FR2809403A1 - New bacteriocin, sakacine G, useful for controlling microbes, particularly Listeria monocytogenes, in foods, is derived from Lactobacillus sakei 2512 - Google Patents

Abstract

Isolated polypeptide (I), designated sakacine G, is a bacteriocin from Lactobacillus sakei 2512. Independent claims are also included for the following: (1) nucleic acid (II) that encodes (I); (2) cloning and/or expression vector containing (II); (3) host cell transformed with the vector of (2); (4) preparation of recombinant (I) by culturing cells of (3); and (5) composition comprising at least one (I) or L. sakei 2512.

Description

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 The present invention relates to a bacteriocin of Lactobacillus sakei and more particularly of Lactobacillus sakei 2512, a nucleotide sequence coding for this bacteriocin, and the industrial use of this bacteriocin as an active agent against pathogenic or undesirable flora in the preparation of food products.

 Lactic acid bacteria are used extensively in food fermentations to not only improve the flavor and texture of foods but especially to extend their shelf life. Many lactic acid bacteria have the ability to inhibit the growth of certain gram-positive bacteria, including pathogenic strains such as Listeria monocytogenes, through the excretion of antagonistic molecules, including peptide compounds. These peptide compounds, called bacteriocins, therefore have an interesting potential for the qualitative and sanitary preservation of fermented food products.

 As a representative of these bacteriocins, there may be mentioned those forming the subclass of polypeptides called bacteriocins anti-Listeria, class IIa bacteriocins (Ennahar S. et al., 2000, FEMS Microbiol., Rev., 24: 85-106 ) and cystibiotics (Jack R. et al., 1995, Microbiol.Rev., 59 (2): 171-200). The potential use of one of these class IIa bacteriocins, divercin V41, has recently been reported to prevent the growth of Listeria monocytogenes in smoked salmon (Duffes, F. et al., 1999, J. Food Prot. ., 62 (12): 1394-1403).

 The sequences of these polypeptides have strong similarities in their N-terminal part, with in particular the presence of a disulfide bridge. The hydrophobic C-terminal part is much more variable, but some of these bacteriocins, known as pediocin (pediocin PA-1, enterocin A and divercin V41), are characterized by a size greater than 40 residues and the presence of a second disulfide bridge on the C-terminal side.

 The present inventors have demonstrated a new class IIa bacteriocin produced from a specific strain of

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 Lactobacillus sakei, which is particularly effective in inhibiting the growth of Listeria, especially Listeria monocytogenes.

 In agreement with Tagg J.R. et al. , Bacteriol. Rev. 40; (1976), the term "Bacteriocin" in the sense of the invention refers to a polypeptide produced, by ribosomal synthesis, from microorganisms, capable of specifically inhibiting the growth of other bacteria.

 The subject of the present invention is therefore a polypeptide derived from the strain Lactobacillus sakei 2512, with a bacteriocin activity.

 The strain Lactobacillus sakei 2512 was deposited on May 25, 2000 with the National Collection of Microorganism Cultures where it is registered under the deposit number I - 2479.

 The bacteriocin which is the subject of the present invention has been called Sakacin G. It is a polypeptide having a molecular mass of about 3700 to 3900 and preferably about 3834 Da determined by mass spectrometry. It has a bacterial inhibition spectrum very similar to that of class IIa bacteriocins. It is thus particularly effective against strains of Lactobacillus sakei other than Lactobacillus sakei 2512, Pediococcus cerevisiae, all Listeria strains and against Enterococcus faecalis and durans. On the other hand, it is inactive against other species of Lactobacillus such as Lactobacillus debrueckii, Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus casei, and a strain of Enterococcus faecium.

 Like the bacteriocins anti-Listeria pediocin type, Sakacin G has in its peptide structure advantageously two disulfide bridges.

 An analysis of the genetic determinants of several class IIa bacteriocins has shown that the genes involved in their production, transport and immunity are organized into one or more operon-like structures. These operons have an often plasmid localization and generally have at least two genes encoding proteins, homologous to an ABC-transporter

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 and an accessory protein, probably involved in the export of bacteriocins.

