WO2008110325A2

WO2008110325A2 - Probiotic

Info

Publication number: WO2008110325A2
Application number: PCT/EP2008/001892
Authority: WO
Inventors: Adriano O. Henriques; Claudia Alexandra Dos Reis-Serra; Ghislain Schyns
Original assignee: DSM IP Assets BV
Current assignee: DSM IP Assets BV
Priority date: 2007-03-09
Filing date: 2008-03-10
Publication date: 2008-09-18
Anticipated expiration: 2009-09-09
Also published as: WO2008110325A3

Abstract

The present invention relates to newly identified probiotics. The invention also relates to polynucleotide sequences comprising genes that encode proteins which are involved into probiotic behavior.

Description

Probiotic

The present invention relates to a novel probiotic usefull in the production of animal feed, functional food (for example yoghurt, cheese) and non-food preparations (as for example tablets, capsules).

Functional food is a foodstuff, consumed additionally to usual food and containing bio-preparations or other components favourably influencing human health or decreasing disease risks.

The term "probiotic" generally refers to a non-pathogenic bacterium fed to animals, including birds, as a way to prevent colonization by pathogenic microorganisms, e.g. protozoa.

Probiotics are defined as preparations of living microorganisms which when administered in adequate amounts confer a health benefit on the host. Probiotics are delivered to farm animals for improving their intestinal microbial balance but also to human in the form of dairy-based foods containing intestinal species of lactobacilli and bifidobacteria.

Most of probiotics used in functional food are lactic acid bacteria, mainly lactobacilli. Lactobacilli are non-pathogenic micro-organisms, colonising the human intestinal and urogenital tract from early childhood to old age. Nowadays, several commercial probiotic lactobacilli are successfully used, among which Lactobacillus rhamnosus is one of the best known.

Several strains of Lactobacillus fermentum are used for correction and stabilisation of intestinal micro-flora in case of dysbacterioses and urogenital infections with different ethiologies.

Examples of probiotics used as feed additive for farm animals are strains from the genera of Bacillus, Lactobacillus, Pediococcus and Propionibacterium.

Probiotic behavior has been generally attributed to the following different mechanisms: Antimicrobial Activity (AA), Immune Stimulation (IS), Competitive Exclusion (CE) and Conversion of Genotoxic Compounds (CGC). 1. Antimicrobial activity (AA):

Many mediators of bacterial probiosis have been described in bacteria, including bacteriophages, but also biosynthetic complex antimicrobial proteic/ peptidic molecules and simple molecules such as hydrogen peroxide, organic acids and ammonia. In particular, Bacillus spp. biosynthetize, for probiosis, a complex variety of bioactive metabolites with very specific spectrum and modes of actions, like lytic enzymes, bacteriocins and antibiotics. Amongst lytic enzymes are the autolysins which are extracellular hydrolases or endopeptidases that can lyse both live and heated bacterial cells. Examples of autolysin are elastases, chitinases, cellulases or laminarinases. Bacteriocin and bacteriocin-like inhibitory substances (BLIS) are another category of very important antimicrobial molecules. Bacteriocins are peptides synthetized by ribosomes and include lantibiotics (examples include but are not limited to nisin, subtilin, sublancin, mersacidin), pediocin-like peptides (example include but are not limited to coagulin), BLIS (example include but are not limited to bacillocin and lichenin) and cyclic peptide (example include but are not limited to subtilosin). Antibiotic are secondary metabilites with either specific or broad activity spectrum. Bacillus strains biosynthetize lipopeptide as well as polypeptides antibiotics. Examples of lipopetides antibiotics produced by Bacillus spp. Include but are not limited to surfactin, iturin, bacillomycin, bacilysin, plipastatins and fengycins. Examples of polypeptides antibiotics include but are not limited to bacitracin, gramicidin, colistins and circulins.

2. Immune stimulation (IS):

Immune stimulation represents another way by which probiotic works. Bacteria within the gastrointestinal tract can bind to receptors expressed at the surface of epithelial cells to promote activation of immunological defense mechanism, such as production of anti-inflammatory cytokines (example of these include but are not limited to TNF- α and IFN-γ) or enhance the production of regulatory cytokines (examples of these are IL-10 or TGF-β). In the context of using preparations of spores produced by Bacillus spp. for probiosis, it is worth mentioning the recently described role of Bacillus subtilis in the development of gut-associated lymphoid tissues (Rhee et al, 2004).

3. Competitive exclusion (CE):

Reducing adhesion and/ or blocking penetration of pathogens across the mucosal layer constitute the basis of competitive exclusion which is also an important way how probiotic deliver their health beneficial effect. Molecules like adhesins are thought to play a role in competitive exclusion mechanism. Microbial pathogens, including those responsible for major enteric infections, exploit oligosaccharides that are displayed on the surface of host cells as receptors for toxins and adhesins. Blocking crucial pathogen ligand-receptor interactions through competition with probiotics expressing more adhesins or adhesin-like molecules constitutes an important option for probiosis.

4. Conversion of Genotoxic Compounds (CGC)

Supernatants from probiotic cultures have been shown to convert genotoxic compounds to unreactive products. Examples from, but not limited to, Lactobacilli spp. and Bifidobacterium spp. have been reported.

Surprisingly, it has now been found that the genome of the Bacillus subtilis undomesticated strain BSPl {Bacillus subtilis strain BSPl has been deposited at Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), Mascheroder Weg IB, D-38124 Braunschweig, Germany according to the Budapest Treaty on March 8, 2007). that presents strong probiotic behavior in vitro (i.e. unique antimicrobial activity and strong biofilm production) comprises a group of genes which is not present on the genome of the non-probiotic counterpart Bacillus subtilis 168 {Bacillus subtilis strain 168 is public available at Bacillus Genetic Stock Center (BGSC), Columbus, Ohio) or divergent from genes present on Bacillus subtilis 168 public genome and that the unique probiotic behavior of the strain BSPl relies on the genes from said group, wherein the activity pattern of the polypeptides encoded by these genes may be attributed to the four different mechanisms defined herein above or interaction with "resident" host genes.

Consequently the invention relates to a probiotic strain which genome comprises at least one polynucleotide encoding a protein which is involved into probiotic behavior and which polynucleotide is substantially identical" to a polynucleotide sequence according to SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO:81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO:181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO:219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO:281, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, SEQ ID NO: 303, SEQ ID NO: 305, SEQ ID NO: 307, SEQ ID NO: 309, SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 319, SEQ ID NO: 321, SEQ ID NO: 323, SEQ ID NO: 325, SEQ ID NO: 327, SEQ ID NO:329, SEQ ID NO: 331 , SEQ ID NO: 337, SEQ ID NO: 339, SEQ ID NO: 341, SEQ ID NO: 343, SEQ ID NO: 345, SEQ ID NO: 347, SEQ ID NO: 349, SEQ ID NO: 351, SEQ ID NO: 353, SEQ ID NO: 355, SEQ ID NO: 357, SEQ ID NO: 359, SEQ ID NO: 361, SEQ ID NO: 363, SEQ ID NO: 365, SEQ ID NO: 367, SEQ ID NO: 369, SEQ ID NO: 371, SEQ ID NO: 373, SEQ ID NO: 375, SEQ ID NO: 377, SEQ ID NO: 379, SEQ ID NO:381, SEQ ID NO: 383, SEQ ID NO: 385, SEQ ID NO: 387, SEQ ID NO: 389, SEQ ID NO: 391, SEQ ID NO: 393, SEQ ID NO: 395, SEQ ID NO: 397, SEQ ID NO: 399, SEQ ID NO: 401 and SEQ ID NO: 403 respectively. More precisely the at least one polynucleotide encodes a protein which is attributed to one of the following subgroup of proteins:

1) proteins which show or support or maintain antimicrobial activity (herein abbreviated by AA) or 2) proteins which stimulate the immune system (herein abbreviated by IS) or

3) proteins which play a role in competitive exclusion mechanism (herein abbreviated by CE) or

4) proteins which convert genotoxic coumpounds in unreactive products.

