screen for antibacterial activity. We have identified a novel protein exhibiting antibacterial activity towards S. aureus within the E. coli host. Our results clearly demonstrate the potential of this technology to find new molecules from endophytic metagenomes.

A listing of the cited references is given below. The contents of all citations are herewith incorporated by reference.

The following non-limiting examples illustrate the invention

EXAMPLES

Example 1. Strains, plasm ids and growth conditions

EPI-300™-Tl^R Phage Tl-resistant E. coli cultures were grown at 37°C on Luria-Bertani (LB) agar or in LB broth + 10 mM MgS0₄ supplemented with the appropriate antibiotics. The following antibiotic concentrations were used for the E. coli strain: chloramphenicol 12.5 μg mF¹ and ampicillin 100 μg ml^-1. Plasmid pCClFOS™ (Epicentre, Madison, Wis.) that carries two origins of replication, a single copy origin (ori2) and an inducible high copy origin (priV) (Wild et al., 2002), was used to construct the metagenomic library from endophytes of Empetrum nigrum and for subcloning genes conferring antibacterial activity.

Example 2. DNA preparation from plant

Fresh and young leaves of Empetrum nigrum L. were used for isolation of genomic DNA according to the protocol described by Pirttila et al. (2001). Briefly, the fresh plant leaves (0.1 g) were ground into a fine powder in liquid nitrogen and mixed with 350 μΐ extraction buffer [2% (w/v) hexadecyltrimethyl-ammonium bromide (CTAB), 100 mM Tris-HCl (pH 8), 20 mM EDTA (pH 8), 1.4 M NaCl, 2% (w/v) polyvinylpyrrolidone Mr 750,000 (PVP), and 2% (v/v) β-mercaptoethanol] and 350 μΐ 8 M LiCl, and heated at 65°C for 10 min. The mixture was extracted twice with an equal volume of 24:1 (v/v) chloroform-isoamyl alcohol, centrifuged at 13,793 x g for 5 min at 4°C, and precipitated with 0.5 volume 3 M potassium acetate (pH 4.8) at -20°C for 30 min. After centrifugation, the supernatant was precipitated with 0.6 volume of cold isopropanol at -20°C for 30 min. The precipitate was collected by centrifugation at 13,793 x g for 10 min at 4°C, air-dried, and dissolved in 0.6 ml sterile distilled water. The DNA was then precipitated with two volumes of cold absolute ethanol at -20°C for 1 h, collected by centrifugation (13,793 x g for 10 min at 4°C), washed with 70% (v/v) ethanol and air dried.

Example 3. General DNA manipulation

Plasmid preparation, restriction endonuclease digestion, DNA ligation, plasmid DNA transformation, agarose gel electrophoresis, and other standard recombinant DNA techniques were carried out by the standard methods described by Sambrook et al. (2001). Plasmid DNA was isolated and sequenced according to the manufacturer's instructions (Abi 3730 DNA Analyser, Abi Prism BigDye Terminator Cycle Sequencing Kit, Applied Biosystems, Warrington, UK). The sequences were analyzed by alignment with all accessible sequences obtained through the Basic Local Alignment Search Tool (BLAST) provided by the National Center for Biotechnology Information. The default parameters with algorithm blastp were used in the search against non-redundant protein sequence database.

Example 4. Metagenomic library construction and screening

The precipitated DNA originating from E. nigrum containing both DNA of the plant and endophytes was dissolved in sterile water to a concentration of 0.1 μg μΓ¹. The DNA was then subjected to preparative pulsed-field gel electrophoresis in a CHEF-DRII (Bio-Rad) system. The electrophoresis conditions (pulse intervals and durations) were: N/S - 60 s and E/W - 60 s for 6 h; N/S - 90 s and E/W - 90 s for 6 h; N/S - 99 s and E/W - 99 s for 6 h, respectively, with a voltage of 6 V/cm, a 120° fixed angle and using a 0.15xTris-borate- EDTA (TBE) buffer. During the electrophoresis, the temperature was maintained at 10°C. After electrophoresis, a strip from each side of the gel was cut off and stained with ethidium bromide to visualize the DNA. The high-molecular- weight DNA was then excised from the remaining unstained part of the gel and electro-eluted for lh at 100 V in a dialysis bag containing 0.5xTBE. After the dialysis, the DNA was subjected to DNA end repair to produce 5'-phosphorylated DNA for the ligation step. The prepared DNA (0.25 μg) was ligated with 0.5 mg of blunt-ended dephosphorylated pCClFOS™ vector and the ligation mixture was packaged into lambda phages using MaxPlax Lambda Packaging Extracts (Epicentre). The packaged library was transduced into E. coli EPI-300, and the transformants were selected on LB agar plates supplemented with chloramphenicol. The packaged fosmid library was stored in cryotubes as clone pools containing approximately 10³ clones per pool until screening (Brady, 2007). For screening, the clone pools were diluted with dilution buffer (10 mM Tris-HCl, pH 8.3, 100 mM NaCl, and 10 mM MgCl₂) and spread onto 150-mm LB agar plates supplemented with chloramphenicol to obtain -1000 colonies per plate. The library plates were incubated at 30 °C overnight and at room temperature (RT) for additional 3-5 days. The plates were then overlaid with top agar containing exponentially growing Staphylococcus aureus and incubated overnight at 37 °C followed by further incubation at RT for 3-5 days. Colonies with antibacterial activity were identified by production of a zone of inhibition of S. aureus growth. Such colonies were picked through the top agar and separated from the chloramphenicol-sensitive assay strain (S. aureus) by streaking onto LB plates containing both ampicillin and chloramphenicol. Restriction digestion analysis of the selected antibacterial fosmid clones were carried out with BamHl and analyzed by electrophoresis.