 Cloning of the nucleotide fragment (SEQ ID No. 9) containing the Sakacin G gene revealed the existence of two complete open reading frames skgAl (SEQ ID No. 1) and skgA2 (SEQ ID No. 3) and two other truncated skgl (SEQ ID No. 5) and skgD (SEQ ID No. 7) located at both ends of the cloned fragment, a schematic representation of which is presented in FIG.

 The products of the skgAl and skgA2 genes, termed pre-bacteriocins, can be processed in which their respective leader peptides are cleaved between residues 18 and 19, thereby releasing active Sakacin G (residues 19-55).

 The present invention therefore also relates to an isolated polypeptide corresponding to a bacteriocin, characterized in that it comprises the sequence ID N 2 and / or the sequence ID N 4. The sequence of the mature bacteriocin comprises the sequence ID N 12.

 The reading frame called skgl encodes a protein of 54 residues. The comparison of this sequence with that of the data banks shows strong similarities of Skgl with so-called immunity proteins. It probably encodes the immunity protein protecting the Sakacin G-producing bacterium.

 The present invention also extends to an isolated polypeptide comprising the ID 6 sequence corresponding to the skgI reading frame.

 Regarding the last gene skgD, it encodes a portion of protein that has homology with proteins of the ABC transporter family, and more particularly of the pediocin PA-1 transporter. The skgD gene presumably codes for the specific ABC-transporter of Sakacin G.

 The present invention also extends to the isolated polypeptide comprising the sequence ID N 8 corresponding to this gene called skgD.

 It is understood that the homologous sequences, defined as

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 i) sequences similar to at least 70% of the sequence SEQ ID N 2, N 4, N 6, N 8 or N 12; or ii) the sequences encoded by a homologous nucleic acid sequence as defined below, that is to say a nucleic acid sequence hybridizing with the sequence SEQ ID N 1, N 3, N 5, N 7 or N 9 or its complementary sequence, under stringent conditions of hybridization.

 Again, the term "similar" refers to the perfect similarity or identity between the amino acids of the homologous sequences compared but also to the non-perfect resemblance that is termed similarity. This search for similarities in a polypeptide sequence takes into account conservative substitutions that are amino acid substitutions of the same class, such as amino acid substitutions to uncharged side chains (such as asparagine, glutamine, serine, threonine, and tyrosine), amino acids with basic side chains (such as lysine, arginine, and histidine), amino acids with acidic side chains (such as aspartic acid and glutamic acid); from amino acids to apolar side chains (such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine).

 More generally, the term "homologous amino acid sequence" therefore means any amino acid sequence which differs from the sequence SEQ ID N 2, N 4, N 6, N 8 or N 12 by substitution, deletion and / or insertion of an amino acid or a reduced number of amino acids, in particular by substitution of natural amino acids by non-natural amino acids or pseudo-amino acids at positions such that these modifications do not significantly affect the Biological activity of isolated polypeptide and preferably Sakacin G.

 Preferably, such a homologous amino acid sequence is similar to at least 85% of the SEQ ID N 2, N 4, N 6, N 8 or N 12 sequence, preferably at least 95%.

 Homology is generally determined using sequence analysis software (eg, Sequence Analysis Software Package of the

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 Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Similar amino acid sequences are aligned to obtain the maximum degree of homology (i.e. identity or similarity, as defined above). For this purpose, it may be necessary to artificially introduce gaps in the sequence. Once the optimal alignment is achieved, the degree of homology is established by recording all the positions for which the amino acids of the two compared sequences are identical, relative to the total number of positions.

 The biological activity of the isolated polypeptide and especially Sakacin G refers to its ability to inhibit the growth of undesirable and / or pathogenic bacterial strains, preferably Listeria bacteria and more particularly Listeria monocytogenes bacteria.

 The present invention also relates to an isolated nucleic acid encoding a polypeptide as defined above.

 More specifically, the subject of the present invention is an isolated nucleic acid comprising the sequence ID N 1 and / or the sequence ID N 3.

 The complete nucleotide sequence of the region involved in the expression of Sakacin G (2042 bp) has been determined, and is represented in sequence ID N 9. The present invention also relates to a nucleic acid comprising such a sequence.

 As previously described, this sequence has two complete open reading frames skgAl and skgA2 and two other truncated skgl and skgD located at both ends of the cloned fragment. The supposed genes skgAl (SEQ ID N 1), skgA2 (SEQ ID N 3) and skgl (SEQ ID N 5) are oriented in opposite directions with respect to skgD (SEQ ID N 7).