In accordance with the invention, the activity of each protein selected from the group consisting of AA, IS, CE or CGC proteins is as such that it leads to an improved probiotic behavior of the microorganisms compared to the counterpart of said organism which does not comprise the polynucleotide encoding said protein.

The probiotic behavior can greatly be improved if more than one, preferably more than 3, or more than 5, for example more than 10 proteins selected from the group consisting of AA, IS, CE or CGC proteins are expressed in the probiotic. Such is herein also referred to as concurrent expression. If the concurrent expression is the result of a genetic manipulation event, this is also referred to as concurrent manipulation.

hi this context it should be mentioned that the expression of "a polynucleotide which is substantially identical" refers with respect to the encoding sequences to a polynucleotide sequence selected from the group consisting of: a) polynucleotides comprising the nucleotide sequence according to any SEQ ID NO listed in column 3 of table 1 ; b) polynucleotides comprising a nucleotide sequence encoding a fragment or derivative of a polypeptide comprising the amino acid sequence according to any

SEQ ID NO listed in column 4 of table 1 or encoding a fragment or derivative of a polypeptide encoded by a polynucleotide of any of (a) wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said fragment or derivative has the activity of a protein involved into probiotic behavior; c) polynucleotides the complementary strand of which hybridizes under stringent conditions to a polynucleotide encoding a polypeptide comprising the amino acid sequence of any of (b) or to a polynucleotide as defined in any one of (a) and which encode a protein involved into probiotic behavior; and d) polynucleotides which are at least 70%, such as 85, 90 or 95% homologous to a polynucleotide encoding a polypeptide comprising the amino acid sequence according to any of (b) or to a polynucleotide as defined in any one of (a) to (c) and which encode a protein involved into probiotic behavior; or the complementary strand of such a polynucleotide.

In another embodiment the genome is genetically engineered by DNA sequences specified hereinabove.

The term "genetically engineered" or "genetically altered" means the scientific alteration of the structure of genetic material in a living organism. It involves the production and use of recombinant DNA. More in particular it is used to delineate the genetically engineered or modified organism from the naturally occurring organism.

Genetic engineering may be done by a number of techniques known in the art, such as e.g. gene replacement, gene amplification, gene disruption, transfection, transformation using plasmids, viruses, or other vectors. A genetically modified organism, e.g. genetically modified microorganism, is also often referred to as a recombinant organism, e.g. recombinant microorganism.

The polypeptides as listed in column 4 of table 1 may be isolated from Bacilus subtilis strain BSPl. The corresponding polynucleotide sequences or genes are absent or divergent in/ from Bacillus subtilis 168 genome which was published by Kunst et al. (Nature. 1997, Nov 20; 390 (6657):249-56).

Table 1 : Genes responsible for probiotic behavior in Bacillus subtilis (divergent gene names are in bold

Examples of known probiotic bacteria able to express the at least one of the DNA sequences specified hereinabove include strains from the genera of Bacillus, Lactobacillus, Pediococcus, Sporolactobacillus and Propionibacterium.

The invention also relates to polynucleotides which are at least 70% identical to a polynucleotide as defined herein above.

The invention also relates to a polynucleotide encoding at least a biologically active fragment or derivative of a polypeptide as shown in column 4 of table 1.

As used herein, "biologically active fragment or derivative" means a polypeptide which retains essentially the same biological function or activity as the polypeptide shown in column 4 of table 1. Examples of biological activity may for instance be enzymatic activity, signaling activity or antibody reactivity. The term "same biological function" or "functional equivalent" as used herein means that the protein has essentially the same biological activity, e.g. enzymatic, signaling or antibody reactivity, as a polypeptide shown in column 4 of table 1.

In the following, the construction and expression of a single gene/polynucleotide and the alteration in the genome of the host cell as exemplified above will be described in more detail on the basis of Bbsp0025. Unless otherwise indicated, the description is also applicable for the construction and expression of all other AA, IS, CE or CGC genes disclosed herein.

A wide variety of host/cloning vector combinations may be employed in cloning the double stranded DNA. Preferred vectors for the expression of the genes of the present invention, i.e. the Bbsp0025 gene, in E. coli may be selected from any vectors usually used in E. coli, such as for instance pQE vectors which can express His-tagged recombinant proteins (QIAGEN AG Switzerland), pBR322 or its derivatives including for instance pUC18 and pBluescript II (Stratagene Cloning Systems, Calif, USA), pACYC177 and pACYC184 and their derivatives, and a vector derived from a broad host range plasmid such as RK2 and RSFl 010. .

The polypeptides and polynucleotides as exemplified herein are preferably provided in an isolated form, and preferably are purified to homogeneity.

The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally- occurring polynucleotide or polypeptide present in a living microorganism is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition and still be isolated in that such vector or composition is not part of its natural environment.

An isolated polynucleotide or nucleic acid as used herein may be a DNA or RNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (one on the 5'-end and one on the 3'-end) in the naturally occurring genome of the organism from which it is derived.

As used herein, the terms "polynucleotide", "gene" and "recombinant gene" refer to nucleic acid molecules which may be isolated from chromosomal DNA, which include an open reading frame encoding BbspOO25 protein. A polynucleotide may include a polynucleotide sequence as shown in SEQ ID NO:1 or fragments thereof and regions upstream and downstream of the gene sequences which may include, for example, promoter regions, regulator regions and terminator regions important for the appropriate expression and stabilization of the polypeptide derived thereof.

A gene may include coding sequences, non-coding sequences such as for instance untranslated sequences located at the 3'- and 5'-ends of the coding region of a gene, and regulatory sequences. Moreover, a gene refers to an isolated nucleic acid molecule as defined herein. It is furthermore appreciated by the skilled person that DNA sequence polymorphisms that lead to changes in the amino acid sequences of BbspOO25 proteins may exist within a population, e.g., the Bacillus subtilis population. Such genetic polymorphism in the Bbsp0025 gene may exist among individuals within a population due to natural variation or in cells from different populations. Such natural variations can typically result in 1-5% variance in the nucleotide sequence of the Bbsp0025 gene. Any and all such nucleotide variations and the resulting amino acid polymorphism in Bbsp0025 are the result of natural variation and that do not alter the functional activity of Bbsp0025 proteins are intended to be within the scope of the invention.

As used herein, the terms "polynucleotide" or "nucleic acid molecule" are intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA. The nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides may be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.

The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases.

Unless otherwise indicated, all nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence may be more precisely determined by other approaches including manual DNA sequencing methods well known in the art. As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.

The person skilled in the art is capable of identifying such erroneously identified bases and knows how to correct for such errors.