Example 5. Subcloning and sequencing of clone pFosSIA

The selected antibacterial fosmid clone was named pFosSl . The metagenomic fosmid DNA was isolated using the Plasmid Midi prep Kit (Qiagen) and subjected to partial digestion with Sau3Al (0.1 U μΓ¹ of DNA, 37 C for 15 min) and electrophoresis for size selection of the DNA and subcloning. Fragments greater than 1.5 kb were extracted from the gel and end-repaired to make 5'-phosphorylated DNA for ligation into blunt-ended dephosphorylated pCClFOS™. The ligation mixture was transformed into E. coli and the trans formants were spread onto LB supplemented with chloramphenicol to select subclones showing clear zones of inhibition of S. aureus. One subclone with insert size of -1.8 kb was selected and named after the initial fosmid clone as pFosSIA. The subclone was sequenced using the primers pCCl forward and pCCl reverse. The nucleotide sequence of the insert DNA of pFosSIA is shown as SEQ ID NO: 44. . Deduced amino acid sequence is shown in Figure 3 and as a SEQ ID NO: 4.

Example 6. Production, digestion and analysis of polypeptide having SEQ ID NO: 4, encoded by SEQ ID NO: 44.

The antibacterial gene from pFosSAl was amplified using a primers with Ndel and Sail restriction enzymes pFosSIF

(C AT ATG AG ACT AGT AGCTC ATC CTGTTC CTG ATGC ; SEQ ID NO: 45) and pFosSIR (GTCGACTTATTAACGAGATGACGTCCTCTGCTGTACG; SEQ ID NO: 46) and was expressed in pET 23(b) vector, transformed into XL1 competent cells and the gene was sequenced to confirm the sequence of the antibacterial gene. The expression studies were done using strains BL 21 pLyseS and BL 21 pRARE, the expression in both strains were good and was expressed as insoluble protein (inclusion bodies). The Inclusion bodies were isolated following the protocol of Lith et al, (2007) and suspended in 5M Guanidine hydrochloride/Phosphate buffer and the protein was folded on column (HisTrap Column) using a linier gradient of 3M Guanidine hydrochloride/200 mM Phosphate buffer using AKTA FPLC and eluted with 50 mM EDTA/Phosphate buffer (20 mM). The quantity of folded protein was about 1 mg and was tested for antibacterial activity and it was not active. The folded protein (native) was digested with trypsin in 0.01 % acetic acid and the activity was tested, the digested protein (peptides) is active. The denatured protein digested with trypsin was not active. Example 7. In silico digestion and analysis

The antimicrobial protein having SEQ ID NO: 4 was in silico trypsin digested (http://au.expasy.org/) and the peptides were predicted

(http://www.bicnirrh.res.in antimicrobial/def/) to be antimicrobial with three different algorithms (Support Vector Machine (SVM) classifier, Random Forest Classifier and Discriminant Analysis Classifier). The peptides which were predicted to be antimicrobial in all the three algorithms are bolded and underlined, and others bolded (predicted in one of the algorithms). Results are shown in Table 1 below. Sequences of tryptic peptides are shown as SEQ ID NO's 1 to 3 and 5 to 43. Sequence of peptide "Trypsin3" is EEK.

Table 1. Antimicrobial prediction of tryptic peptides.

Support Vector

Machine (SVM) Random Forest Discriminant classifier^ Classifier⁽²⁾ Analysis Classifier¹^

SEQ

ID ProbaProbaDiscr.