 Also claimed in the context of the present invention, the nucleic acid comprising the sequence ID N 5 as well as the nucleic acid comprising the sequence ID N 7.

 It is understood that homologous sequences, defined as:

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 i) sequences similar to at least 70% of the sequence SEQ ID N1, N3, N5, N7 or N9; or ii) sequences hybridizing with the sequence SEQ ID N1, N3, N5, N7 or N9 or their complementary sequence, under stringent conditions of hybridization, or iii) sequences encoding the polypeptide designated Sakacin G as previously defined.

 Preferably, a homologous nucleotide sequence according to the invention is similar to at least 75% of the sequences SEQ ID N 1, N 3, N 5, N 7 or N 9, more preferably at least 85%, or at least 90% .

 Preferably, such a homologous nucleotide sequence hybridizes specifically to the sequences complementary to the sequence SEQ ID No. 1, N 3, N 5, N 7 or N 9 under stringent conditions. The parameters defining the stringency conditions depend on the temperature at which 50% of the paired strands separate (Tm).

For sequences comprising more than 30 bases, Tm is defined by the relationship (Sambrook et al., 1989, NY: Cold Spring Harbor Laboratory):
Tm = 81.5 + 0.41 (% G + C) + 16.6 Log (cation concentration) - 0.63 (% formamide) - (600 / number of bases)
For sequences shorter than 30 bases, Tm is defined by the relation: Tm = 4 (G + C) 2 (A + T).

 Under appropriate stringency conditions, at which the aspecific sequences do not hybridize, the hybridization temperature may preferably be from 5 to 10 C below Tm, and the hybridization buffers used are preferably ionic strength solutions. such as a 6xSSC solution for example.

 The term "similar sequences" used above refers to the perfect similarity or identity between the compared nucleotides but also to the non-perfect similarity that is described as similarity. This search for similarities in the nucleic sequences distinguishes, for example, purines and

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 pyrimidine.

 A nucleotide sequence homologous to the open reading frames represented in SEQ ID N1, N3, N5, N7 or N9 therefore includes any nucleotide sequence which differs from the sequence SEQ ID N1, N3, N5, N7. or N 9 by mutation, insertion, deletion or substitution of one or more bases, or by degeneracy of the genetic code, provided that it encodes a polypeptide having the biological activity of Sakacin G, as defined below .

 Among such homologous sequences are the sequences of the genes of bacteria other than Lactobacillus, coding for Sakacin G.

 The polypeptides of the present invention can be synthesized by any of the methods well known to those skilled in the art. The polypeptides of the invention may for example be synthesized by synthetic chemistry techniques, such as Merrifield-type synthesis which is advantageous for reasons of purity, antigenic specificity, absence of undesired side products and for its ease of production.

 The present invention also relates to a process for producing a recombinant polypeptide in which a vector comprising a nucleic acid according to the present invention is transferred into a host cell which is cultured under conditions allowing the expression of a polypeptide according to the present invention or a polypeptide encoded by a nucleic acid sequence according to the present invention.

 The recombinant bacteriocin can also be produced by a method, in which a vector containing a nucleic acid comprising a nucleotide sequence in accordance with the invention and preferably the sequences SEQ ID N 1 and / or N 3 where a homologous sequence is transferred into a host cell which is cultured under conditions permitting the expression of the corresponding polypeptide. The protein produced can then be recovered and purified. The purification methods used are known to those skilled in the art.

The recombinant polypeptide obtained can be purified from lysates and cell extracts, from the supernatant of the culture medium, by methods used individually or in combination, such as fractionation, methods of

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 chromatography, immunoaffinity techniques using specific mono- or polyclonal antibodies, etc.

 The nucleic acid sequence of interest, encoding Sakacin G, may be inserted into an expression vector, in which it is operably linked to elements allowing the regulation of its expression, such as, in particular, promoters. activators and / or transcription terminators. The signals controlling the expression of the nucleotide sequences (promoters, activators, termination sequences, etc.) are chosen as a function of the cellular host used. For this purpose, the nucleotide sequences according to the invention may be inserted into autonomously replicating vectors within the chosen host, or integrative vectors of the chosen host. Such vectors will be prepared according to methods commonly used by those skilled in the art, and the resulting clones can be introduced into a suitable host by standard methods, such as for example electroporation or calcium phosphate precipitation.