A nucleic acid molecule according to the invention may comprise only a portion or a fragment of the nucleic acid sequence provided by the present invention, such as for instance the sequence shown in SEQ ID NO:1, for example a fragment which may be used as a probe or primer or a fragment encoding a portion of a protein according to the invention. The nucleotide sequence determined from the cloning of the Bbsp0025 gene allows for the generation of probes and primers designed for use in identifying and/or cloning other Bbsp0025 family members, as well as Bbsp0025 homologues from other species. The probe/primer typically comprises substantially purified Case 26054WQ

oligonucleotides which typically comprises a region of nucleotide sequence that hybridizes preferably under highly stringent conditions to at least about 12 or 15, preferably about 18 or 20, more preferably about 22 or 25, even more preferably about 30, 35, 40, 45, 50, 55, 60, 65, or 75 or more consecutive nucleotides of a nucleotide sequence shown in SEQ ID NO: 1 or a fragment or derivative thereof.

A nucleic acid molecule encompassing all or a portion of the nucleic acid sequence of SEQ ID NO:1 maybe isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence information contained herein.

A nucleic acid of the invention may be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid thus amplified may be cloned into an appropriate vector and characterized by DNA sequence analysis.

Fragments of a polynucleotide may also comprise polynucleotides not encoding functional polypeptides. Such polynucleotides may function as probes or primers for a PCR reaction.

Nucleic acids irrespective of whether they encode functional or non-functional polypeptides may be used as hybridization probes or polymerase chain reaction (PCR) primers. Uses of the nucleic acid molecules of the present invention that do not encode a polypeptide having a Bbsp0025 activity include, inter alia, (1) isolating the gene encoding the protein of the present invention, or allelic variants thereof from a cDNA library, e.g., from other organisms than Bacillus subtilis and (2) Northern blot analysis for detecting expression of mRNA of said protein in specific cells or (3) use in enhancing and/or improving the function or activity of homologous BbspOO25 genes in said other organisms.

Probes based on the nucleotide sequences provided herein may be used to detect transcripts or genomic sequences encoding the same or homologous proteins for instance in other organisms. Nucleic acid molecules corresponding to natural variants and non-Bacillus subtilis homologues of the Bacillus subtilis Bbsp0025 DNA of the invention which are also embraced by the present invention may be isolated based on their homology to the Bacillus subtilis BbspOO25 nucleic acid disclosed herein using the Bacillus subtilis DNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques, preferably under highly stringent hybridization conditions.

In preferred embodiments, the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme cofactor.

Homologous or substantially identical gene sequences may be isolated, for example, by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of nucleotide sequences as taught herein.

The template for the reaction may be cDNA obtained by reverse transcription of mRNA prepared from strains known or suspected to express a polynucleotide according to the invention. The PCR product may be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a new nucleic acid sequence as described herein, or a functional equivalent thereof.

The PCR fragment may then be used to isolate a full length cDNA clone by a variety of known methods. For example, the amplified fragment may be labeled and used to screen a bacteriophage or cosmid cDNA library. Alternatively, the labeled fragment may be used to screen a genomic library.

PCR technology can also be used to isolate full-length cDNA sequences from other organisms. For example, RNA may be isolated, following standard procedures, from an appropriate cellular or tissue source. A reverse transcription reaction may be performed on the RNA using an oligonucleotide primer specific for the most 5'-end of the amplified fragment for the priming of first strand synthesis.

The resulting RNA/DNA hybrid may then be "tailed" {e.g., with guanines) using a standard terminal transferase reaction, the hybrid may be digested with RNaseH, and second strand synthesis may then be primed {e.g., with a poly-C primer). Thus, cDNA sequences upstream of the amplified fragment may easily be isolated. For a review of useful cloning strategies, see e.g., Sambrook et al., supra; and Ausubel et al., supra.

Homologues, substantially identical sequences, functional equivalents, and orthologs of the genes exemplified herein, such as the Bbsp0025 gene according to SEQ ID NO:1 may be obtained from a number of different microorganisms. The procedures for the isolation of specific genes and/or fragments thereof are exemplified herein. Following these procedures, Bbsp0025 genes have successfully been isolated from Bacillus subtilis. Accordingly, nucleic acids encoding other Bbsp0025 family members, which thus have a nucleotide sequence that differs from a nucleotide sequence according to SEQ ID NO: 1 , are within the scope of the invention.

Moreover, nucleic acids encoding Bbsp0025 proteins from different species which thus have a nucleotide sequence which differs from a nucleotide sequence shown in SEQ ID NO:1 are within the scope of the invention.

The invention also discloses an isolated polynucleotide hybridisable under stringent conditions, preferably under highly stringent conditions, to a polynucleotide according to the present invention, such as for instance a polynucleotide shown in SEQ ID NO:1.

As used herein, the term "hybridizing" is intended to describe conditions for hybridization and washing under which nucleotide sequences at least about 50%, at least about 60%, at least about 70%, more preferably at least about 80%, even more preferably at least about 85% to 90%, most preferably at least 95% homologous to each other typically remain hybridized to each other.

In one embodiment, a nucleic acid of the invention is at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homologous to a nucleic acid sequence shown in SEQ ID NO:1 or the complement thereof.

A preferred, non-limiting example of such hybridization conditions are hybridization in 6x sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in Ix SSC, 0.1% SDS at 50°C, preferably at 55°C, more preferably at 60°C and even more preferably at 65°C .

Highly stringent conditions include, for example, 2 h to 4 days incubation at 42 °C using a digoxigenin (DIG)-labeled DNA probe (prepared by using a DIG labeling system; Roche Diagnostics GmbH, 68298 Mannheim, Germany) in a solution such as DigEasyHyb solution (Roche Diagnostics GmbH) with or without 100 μg/ml salmon sperm DNA, or a solution comprising 50% formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 0.02% sodium dodecyl sulfate, 0.1% N-lauroylsarcosine, and 2% blocking reagent (Roche Diagnostics GmbH), followed by washing the filters twice for 5 to 15 minutes in 2x SSC and 0.1% SDS at room temperature and then washing twice for 15-30 minutes in 0.5x SSC and 0.1% SDS or 0.1 x SSC and 0.1% SDS at 65-68⁰C.

Preferably, an isolated nucleic acid molecule of the invention that hybridizes under preferably highly stringent conditions to a nucleotide sequence of the invention corresponds to a naturally-occurring nucleic acid molecule. As used herein, a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature {e.g., encodes a natural protein). In one embodiment, the nucleic acid encodes a natural Bacillus subtilis Bbsp0025 protein.

The skilled artisan will know which conditions to apply for stringent and highly stringent hybridization conditions. Additional guidance regarding such conditions is readily available in the art, for example, in Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, N. Y.; and Ausubel et al. (eds. ), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N. Y.). Of course, a polynucleotide which hybridizes only to a poly (A) sequence (such as the 3'- terminal poly (A) tract of mRNAs), or to a complementary stretch of T (or U) residues, would not be included in a polynucleotide of the invention used to specifically hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g. , practically any double-stranded cDNA clone).

In a typical approach, genomic DNA or cDNA libraries constructed from other organisms, in particular other Bacillus species may be screened.