NO: Name Class bility Class bility Class Score

1 Trypsin 1 Non-AMP 0.944 Non-AMP 0.936 Non-AMP 0.245

2 Trypsin2 Non-AMP 0.887 Non-AMP 0.928 Non-AMP 0.811

3 Trypsin3 Non-AMP 0.671 AMP 0.69 AMP -1.860

- Trypsin4 Non-AMP 0.918 AMP 0.79 Non-AMP 3.515

5 Trypsins Non-AMP 0.971 AMP 0.896 Non-AMP 3.259

6 Trvpsin6 AMP 0.794 AMP 0.754 AMP -0.801

7 Trypsin7 AMP 0.995 AMP 0.862 Non-AMP 0.306

8 Trypsin8 Non-AMP 0.711 Non-AMP 0.552 AMP -0.371

9 Trypsin9 AMP 1.000 AMP 0.932 Non-AMP 0.278

10 Trypsin 10 AMP 0.998 AMP 0.816 AMP -3.747

11 Trypsin 11 AMP 0.555 AMP 0.868 Non-AMP 1.654

12 Trypsin 12 Non-AMP 0.980 Non-AMP 0.982 Non-AMP 2.389 Trypsin 13 Non-AMP 0.840 AMP 0.618 AMP -1.033

Trvpsinl4 AMP 0.732 AMP 0.654 AMP -1.044

Trypsin 15 AMP 0.968 AMP 0.656 AMP -0.651

Trypsin 16 AMP 0.859 AMP 0.914 Non-AMP 0.612

Trypsin 17 Non-AMP 0.906 Non-AMP 0.5 Non-AMP 1.814

Trypsin 18 Non-AMP 0.999 Non-AMP 0.962 Non-AMP 1.986

Trypsin 19 Non-AMP 0.956 AMP 0.91 Non-AMP 1.922

Trypsin20 AMP 0.992 AMP 0.758 Non-AMP 1.634

Trypsin21 Non-AMP 0.987 Non-AMP 0.944 Non-AMP 3.320

Trypsin22 Non-AMP 0.966 Non-AMP 0.728 Non-AMP 0.264

Trypsin23 Non-AMP 0.816 AMP 0.884 Non-AMP 1.668

Trypsin24 Non-AMP 0.978 Non-AMP 0.884 Non-AMP 1.670

Trypsin25 Non-AMP 0.612 AMP 0.608 AMP -0.602

Trypsin26 Non-AMP 0.999 AMP 0.696 Non-AMP 1.812

Trypsin27 Non-AMP 0.761 AMP 0.552 Non-AMP -0.128

Trypsin28 Non-AMP 0.998 Non-AMP 1 Non-AMP 2.074

Trypsin29 Non-AMP 0.941 Non-AMP 0.968 Non-AMP 0.308

Trypsin30 AMP 0.597 AMP 0.744 Non-AMP 0.794

Trypsin31 Non-AMP 0.850 Non-AMP 0.668 Non-AMP 0.491

Trypsin32 AMP 0.561 AMP 0.878 Non-AMP 0.718

Trypsin33 Non-AMP 0.831 AMP 0.834 AMP -1.020

Trypsin34 Non-AMP 0.999 Non-AMP 0.956 Non-AMP 2.402

Trypsin35 Non-AMP 0.997 Non-AMP 0.906 Non-AMP 0.907

Trypsin36 Non-AMP 0.996 Non-AMP 0.974 Non-AMP 1.987

Trypsin37 Non-AMP 0.533 AMP 0.736 AMP -1.975

Trypsin38 Non-AMP 0.997 AMP 0.786 Non-AMP 1.334

Trypsin39 Non-AMP 0.929 AMP 0.564 AMP -0.990

Trypsin40 AMP 0.636 AMP 0.886 Non-AMP 0.469

Trypsin41 Non-AMP 0.996 AMP 0.64 Non-AMP 1.51 1

Trvpsin42 AMP 0.825 AMP 0.78 AMP -1.884

Trypsin43 AMP 0.994 AMP 0.72 AMP -1.417 out of 43 sequences are AMP(s)

out of 43 sequences are AMP(s) ^ A peptide is predicted to be antimicrobial if the discriminant score is less than -0.251; 13 out of 43 sequences are AMP(s).

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Claims

1. A polypeptide, a fragment or fragments thereof comprising an amino acid sequence which has at least 31 % identity with SEQ ID NO: 4 or a fragment or fragments thereof.

2. The polypeptide, fragment or fragments thereof according to claim 1, wherein said polypeptide has an identity of at least 40 %, in particular at least 50 %, preferably at least 60 %, advantageously at least 70 % with the amino acids shown as SEQ ID NO: 4.