 The cloning and / or expression vectors as described above, containing a nucleotide sequence defined according to the invention also form part of the present invention.

 The invention is further directed to host cells transiently or stably transformed by these expression vectors. These cells can be obtained by introducing into a host cell, preferably procaryotic, a nucleotide sequence inserted into a vector as defined above, then culturing said cells under conditions allowing replication and / or the expression of the transferred nucleotide sequence.

 Examples of host cells include such bacteria as Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, Pediococcus, Escherichia and yeasts.

 The nucleotide sequences of the invention may be of artificial origin or not. It can be DNA or RNA sequences obtained by

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 screening of libraries of sequences using probes prepared on the basis of SEQ ID No. 1, N 3, N 5 and / or N 7 or N 9 sequences. Such libraries can be prepared by known conventional molecular biology techniques. of the man of the art.

 The nucleotide sequences according to the invention can also be prepared by chemical synthesis, or by mixed methods including the chemical or enzymatic modification of sequences obtained by screening the libraries.

 The present invention also relates to a method for inhibiting the growth of Listeria, more particularly Listeria monocytogenes in an environment which may or may not be food and which is susceptible to contamination with Listeria monocytogenes.

 Listeria monocytogenes are pathogenic microorganisms that cause severe diseases in humans and animals, and which can be easily transmitted by contaminated food, especially meat, meat products, marine products, food and feed. milk and derived products. The present invention therefore proposes a method for inhibiting the growth of Listeria monocytogenes in a food likely to contain Listeria monocytogenes as a contaminant, said process comprising the addition of a polypeptide according to the invention in said food in a sufficient quantity. to inhibit the growth of Listeria monocytogenes.

 The bacteriocins according to the invention are preferably used in any food system in an amount of between 1 and 100,000 arbitrary units (AU) of bacteriocins per gram of food.

 A bacteriocin AU is defined as 5 μl of the highest dilution of the culture supernatant leading to a defined growth inhibition zone relative to a control strain of a gram-positive bacterium on agar medium.

 Although foods are most affected by Listeria monocytogenes contamination, veterinary and medical products can also

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 be contaminated with this type of bacteria, as well as cosmetics or related products.

 The bacteriocins according to the present invention, and in particular Sakacin G, are therefore also useful for inhibiting the growth of this type of pathogen in these products.

 The subject of the present invention is therefore the use of a bacteriocin according to the present invention as an active agent against pathogenic or undesirable flora especially in the preparation of food products and more specifically to inhibit the growth and propagation of Listeria, more particularly of Listeria monocytogenes, in food products.

 The polypeptide may be incorporated as such in the food product under consideration or may be produced therefrom from the strain Lactobacillus Sakei 2512.

 The present invention thus also relates to the use of the strain Lactobacillus Sakei 2512 in a food product to generate a bacteriocin polypeptide according to the invention.

 The invention also relates to a bacteriocin composition, characterized in that it comprises at least one polypeptide according to the present invention, that is to say derived from the strain Lactobacillus Sakei 2512 or comprising the sequence SEQ ID N 2, or N 4, or N 12 or the strain Lactobacillus Sakei 2512.

 The invention also extends to the use of the strain Lactobacillus sakei 2512 intended to produce a polypeptide as defined above, for inhibiting the growth and propagation of Listeria, more particularly of Listeria monocytogenes, in food products as well as compositions comprising such strain.

 The examples and the figure below are presented as non-limiting illustrations of the object of the present invention.

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 FIGURE: Figure 1: Schematic of the genetic locus involved in the production of sakacin G.

 MATERIALS AND METHODS # Bacterial strains and culture media. Lactobacillus sakei 2512 is cultured at 30 ° C. in MRS medium (DIFCO Laboratories) sterilized for 12 min at 110 ° C. The indicator strains are cultured in BIF ("brain-heart infusion", DIFCO Laboratories) medium at 37 ° C.

 # Activity test. BHI medium, agar at 10 g / l, is inoculated at 1% by a preculture of indicator strain in stationary phase before being poured into a Petri dish. Fifty microliters of sakacin G solution are deposited in wells drilled in the die-cut agar. The bacteriocin activity results in the appearance of zones of inhibition around the wells after incubation overnight at 37 C.