For example, Bacillus strains may be screened for homologous polynucleotides by Northern blot analysis. Upon detection of transcripts homologous to polynucleotides according to the invention, DNA libraries may be constructed from RNA isolated from the appropriate strain, utilizing standard techniques well known to those of skill in the art. Alternatively, a total genomic DNA library may be screened using a probe hybridisable to a polynucleotide according to the invention.

A nucleic acid molecule of the present invention, such as for instance a nucleic acid molecule shown in SEQ ID NO: 1 or a fragment or derivative thereof, may be isolated using standard molecular biology techniques and the sequence information provided herein. For example, using all or portion of the nucleic acid sequence shown in SEQ ID NO:1 as a hybridization probe, nucleic acid molecules according to the invention may be isolated using standard hybridization and cloning techniques (e.g. , as described in Sambrook, J., Fritsh, E. F. , and Maniatis, T. Molecular Cloning : A Laboratory Manual. 2nd, ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

Furthermore, oligonucleotides corresponding to or hybridisable to nucleotide sequences according to the invention may be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

The terms "homology", "identically" or "percent identity" are used interchangeably herein. For the purpose of this invention, it is defined here that in order to determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g. , gaps may be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions/total number of positions (i.e., overlapping positions) x 100). Preferably, the two sequences are the same length.

The skilled person will be aware of the fact that several different computer programs are available to determine the homology between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences may be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. MoI. Biol. (48): 444-453 (1970) ) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.accelrys.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms. In yet another embodiment, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.accelrys.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1 , 2, 3, 4, 5 or 6. In another embodiment, the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4: 11-17 (1989) which has been incorporated into the ALIGN program (version 2.0) (available at http://vega.igh.cnrs.fr/bin/align-guess.cgi) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention may further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches may be performed using the BLASTN and BLASTX programs (version 2.0) of Altschul, et al. (1990) J. MoI. Biol. 215:403-10. BLAST nucleotide searches may be performed with the BLASTN program, score = 100, word length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the present invention. BLAST protein searches may be performed with the BLASTX program, score = 50, word length = 3 to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST maybe utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25 (17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) may be used. See http://www.ncbi.nim.nih.gov.

In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is the complement of a nucleotide sequence as of the present invention, such as for instance the sequence shown in SEQ ID NO:1. A nucleic acid molecule, which is complementary to a nucleotide sequence disclosed herein, is one that is sufficiently complementary to a nucleotide sequence shown in SEQ ID NO: 1 such that it may hybridize to said nucleotide sequence thereby forming a stable duplex.

hi a further embodiment, a nucleic acid of the invention as shown in SEQ ID NO:1 or the complement thereof contains at least one mutation leading to a gene product with modified function/activity. The at least one mutation may be introduced by methods known in the art or described herein. In regard to BbspOO25, the at least one mutation leads to a Bbsp0025 protein whose function compared to the wild type counterpart is enhanced or improved. The activity of the Bbsp0025 protein is thereby increased. Methods for introducing such mutations are well known in the art.

Cells with Bbsp0025 activity or optionally an increased activity are preferred, since such cells will support or enhance probiotic behavior.

The invention provides also an isolated polypeptide having the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence obtainable by expressing a polynucleotide of the present invention, such as for instance a polynucleotide sequence shown in SEQ ID NO: 1 in an appropriate host.

Functional equivalents of polypeptides as exemplified herein may be also part of the present invention and are defined on the basis of the amino acid sequences of the present invention by addition, insertion, deletion and/or substitution of one or more amino acid residues of such sequences. Amino acid exchanges in proteins and peptides which do not generally alter the activity of such molecules are known.

Polypeptides according to the invention may contain only conservative substitutions of one or more amino acids in the amino acid sequence represented by SEQ ID NO:2 or substitutions, insertions or deletions of non-essential amino acids. Accordingly, a non-essential amino acid is a residue that may be altered in the amino acid sequences shown in SEQ ID NO:2 without substantially altering the biological function. For example, amino acid residues that are conserved among the proteins of the present invention, are predicted to be particularly unamenable to alteration. Furthermore, amino acids conserved among the proteins according to the present invention and other Bbsp0025 proteins are not likely to be amenable to alteration.

The term "conservative substitution" is intended to mean that a substitution in which the amino acid residue is replaced with an amino acid residue having a similar side chain. These families are known in the art and include amino acids with basic side chains (e.g., lysine, arginine and histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar 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, histidine). This invention is further illustrated by the following examples which should not be construed as limiting. The contents of all references cited throughout this application are hereby incorporated by reference.

EXAMPLES

Hereinafter the identification in the genome of BSP 1 of a homolog of the bsmA gene is described, which promotes bacterial aggregation and biofϊlm formation in Serratia marcescens MGl and which has been shown to be involved in biotic surface adhesion (Labbate et al., J Bacterid. 2004 Feb;186(3):692-8; Labbate et al., 2007, J. Bacteriol. In press). Applicant has found in silico that this gene has homologs in other Bacilus species, like Bacillus cereus but also in other genuses like Gluconobacter sp. oxydans. Applications of the discovery of the bsmA gene/ protein are multiple. First, the protein encoded by bsmA, or the mechanism in which BsmA is involved, represents a target for molecular inhibition of the biofϊlm process. Biofilms generated by pathogens microorganisms are indeed of major concern in medicine. A second application possibility resides in screening for presence of homolog of the bsmA gene while selecting microbial strain with optimal probiotic or manipulating the copy number of the bsmA gene, as shown hereafter in a Bacillus subtilis BSPl recombinant strain. Better adhesion ability is of interest for creating better probiotic strains but also, through better biofϊlm formation, in a variety of industrial applications that range from reduction of corrosion to bioremediation. Finally, deletion of the bsmA gene is of importance in applications to reduce biofilm formation and adhesion ability, which is desirable not only in fermentation processes based on microorganisms harboring that gene, like Bacillus sp. or Gluconobacter oxydans, in bio-fouling, but also for medical applications where biofilms of potentially pathogens strains represent a major threat.

General Methodology

In silico procedure

In silico procedures like gene mining or comparative alignments were made by classical tools know from people skilled in the art. The alignment ClustalW software was available from http://www.ebi.ac.uk/clustalw/. Strains and plasmids:

Bacillus subtilis strains of the present invention are the undomesticated isolate BSPl and its derivatives. The genome of BSPl has been sequenced at DSM and is herein used to derive the sequences of all primers.

Media:

Standard liquid complete medium is Luria-Bertani broth, Miller (LB broth, MercK), while standard solid complete medium is Luria-Bertani agar, Miller (LB agar, Difco). Standard sporulation medium is Difco Sporulation medium (DSM, Difco). Medium for biofilm formation is MSgg minimal medium. The compositions of these media are described bellow:

- LB broth medium: 25g of LB broth (MercK cataloge # 1.10285), IL water. Autoclave.

- LB agar medium: 4Og of LB agar (Difco Catalog # 244510), IL water. Autoclave.

- DSM medium: 8g Bacto Nutrient Broth (Difco Catalog # 234000), Ig KCl , 0.25g MgSO₄, water to IL. Autoclave. Just prior to use add ImL of each following solutions: IM Ca(NO₃)₂, 0.01M MnCl₂, 0.001M FeSO₄.