3. The fragment(s) according to claim 1 or 2, said fragment being obtainable by digesting the polypeptide according to claim 1 or 2.

4. The fragment(s) according to claim 3, said fragment being obtainable by trypsinating the polypeptide according to claim 1 or 2.

5. The fragment(s) according to claim 3 or 4, wherein said fragment has a length of 5 to 41 amino acids, preferably 5 to 26 amino acids, more preferably 5 to 21 amino acids and most preferably 5 to 19 amino acids.

6. The fragment(s) according to any of the preceding claims comprising at least one fragment selected from fragment having essentially SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14; SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42 and SEQ ID NO: 42 or a fragment having at least 80% identity to said sequence

7. The fragment(s) of claim 6 containing at least one fragment selected from fragment having essentially SEQ ID NO: 6 (encoding Trypsin6), SEQ ID NO: 14 (encoding Trypsinl4), SEQ ID NO: 15 encoding (encoding Trypsinl5), SEQ ID NO: 42 (encoding Trypsin42) and SEQ ID NO: 43 (encoding Trypsin43) 42 or a fragment having at least 80% identity to said sequence.

8. The polypeptide according to any of the preceding claims said polypeptide being isolated or genetically originating from an endophyte of Empetrum nigrum.

9. The polypeptide, a fragment or fragments thereof according to any of the preceding claims said polypeptide being recombinantly produced.

10. The polypeptide, a fragment or fragments thereof according to any of the preceding claims said polypeptide being chemically synthesized.

11. The polypeptide, a fragment or fragments thereof according to any of the preceding claims, having an activity against microorganisms of the family Staphylococcusa in particular against microorganisms of the genus Staphylococcus, more particularly against microorganisms of the species S. aureus.

12. A polynucleotide having at least part of the sequence shown as SEQ ID NO: 44, encoding a polypeptide or a fragment according to any of the preceding claims.

13. The polynucleotide according to claim 12, having a sequence of a sufficient length of base pairs for being able to encode said polypeptide or an active fragment thereof.

14. The polynucleotide according to claim 12 or 13 wherein the fragment encodes one or more of the sequence(s) selected from SEQ ID NO: 2; SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14; SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 20, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41 SEQ ID NO: 42 and SEQ ID NO: 42 or a fragment having at least 80% identity to said sequence.

15. The polynucleotide according to claim 14 wherein the fragment encodes one or more of the sequence(s) selected from SEQ ID NO: 6 encoding Trypsin6, SEQ ID NO: 14 encoding Trypsinl4, SEQ ID NO: 15 encoding Trypsinl5, SEQ ID NO: 42 encoding Trypsin42 and SEQ ID NO: 43 encoding Trypsin43 or a fragment having at least 80% identity to said sequence.

16. A method for isolating genes encoding microbial metabolites, comprising the steps of

- grinding plant material containing the microbe(s);

- isolating total DNA of the ground plant material;

- separating DNA from the plant DNA using pulse-field gel electrophoresis;

- digesting DNA and constructing a genomic library based on the digested DNA;

- screening the library for a preselected activity; and

- isolating clones carrying the preselected activity.

17. The method of claim 16 wherein the pulse-field gel electrophoresis includes the following steps:

- N/S - 60 s and E/W - 60 s for 6 h;

- N/S - 90 s and E/W - 90 s for 6 h;

- N/S - 99 s and E/W - 99 s for 6 h, respectively,

- a voltage of 6 V/cm,

- a 120° fixed angle and

- using a 0.15xTris-borate-EDTA (TBE) buffer.

18. The method according to claim 16 or 17, wherein the gene encoding the antimicrobial activity is further isolated and cloned for production.

19. A polypeptide obtained by the process of any one of claims 17 to 18.

20. An antimicrobial composition comprising an antimicrobial polypeptide or a fragment thereof according to any of claims 1 to 15 or obtained by a method of any of claims 16 to 19.

21. A polypeptide or a fragment thereof, as defined in any of claims 1 to 15 or claim 19, for use in therapy, particularly for use as an antimicrobial agent.

22. A method of killing or inhibiting growth of microbes, comprising the step of contacting said microbes with a polypeptide or a fragment thereof according to any of claims 1 to 15 or polypeptide of fragment obtained by a method of any of claims 16 to 19.

23. The method according to claim 22, wherein the polypeptide or fragment thereof exhibits an activity against microorganisms of the family Staphylococcusa in particular against microorganisms of the species S. aureus.

24. Use of a polypeptide or fragment thereof according to claims 1 to 15 or 19, or polypeptide of fragment obtained by a method of any of claims 16 to 18, as a medicament, feed additive, preservative or surfactant.