 # Protein analysis. The sakacin G is analyzed by mass spectrometry on a Perkin-Elmer Sciex API 165 instrument equipped with an Ionspray ionization source. After lyophilization, the active HPLC fraction is taken up with an acetonitrile / water solution (1: 1) containing 0.1% of formic acid and then injected by infusion at a flow rate of 5 l / min.

 The protein concentration is determined by the bicinchoninic acid method using the BCA kit (Sigma) according to the manufacturer's instructions.

 Protein sequence comparisons are made using the BLAST (1) program, accessible from the ExPASy server of the "Swiss Institute of Bioinformatics".

 # Molecular cloning and transformation. The plasmids are extracted and purified from Escherichia strains. coli and Lactobacillus sakei 2512 according to the methods previously described by Sambrook et al., 1989, NY:

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 Spring Harbor Laboratory and Muriana and Klaenhammer, 1987, Appl. About.

Microbiol., 53: 553-560 respectively.

 The restriction and modification enzymes of the DNA are used according to the indications of the supplier (Gibco-BRL). The agarose gel electrophoreses, analytical and preparative, are conducted in Tris / borate / EDTA buffer (pH 8.3) according to the methods described by Sambrook et al., 1989, NY: Spring Harbor Laboratory. The digested DNA fragments are purified from the agarose gels using the "Prep-a-Gene" kit (Bio-Rad). Cloning in the plasmids pGEM-T (Promega) and pZER02 (Invitrogen) are performed according to the recommendations of the suppliers. Southern blotting is performed on nylon membrane (Hybond-N +, Amersham) according to Sambrook et al., 1989, NY: Cold Spring Harbor Laboratory. The blot is followed by hybridization with a 32P-labeled radioactive probe using the random primers DNA labeling system kit (Gibco-BRL). E. coli bacteria are made competent and transformed according to the method of Hanahan, 1983. J. Mol. Biol.

 166: 557-80.

 Taq polymerase (Gibco-BRL) is used according to the supplier's recommendations. The amplification of the DNA fragment encoding Sakacin G was carried out using a "Geneamp 9700" apparatus (Perkin-Elmer) according to the following conditions: 35 denaturation cycles at 94 ° C. for 30 s, hybridization at 45 C for 30 s and elongation at 72 C for 1 min followed by an additional cycle of elongation at 72 C for 5 min.

 The DNA fragment carrying the sakacin G locus is sequenced using an ABI Prism 310 # automatic sequencer (Perkin-Elmer) using the "Big-dye terminator" sequencing kit (Perkin-Elmer) and the primers nucleotides.

EXAMPLE 1
Isolation and purification of Sakacin G.

 A 16-hour culture of Lactobacillus. sakei 2512 (100 ml) is centrifuged at 6000 g for 15 min. The culture supernatant is then heated to 70 ° C.

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 for 20 minutes. The cooled supernatant is then diluted with 1 volume of water (the pH of the diluted solution must be less than 6, by addition of 1M HCl if necessary) before being passed over a column (2.5 x 18 cm). containing a cation exchange resin (carboxymethylcellulose, Cellufine C-200, Amicon) equilibrated with water. After successive washings with water (100 ml) and then 0.1M NaCl solution (150 ml), Sakacin G is eluted with 0.5M NaCl solution (200 ml). The pH of all the solutions must be less than 6. The active fraction is then deposited on a solid phase extraction cartridge (Seppak plus CI 8, Waters) equilibrated in water. After successive washings with 5 ml of 20 mM ammonium acetate solutions containing 0.10, 20 and 30% of acetonitrile, Sakacin G is eluted with 10 ml of 20 mM ammonium acetate containing 80% of acetonitrile. After lyophilization, the extract is solubilized in 1 ml of 40% aqueous acetonitrile solution and then injected onto a C8 reverse phase analytical HPLC column (Kromasil, 5 m, 100 Å, 4.6 × 250 mm, A.I.T.). HPLC was performed on an apparatus comprising a Perkin-Elmer series 200 LC pump connected to a Perkin-Elmer 785A detector. The absorption chromatogram is recorded at 220 nm. The separation is carried out at a flow rate of 0.8 ml / min according to the following gradient: Solvent A = water / trifluoroacetic acid 0.1%; solvent B = acetonitrile / water / trifluoroacetic acid 0.07%. After washing for 5 min with 20% of solvent B, the elution is carried out by a gradient of 40% of solvent B in 10 min and then 40 to 55% of solvent B in 20 min.