- MSgg broth medium: 2.5 mM KH₂PO₄, 2.5mM K₂HPO₄, 10OmM MOPS, 2mM MgCl₂, 700 μM CaCl₂, 50μM MnCl₂, 1 μM ZnCl₂, 50μM FeCl₃, 2μM Thiamine, 0.5% glycerol, 0.5% glutamic acid, 50μg/ml tryptophan, 50 μg/ml phenylalanine and water to 1 L. Autoclave

- MSgg solid medium: 2.5 mM KH₂PO₄, 2.5mM K₂HPO₄, 10OmM MOPS, 2mM MgCl₂, 700 μM CaCl₂, 50μM MnCl₂, 1 μM ZnCl₂, 50μM FeCl₃, 2μM Thiamine, 0.5% glycerol, 0.5% glutamic acid, 50μg/ml tryptophan, 50 μg/ml phenylalanine, water to IL and 16g of Bactor agar (Difco Catalog # 214010). Autoclave

Molecular and senetic techniques:

Standard genetic and molecular biology techniques, including Polymerase Chain Reaction (PCR) procedures are generally know in the art and have been previously described. DNA extraction and other standard B. subtilis genetic techniques are also generally know in the art and have been described previously (Harwood and Cutting, 1992, Molecular Biological Methods for Bacillus, 1990, John Wiley & Sons, New York).

Biofilm assay:

From a fresh isolated colony each strain was inoculated in 10 ml LB and incubated at 37°C, 150 rpm. After 16h of growth, each strain was diluted 1 : 100 in 10 ml MSgg medium (6 well plates) and let grow without agitation at 30°C.

EXAMPLE 1

This example provides the deoxynucleotide and amino acid sequences of the Bacillus subtilis BSPl homolog of the bsmA gene (Tables 2 and 4). It also provides amino acid sequences of homolog proteins from Bacillus cereus, Gluconobacter oxydans and Serratia marcescens (Table 7, 5, 6, respectively). Dual alignments between the BSPl BsmA protein and each of these homologs are provided in Table 8, 9 and 10. A multiple alignment comprising BsmA from Bacillus subtilis BSP 1, Bacillus cereus, Serratia marcescens and Gluconobacter oxydans is presented on Table 11. In Serratia marcescens MGl, bsmA has been involved into biotic surface adhesion. In silico mining however classifies BsmA into oxido-reductases.

Table 2: DNA sequence of the bsmA gene identified in Bacilus subtilis BSPl

ATGACATCCGATATTACTAAATATGCTTCTTTTTTAAAGGAAAACTGCTATTCGTAT ATCCCAGCAGATTTCTACCGGCAAAAAACTACAGACGCCGCAGTCCGCAAATTACAG CTTACATATGATGCTTTGAAAGCTGATCCTAAAGGAGGCGGCCGTTACAGAGCGCAT TCCCGCTACATTTTGGCTCCTCACTCCGACACTCTTGAATTAGATCCGGATAATGGA TATTTCCAATCAAAAGAATATAATTATGATGATGGCGGTATCGTCAGACAATTTGAA AAAATATCAGATGAGTTTCTACAGCATCCTGTAACGCGGCACATGATACATTCAAAT GTAGAAATGGCCCGCCAAACCGATTTTGTAGATTGGGAGAAAGAAGTTATCGTCGGG CTTCACCAAGTCAGATATCAAGTGACACCTGACGCGCCTTCATACAGCTCACCCATT TGGCTGCATCGTGACGATGAACCGCTTGTTTTCGTCCATCTGTTTAAACTGAGTGAG GATGCCATCGGAGGAGACAACTTAATTGCTCCGTCTGTCAAGCAAATTGATAAAGTG ATACGGCTGACGGACCCGCTTGAAACACTTGCTCTCGGGCAAAAGGTCTTTCATGCG GTTACACCGGTCGGCACTGCCAATGCAGATGGCGCGCATCGAGATATATTATTAGTT ACAtTTTCTAATAG

Table 3: DNA sequence of the bsmA gene identified in Gluconobacter oxydans N441 ATGAACCCTACCGATCTCTCCCCGCCCTTTGACAGGGTTCTGACCGGCATCGAAGAT ACGCTGCTGGAAAAAGGGTTTTCCTTCCTGCAAGCCGACGTCACGCGCCCTCTGCTG GAGCATGAAGGCCTGACAGACTGGCAGGGATTTGCCGAGAGCTGGAACCATCTGGGT CTTGACCGCTACATGGCGGATGGCGGCCGCTACCGCCGCCGCCGTTATGCGACCTTC GCCGTCTCGGGCGACACCATCACCCGCAAGAAACACCAGCCCCATTACCAGAGCCGG GATTACAACCTGCTCAATGGCGGCATCGAGCGCTGGTTTCCGGCCGTAACGGACGCC ATCGCCACCCATCCCGCCATGACCGCCGCCATCCGCACCGTCAGCCGGATCGCGGAT GCCCTGACGCCGCCGAAGGAAAAACCCCCGGTCTGGCATGTCGAGGTGCATCAGTTC CGCATCGAGGCCCGTCCGGGCAATGAAGGCCACCCCACACCCGAGGGCCTGCATCGC GATGGCGTGGACTGGGTTCTCGTGCTGATGGTCGCCCGCCACAACGTCGAACAGGGC GTTACCACGATCCACGGCATGCGCAAGGACCCGCTCGGCTCCTTCACGCTCACCAAC CCGCTGGATGCCGCCATCGTGGACGACCACCGCGTCTATCATGGCGTCACGGCCGTC CAGCCCGTGGATCCGTCACAGCCGGCCTATCGGGACGTGCTGGTGGTGACGCTCCGT CACGAA

Table 4: Amino acid sequence of the BsmA protein from Bacillus subtilis strain BSPl

MMTSDITKYASFLKENCYSYIPADFYRQKTTDAAVRKLQLTYDALKADPKGGGRYRA HSRYILAPHSDTLELDPDNGYFQSKEYNYDDGGIVRQFEKISDEFLQHPVTRHMIHS NVEMARQTDFVDWEKEVIVGLHQVRYQVTPDAPSYSSPIWLHRDDEPLVFVHLFKLS EDAIGGDNLIAPSVKQIDKVIRLTDPLETLALGQKVFHAVTPVGTANADGAHRDILL VTFSN

Table 5: Amino acid sequence of the BsmA protein from Gluconobacter oxydans N441

MNPTDLSPPFDRVLTGIEDTLLEKGFSFLQADVTRPLLEHEGLTDWQGFAESWNHLG LDRYMADGGRYRRRRYATFAVSGDTITRKKHQPHYQSRDYNLLNGGIERWFPAVTDA IATHPAMTAAIRTVSRIADALTPPKEKPPVWHVEVHQFRIEARPGNEGHPTPEGLHR DGVDWVLVLMVARHNVEQGVTTIHGMRKDPLGSFTLTNPLDAAIVDDHRVYHGVTAV QPVDPSQPAYRDVLWTLRHE

Table 6: Amino acid sequence of the BsmA protein from Serratia marcescens MGl MLQKHENVISMTVLSPQATQQLMPSFSSLPHTQHADGKYRLRRYSWKYLNPNVAEV GPRTFMQSDQINHFQGNWRRFEPIDSAVLQSAGMAEMCNLFAESNELPEGAEIEIH QMRWAIEKDTQVAPEGIHQDGFDHIAMIAIHRHNIVGGEIMLYDDSHHDPFFKKAL ADGEAVLLADSKLWHNATPINAVSPAEEGHLDLFVLTARGEKA

Table 7: Amino acid sequence of the BsmA protein from Bacillus cereus ATCC 14579 / DSM 31 MEVFIMTFVLSKMNGFSIEEKVHEFESKGFLEISNEIFLQEEENHRLLTQAQLDYYN LEDDAYGECRARSYSRYIKYVDSPDYILDNSNDYFQSKEYNYDDGGKVRQFNSINDS FLCNPLIQNIVRFDTEFAFKTNIIDTSKDLIIGLHQVRYKATKERPSFSSPIWLHKD DEPWFLHLMNLSNTAIGGDNLIANSPREINQFISLKEPLETLVFGQKVFHAVTPLG TECSTEAFRDILLVTFSYKETK

Table 8: Clustal multiple sequence alignment of amino acid sequences of the BsmA proteins from Bacillus subtilis strain BSPl and Gluconobacter oxydans N441.