 The fraction corresponding to the peak at 23 min which proved to be active against Listeria ivanovii BUG 496 was analyzed by ion-ion mass spectrometry. The molecule appears at least 95% pure and has a molecular weight of 3834.32 0.31 Da. The amount of Sakacin G thus purified was estimated at 120 μg from 100 ml of culture. The purification yield was estimated at 55% of activity found. Part of the Sakacin G primary sequence was determined by microsequencing and two degenerate oligonucleotides were derived from this sequence.

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EXAMPLE 2
Cloning of the genetic locus involved in the production of sakacin G.

 By reverse genetics, the two degenerate oligonucleotides SakGOl (5 'AARTATTATGGNAAYGGNGT 3') (SEQ ID N 10) and SakG02 (5 'ACATGATGNCCNCCRTTNGC 3') (SEQ ID NO: 11) were chosen to amplify the corresponding DNA fragment. to the structural gene of mature Sakacin G (SEQ ID No. 12) by polymerase chain reaction (PCR).

The amplification thus obtained, of an approximate size of 100 bp was cloned into the plasmid pGEM-T to form the plasmid pJMBYCOl. The 560 bp Pvull restriction fragment from pJMBYC01, including the inserted fragment, served as a hybridization probe, during a Southern blot, to locate the structural gene on the Lactobacillus sakei 2512 genome. of a plasmid extract of Lactobacillus sakei 2512 digested with restriction enzymes HindIII and EcoRI, the probe revealed fragments of respective sizes of about 2.1 and 9 kbp. The 2.1 kbp HindIII fragment was purified and inserted into the vector pZER02 to give the plasmid pJMBYCO2. The presence of the Sakacin G structural gene in pJMBYC02 was demonstrated by PCR amplification with primers SakGO1 and SakG02 and then by nucleotide sequencing of the fragment inserted into pJMBYCO2.

 The complete nucleotide sequence (2042 bp) of the HindIII restriction fragment inserted in pJMBYCO2 was determined. Analysis of this sequence revealed the existence of two complete open reading frames skgAl and skgA2 and two other truncated skgI and skgD located at both ends of the cloned fragment.

The supposed genes skgAl skgA2 and skgI are oriented in opposite directions with respect to skgD.

 Each of the open reading frames is preceded by a potential ribosome binding site. The skgAl and skgA2 genes both encode proteins of 55 amino acid residues whose 19-55 sequences are completely identical. The sequence 19-52 corresponds to the sequence of sakacin G obtained

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 by microsequencing. The presumed presence of 3 cysteine residues at positions 9, 14 and 24 is confirmed and three additional residues of glycine, valine and cysteine appear at the C-terminus.

 The 1-18 sequences of the SkgA1 and SkgA2 proteins differ only by 3 residues and show strong similarities with the "leader" peptides of class II bacteriocins, which are involved in the transport of these peptides by specific ABC-transporters. In particular, the terminal GG motif (17-18) is characteristic of these leader sequences and constitutes the site of maturation of these bacteriocins. Comparison of the nucleotide sequences of the skgAl and skgA2 genes also shows a sequence identity of greater than 95% for the part of the genes encoding mature bacteriocin. The incomplete open reading frame called skgl encodes a 54-residue protein. The comparison of this sequence with those of the data banks shows strong homologies of SkgI with the so-called immunity proteins LccI and MesI. The involvement of Mesl in protection against mesentericin Y105 has been demonstrated. It can be assumed that skgI encodes the sakacin G immunity protein.

 The last skgD gene encodes a protein portion of 394 amino acids.

According to the databases, SkgD is highly homologous to proteins of the ABC-transporter family and more particularly of PA-1: PedD or PapD transporters (Marugg J.D. et al., 1992, Appl.

Microbiol. 58 (8): 2360-7; Motlagh A. et al., 1994, Lett. Appl. Microbiol.

18 (6): 305-12), sakacin P: (Huhne K. et al., 1996, Microbiology.

142 (Pt6): 1437-48), sakacin A: (Axelsson L. et al., 1995, J.

Bacteriol. 117 (8): 2125-37) and mesentericin Y105: MesD (Fremaux, C. et al., 1995, Microbiology, 141 (Pt 7): 1637-45).

EXAMPLE 3
Inhibition spectrum.

 The sensitivity to Sakacin G of 17 bacterial strains was tested by the well test method (cfMaterials and Methods). The results are shown in Table 1 below:

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TABLE 1

<Tb>
<tb> Radius <SEP> of the <SEP> halos
<tb> inhibition <SEP> (mm)
<tb> The. <SEP> lactis <SEP> ATCC11454 <SEP> 0
<tb> Ln. <SEP> Paramesenteroides <SEP> DSM <SEP> 20288 <SEP> 0
<tb> Ln. <SEP> Mesenteroides <SEP> DSM <SEP> 20484 <SEP> 0
<tb> Ln. <SEP> Mesenteroides <SEP> DSM <SEP> 20240 <SEP> 0
<tb> Lb. <SEP> delbrueckii <SEP> DSM <SEP> 20081 <SEP> 0
<tb> Lb. <SEP> plantarum <SEP> DSM <SEP> 20174 <SEP> 0
<tb> Lb <SEP> brevis <SEP> DSM <SEP> 20054 <SEP> 0
<tb> Lb. <SEP> casei <SEP> DSM <SEP> 20011 <SEP> 0
<tb> Lb. <SEP> sakei <SEP> 2515 <SEP> 1
<tb> P. <SEP> acidilactici <SEP> ENSAIA <SEP> 583 <SEP> 0
<tb> P. <SEP> cerevisiae <SEP> IP <SEP> 5492 <SEP> 1
<tb> E. <SEP> faecium <SEP> ENSAIA <SEP> 631 <SEP> 0
<tb> E. <SEP> faecalis <SEP> IP <SEP> 5430 <SEP> 2
<tb> E. <SEP> faecalis <SEP> ENSAIA <SEP> 636 <SEP> 1
<tb> E. <SEP> durans <SEP> ENSAIA <SEP> 630 <SEP> 2
<tb> L. <SEP> inocua <SEP> 8811 <SEP> 3
<tb> L. <SEP> ivanovi <SEP> BUG <SEP> 496 <SEP> 6
<Tb>

The spectrum of inhibition of this bacteriocin appears to be rather narrow and limited to strains of Lactobacillus sakei and Pediococcus cerevisiae for lactic acid bacteria. This peptide appears, like the other class IIa bacteriocins, active against all the strains of Listeria tested, as well as against Enterococcus faecalis and durans but not against Enterococcus faecium.

Claims (12)

1. Isolated polypeptide, characterized in that it is a bacteriocin, called Sakacin G, from Lactobacillus sakei 2512.  2. isolated polypeptide according to claim 1, characterized in that it is a class IIa bacteriocin.  Nucleic acid comprising a nucleotide sequence encoding a polypeptide according to claim 1 or 2.  A cloning and / or expression vector comprising a nucleic acid according to claim 3.  Vector-transformed host cell according to claim 4.  6. Host cell according to claim 5, characterized in that it is a microorganism selected from Lactococcus, Lactobacillus, Leuconostoc, Streptococcus, Pediococcus, Escherichia or a yeast.  A process for producing a recombinant polypeptide in which a vector comprising a nucleic acid according to claim 3 is transferred into a host cell which is cultured conditions for expression of a polypeptide according to claim 1 or 2.  8. Use of a polypeptide according to any one of claims 1 or 2 as active agent against pathogenic or undesirable flora in the preparation of food products.  9. Use according to claim 8, characterized in that said polypeptide is used to inhibit the growth and propagation of Listeria, more particularly Listeria monocytogenes, in food products. <Desc / Clms Page number 18>  10. Use according to claim 8 or 9, characterized in that said polypeptide is produced in the food product from the strain Lactobacillus Sakei 2512.  11. Use of the strain Lactobacillus Sakei 2512 in food products to produce a bacteriocin polypeptide according to claim 1 or 2.  12. Bacteriocin composition characterized in that it comprises at least one polypeptide according to any one of claims 1 or 2 or the strain of Lactobacillus Sakei 2512.