BS200 MMTSDITKYASFLKENCYSYIPADFYRQKTTDAAVRKLQ LTYDALKAD- 48 GOx MNPTDLSPPFDRVLTGIEDTLLEKGFSFLQADVTRPLLEHEGLTDWQGFAESWNHLGLDR 60

BS200 -PKGGGRYRAHSRYILAPHSDTLELDPDNGYFQSKEYNYDDGGIVRQFEKISDEFLQHPV 107 GOx YMADGGRYRRRRYATFAVSGDTITRKKHQPHYQSRDYNLLNGGIERWFPAVTDAIATHPA 120

+**** . . * **. . ..**..** .*** * * ..* . **

BS200 TRHMIHSNVEMAR-QTDFVDWEKEVIVGLHQVRYQVTPDAPSYSSPIWLHRDDEPLVFVH 166 GOx MTAAIRTVSRIADALTPPKEKPPVWHVEVHQFRIEARPGNEGHPTPEGLHRDGVDWVLVL 180

* .. . * * . * .** * . + . * ** ** * . *

BS200 LFKLSEDAIGGDNLIAPSVKQIDKVIRLTDPLETLALG-QKVFHAVTPVGTAN-ADGAHR 224 GOx MVAR-HNVEQGVTTIHGMRKDPLGSFTLTNPLDAAIVDDHRVYHGVTAVQPVDPSQPAYR 239

**.**.. . * . * **

BS200 DILLVTFSN- 233 GOx DVLWTLRHE 249

Table 9: Clustal multiple sequence alignment of amino acid sequences of the BsmA proteins from Bacillus subtilis strain BSPl and Serratia marcescens MGl

BS200 MMTSDITKYASFLKENCYSYIPADFYRQKTTDAAVRKLQLTYDALKADPKGGGRYRAHSR 60 SL MLQKHENVISMTVLS PQATQQLMPSFSSLPHTQHADGKYRLRRY 44 BS200 YILAPHSDTLELDPDNGYFQSKEYNYDDGGIVRQFEKISDEFLQHPVTRHMIHSNVEMAR 120

SL SWKYLNPNVAEVGPRTFMQSDQINHFQGNWRRFEPIDSAVLQSAGMAEMCNLFAESN- 103

BS200 QTDFVDWEKEVIVGLHQVRYQVTPDAPSYSSPIWLHRDDEPLVFVHLFKLSEDAIGGDNL 180 SL ELPEGAEIEIHQMR-WAIEKDTQVAPEGIHQDG--FDHIAMIAIHRHNIVGGEI 155

. *# . * * . . . . * . * . * _m * * _ . .

BS200 IAPSVKQIDKVIR--LTDPLETLALGQKVFHAVTPVGTAN-ADGAHRDILLVTFSN 233

SL MLYDDSHHDPFFKKALADGEAVLLADSKLWHNATPINAVSPAEEGHLDLFVLTARGEKA 214 Table 10: Clustal multiple sequence alignment of amino acid sequences of the BsmA proteins from Bacillus subtilis strain BSPl and Bacillus cereus strain ATCC 14579 / DSM 31.

BS200 MMTSDITKYASF-LKENCYSY IPADFYRQKT-TDAAVRKLQLTYDALKA 47 BC MEVFIMTFVLSKMNGFSIEEKVHEFESKGFLEISNEIFLQEEENHRLLTQAQLDYYNLED 60

BS200 DPKGGGRYRAHSRYILAPHSDTLELDPDNGYFQSKEYNYDDGGIVRQFEKISDEFLQHPV 107 BC DAYGECRARSYSRYIKYVDSPDYILDNSNDYFQSKEYNYDDGGKVRQFNSINDSFLCNPL 120 *_ * * *..**** * ** * ************* ****._*_*_** .*.

BS200 TRHMIHSNVEMARQTDFVDWEKEVIVGLHQVRYQVTPDAPSYSSPIWLHRDDEPLVFVHL 167 BC IQNIVRFDTEFAFKTNIIDTSKDLIIGLHQVRYKATKERPSFSSPIWLHKDDEPWFLHL 180

BS200 FKLSEDAIGGDNLIAPSVKQIDKVIRLTDPLETLALGQKVFHAVTPVGTANADGAHRDIL 227 BC MNLSNTAIGGDNLIANSPREINQFISLKEPLETLVFGQKVFHAVTPLGTECSTEAFRDIL 240

BS200 LVTFSN 233 BC LVTFSYKETK 250

Table 11 : Clustal multiple sequence alignment of amino acid sequences of the BsmA proteins from Bacillus subtilis strain BSPl, Serratia marcescens MGl, Gluconobacter oxydans N441 and Bacillus cereus ATCC 14579 / DSM 31.

GOx MNPTDLSPPFDRVLTGIEDTLLEKGFSFLQADVTRPLLEHEGLTDWQGFAESWNHLGLDR 60

SL MLQKHENVISMTVLSPQATQQLMPSFSSLPH 31

BS200 DPKGGGRYRAHSRYILAPHSDTLELDPDNGYFQSKEYNYDDGGIVRQFEKISDEFLQHPV 107

BC DAYGECRARSYSRYIKYVDSPDYILDNSNDYFQSKEYNYDDGGKVRQFNSINDSFLCNPL 120

GOx YMADGGRYRRRRYATFAVSGDTITRKKHQPHYQSRDYNLLNGGIERWFPAVTDAIATHPA 120

SL TQHADGKYRLRRYSWKYLNPNVAEVGPRTFMQSDQINHFQGNWRRFEPIDSAVLQSAG 91 . * ** . * .* * * .

BS200 TRHMIHSNVEMARQTD-FVDWEKEVIVGLHQVRYQVTPDAPSYSSPIWLHRDDEPLVFVH 166

BC IQNIVRFDTEFAFKTN-IIDTSKDLIIGLHQVRYKATKERPSFSSPIWLHKDDEPWFLH 179

GOx MTAAIRTVSRIADALTPPKEKPPVWHVEVHQFRIEARPGNEGHPTPEGLHRDGVDWVLVL 180

SL MAEMCNLFAESN ELPEGAEIEIHQMRWAIEKDT-QVAPEGIHQDGFDHIAMI 143 • • • _{** *} .* ._*._* .

BS200 LFKLSEDAIGGDNLIAPSVKQIDKVIRLTDPLETLALG-QKVFHAVTPVG-TANADGAHR 224

BC LMNLSNTAIGGDNLIANSPREINQFISLKEPLETLVFG-QKVFHAVTPLG-TECSTEAFR 237

GOx MVARHNVEQGVTTIHGMRKDPLGSFT-LTNPLDAAIVDDHRVYHGVTAVQPVDPSQPAYR 239 SL AIHRHNIVGGEIMLYDDSHHDP-FFKKALADGEAVLLADSKLWHNATPINAVSPAEEGHL 202

* , .. ...* * .

BS200 DILLVTFSN 233

BC DILLVTFSYKETK 250 GOx DVLWTLRHE 249

SL DLFVLTARGEKA- 214 EXAMPLE 2

This example describes the screening for the presence of the bsmA gene from BSPl among different Bacillus strains by PCR using the pair of primers listed in Table 12 (403+404, 405+406, 407+408). Although absent form the genomes of laboratory strains PY79 and 168, bsmA is present in different Bacillus species, including, but not limited to, strains of the B. cereus group. Wild strains tested are referred in Barbosa et al (2005).

Table 12. Primers used to screen for bsmA gene

SEQ

Name Nucleotide sequence (5' —> 3') H ID No: fDl CAACAGAGTTTGATCCTGGCTCAG 405 rDl GCTTAAGGAGGTGATCCAGCC 406 bsmA IF CGGGATCCCATGACATCCGATATTACTAAATATGC 407 bsmA 698R GGAATTCCTATTAGAAAATGTAACTAATAA 408 bsmA -55F GGAATTCCGTGTCTCAATTATCAACATGCC 409 bsmA +185R CGGGATCCCTTCATCATTTTACATTCTGCC 410

The results are shown in figure 1. (bsmA presence among Bacillus strains) Figure 1 shows the PCR analysis of different Bacillus strains (Table 2) using oligonucleotides that specifically amplify a region internal to the bsmA gene. As control for DNA quality, universal primers for the small subunit 16SrRNA where used. Expected PCR size: bsmA - 714 bp, 16S RNA - 1500 bp.

EXAMPLE 3

This example describes the effect of a bsmA disruption in biofilm formation of strain BSPl after 24h incubation at 37C in MSgg medium. To disrupt the bsmA gene two 300bp PCR fragments of bsmA, one at the 5' and the other at the 3' end respectively, were amplified from the chromosomal DNA of BSPl . Subsequently both fragments were annealed by Long Flanking PCR to the 5' and 3' ends of a chloramphenicol cassette, amplified together through Long Flanking PCR and cloned into TOPO™XL from Invitrogen, originating pSD23. This later was inserted into the genome of BSPl originating the bsmA::cat chloramphenicol resistant strain BSP 1-2. Figure 2 (Disruption of bsmA make BSPl biofilms less structured.) shows the effect of bsmA disruption on biofilm development by BSPl, i.e. significant loss of biofilm structure in the BSPl -2 derivative in comparison to the wild type BSPl strain. The biofilm development of both BSPl (Panel A and C) and its derivative BSP 1-2 with disrupted bsmA (Panel B and D) was studied in standing cultures of 1 OmI MSgg medium. After 24 h incubation at 37C, biofilms developed by both strains were photographed using a MultilmageTM Light Cabinet from Alpha Innotech Corporation (Panel A and B) and a Nikon CoolPix 995 at maximum amplification (Panel C and D).

EXAMPLE 4

This example describes the effect of inserting an extra copy of bsmA (at the nonessential amyE locus) on biofilm formation in strain BSPl. To insert the bsmA gene into BSPl amyE, a 1000 bp PCR fragment containing both the promoter and coding regions of bsmA was amplified from the chromosomal DNA of BSPl, using primers bsmA-55F and bsmA+185R (Table 12), which generated an EcoRI and a BamHI restriction sites at the 5 'and 3 ' ends respectively. The digested PCR fragment was inserted into pGD364 cut with same enzymes, yielding pBSPl . Following transformation of BSPl to chloramphenicol resistance (Cmr) with Pstl-linearized pBSPl, an AmyE- transformant was identified and named BSP 1-3. As illustrated in Figure 3 the introduction of an additional copy of bsmA into BSPl genome results in the development of more structured biofilms with significantly increased aerial formations or fruiting bodies. The biofilm development of both BSPl (Panel A and C) and its derivative containing an extra copy at the non-essential amyE locus (BSP 1-3, Panel B and D) was studied in standing cultures of 10ml MSgg medium After 24 h incubation at 37C, biofilms developed by both strains were photographed using a MultilmageTM Light Cabinet from Alpha Innotech Corporation (Panels A and B) and a Nikon CoolPix 995 at maximum amplification (Panel C and D).

REFERENCES

Branda, S. S., Gonzalez-Pastor, J. E., Ben-Yehuda, S., Losick, R. and Kolter, R. (2001) Fruiting body formation by Bacillus subtilis. Proc. Natl. Acad. Sci. USA 98: 11621-11626 Labbate, M., Queck, S.Y., Koh, K. S., Rice, S.A., Givsbov, M., and Kjelleberg, S. (2004) Quorum sensing-controlled biofilm development in Serratia liquefaciens MGl. J. Bacterid. 186: 692-698.

Tarn, N.K.M., Uyen, T.Q., Hong, H.A., Due, L.H., Hoa, T.T., Serra, C.R., Henriques, A.O. and Cutting, S. M. (2006) The intestinal life cycle of Bacillus subtilis and close relatives. J. Bacteriol. 188: 2692-2700.

Rhee, K-J., Sethupathi, P., Driks, A., Lanning, D.K. and Knight, K.L. (2004) Role of commensal bacteria in development of gut-associated lymphoid tissue and preimmune antibody repertoire. J. Immunol. 172 (2): 1118-1124.

Claims

1. Probiotic strain which genome comprises at least one polynucleotide encoding a protein which is involved into probiotic behavior and which polynucleotide is "substantially identical" to a polynucleotide sequence according to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO:81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 1 17, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO:129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, SEQ ID NO: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO:219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241 , SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251, SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO:281, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 291 , SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, SEQ ID NO: 303, SEQ ID NO: 305, SEQ ID NO: 307, SEQ ID NO: 309, SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 319, SEQ ID NO: 321, SEQ ID NO: 323, SEQ ID NO: 325, SEQ ID NO: 327, SEQ ID NO:329, SEQ ID NO: 331 , SEQ ID NO: 337, SEQ ID NO: 339, SEQ ID NO: 341 , SEQ ID NO: 343, SEQ ID NO: 345, SEQ ID NO: 347, SEQ ID NO: 349, SEQ ID NO: 351, SEQ ID NO: 353, SEQ ID NO: 355, SEQ ID NO: 357, SEQ ID NO: 359, SEQ ID NO: 361, SEQ ID NO: 363, SEQ ID NO: 365, SEQ ID NO: 367, SEQ ID NO: 369, SEQ ID NO: 371, SEQ ID NO: 373, SEQ ID NO: 375, SEQ ID NO: 377, SEQ ID NO: 379, SEQ ID NO:381, SEQ ID NO: 383, SEQ ID NO: 385, SEQ ID NO: 387, SEQ ID NO: 389, SEQ ID NO: 391, SEQ ID NO: 393, SEQ ID NO: 395, SEQ ID NO: 397, SEQ ID NO: 399, SEQ ID NO: 401 and SEQ ID NO: 403 respectively.

2. Probiotic strain according to claim 1 wherein the at least one polynucleotide encodes a protein which is attributed to one of the following subgroup of proteins: 1) proteins which show or support or maintain antimicrobial activity (herein abbreviated by AA) or

2) proteins which stimulate the immune system (herein abbreviated by IS) or

3) proteins which play a role in competitive exclusion mechanism (herein abbreviated by CE) or 4) proteins which convert genotoxic compounds in unreactive product (herein abbreviated by CGC).

3. Probiotic strain according to claim 1 or 2, which genome comprises at least three, preferably at least five polynucleotides each encoding a protein which is involved into probiotic behavior.

4. Probiotic strain according to claim 3, which genome comprises at least ten, polynucleotides each encoding a protein which is involved into probiotic behavior.

5. Probiotic strain according to any of claims 1 to 4, wherein the expression of "a polynucleotide which is substantially identical" refers with respect to the encoding sequences to a polynucleotide sequence selected from the group consisting of: a) polynucleotides comprising the nucleotide sequence according to any SEQ ID NO listed in column 3 of table 1; b) polynucleotides comprising a nucleotide sequence encoding a fragment or derivative of a polypeptide comprising the amino acid sequence according to any SEQ ID NO listed in column 4 of table 1 or encoding a fragment or derivative of a polypeptide encoded by a polynucleotide of any of (a) wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said fragment or derivative has the activity of a protein involved into probiotic behavior; c) polynucleotides the complementary strand of which hybridizes under stringent conditions to a polynucleotide encoding a polypeptide comprising the amino acid sequence of any of (b) or to a polynucleotide as defined in any one of (a) and which encode a protein involved into probiotic behavior; and d) polynucleotides which are at least 70%, such as 85, 90 or 95% homologous to a polynucleotide encoding a polypeptide comprising the amino acid sequence according to any of (b) or to a polynucleotide as defined in any one of (a) to (c) and which encode a protein involved into probiotic behavior; or the complementary strand of such a polynucleotide.

6. Probiotic strain according to any of claims 1 to 5, which is a procaryotic cell selected from the group consisting of hereinabove include strains from the genera of Bacillus, Lactobacillus, Sporolactobacillus, Pediococcus and Propionibacterium.

7. A polynucleotide sequence, which codes for a protein involved into probiotic behavior and selected from the group comprising

1. proteins which show or support or maintain antimicrobial activity (herein abbreviated by AA) or

2. proteins which stimulate the immune system (herein abbreviated by IS) or 3. proteins which play a role in competitive exclusion mechanism (herein abbreviated by CE) or

4. proteins which convert genotoxic compounds in unreactive products (herein abbreviated by CGC); and which polynucleotide is "substantially identical" to a polynucleotide sequence according to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 1 1 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21 , SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41 , SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO:81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 161, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID N0:181, SEQ ID N0: 183, SEQ ID NO: 185, SEQ ID NO: 187, SEQ ID NO: 189, SEQ ID NO: 191, SEQ ID NO: 193, SEQ ID NO: 195, SEQ ID NO: 197, SEQ ID NO: 199, SEQ ID NO: 201, SEQ ID NO: 203, SEQ ID NO: 205, SEQ ID NO: 207, SEQ ID NO: 209, SEQ ID NO: 211, SEQ ID NO: 213, SEQ ID NO: 215, SEQ ID NO: 217, SEQ ID NO:219, SEQ ID NO: 221, SEQ ID NO: 223, SEQ ID NO: 225, SEQ ID NO: 227, SEQ ID NO: 229, SEQ ID NO: 231, SEQ ID NO: 233, SEQ ID NO: 235, SEQ ID NO: 237, SEQ ID NO: 239, SEQ ID NO: 241, SEQ ID NO: 243, SEQ ID NO: 245, SEQ ID NO: 247, SEQ ID NO: 249, SEQ ID NO: 251 , SEQ ID NO: 253, SEQ ID NO: 255, SEQ ID NO: 257, SEQ ID NO: 259, SEQ ID NO: 261, SEQ ID NO: 263, SEQ ID NO: 265, SEQ ID NO: 267, SEQ ID NO: 269, SEQ ID NO: 271, SEQ ID NO: 273, SEQ ID NO: 275, SEQ ID NO: 277, SEQ ID NO: 279, SEQ ID NO:281, SEQ ID NO: 283, SEQ ID NO: 285, SEQ ID NO: 287, SEQ ID NO: 289, SEQ ID NO: 291, SEQ ID NO: 293, SEQ ID NO: 295, SEQ ID NO: 297, SEQ ID NO: 299, SEQ ID NO: 301, SEQ ID NO: 303, SEQ ID NO: 305, SEQ ID NO: 307, SEQ ID NO: 309, SEQ ID NO: 311, SEQ ID NO: 313, SEQ ID NO: 315, SEQ ID NO: 317, SEQ ID NO: 319, SEQ ID NO: 321, SEQ ID NO: 323, SEQ ID NO: 325, SEQ ID NO: 327, SEQ ID NO:329, SEQ ID NO: 331, SEQ ID NO: 337, SEQ ID NO: 339, SEQ ID NO: 341, SEQ ID NO: 343, SEQ ID NO: 345, SEQ ID NO: 347, SEQ ID NO: 349, SEQ ID NO: 351, SEQ ID NO: 353, SEQ ID NO: 355, SEQ ID NO: 357, SEQ ID NO: 359, SEQ ID NO: 361, SEQ ID NO: 363, SEQ ID NO: 365, SEQ ID NO: 367, SEQ ID NO: 369, SEQ ID NO: 371, SEQ ID NO: 373, SEQ ID NO: 375, SEQ ID NO: 377, SEQ ID NO: 379, SEQ ID NO:381, SEQ ID NO: 383, SEQ ID NO: 385, SEQ ID NO: 387, SEQ ID NO: 389, SEQ ID NO: 391, SEQ ID NO: 393, SEQ ID NO: 395, SEQ ID NO: 397, SEQ ID NO: 399, SEQ ID NO: 401 and SEQ ID NO: 403 respectively.

8. An isolated DNA sequence according to claim 6, wherein the expression of "a polynucleotide which is substantially identical" refers with respect to the encoding sequence to a polynucleotide sequence selected from the group consisting of: a) polynucleotides comprising the nucleotide sequence according to any SEQ ID NO listed in column 3 of table 1; b) polynucleotides comprising a nucleotide sequence encoding a fragment or derivative of a polypeptide comprising the amino acid sequence according to any SEQ ID NO listed in column 4 of table 1 or encoding a fragment or derivative of a polypeptide encoded by a polynucleotide of any of (a) wherein in said derivative one or more amino acid residues are conservatively substituted compared to said polypeptide, and said fragment or derivative has the activity of a protein involved into probiotic behavior; c) polynucleotides the complementary strand of which hybridizes under stringent conditions to a polynucleotide encoding a polypeptide comprising the amino acid sequence of any of (b) or to a polynucleotide as defined in any one of (a) and which encode a protein involved into probiotic behavior; and d) polynucleotides which are at least 70%, such as 85, 90 or 95% homologous to a polynucleotide encoding a polypeptide comprising the amino acid sequence according to any of (b) or to a polynucleotide as defined in any one of (a) to (c) and which encode a protein involved into probiotic behavior; or the complementary strand of such a polynucleotide.

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