EP1007701A1 - Resistance mechanisms to ic type r/m bacteriophages of lactic acid bacteria - Google Patents

Abstract

The invention concerns polypeptides constituting subunits of a resistance mechanism to Ic type R/M bacteriophages, active against the phages of lactic acid bacteria, and nucleic acid sequences coding for said polypeptides. The expression of said polypeptides in lactic acid bacteria enables to increase resistance to the attacks of bacteriophages.

Description

 RESISTANCE MECHANISMS FOR R / M BACTERIOPHAGES OF THE LACTIC BACTERIUM TYPE.

The invention relates to systems for resistance to bacteriophages R / M of the le type, active on bacteriophages of lactic acid bacteria. Obtaining lactic acid bacteria resistant to attack by bacteriophages is of great interest in the field of industrial fermentations.

To this end, it is generally proposed:

- either to select mutants, natural or obtained by mutagenesis, resistant to bacteriophages; - Or to use vectors carrying genes coding for phage resistance mechanisms to transfer these to initially non-resistant strains.

The phage resistance mechanisms are grouped into 3 main classes, depending on the stage of the bacteriophage reproduction cycle with which they interfere:

- the mechanisms grouped under the name "mechanisms of interference with adsorption", delay the adsorption of phage on the bacteria;

- the mechanisms called "abortive infection mechanisms" (Abi), block the multiplication of phages, and cause the death of the infected bacterium before it produces viral particles capable of infecting other cells; the mechanisms known as "restriction / modification mechanisms" (R / M), degrade phage DNA as soon as it enters the bacteria. These mechanisms combine a restriction endonuclease, and a methylase, which has the function of protecting the bacterial genome, by modifying the sequences thereof which could constitute target sequences of the endonuclease.

3 main types of R M mechanisms (types I. II, and III) have been described [for review, cf. for example BICKLE and KRUGER, Microbiological Reviews, 57, pp. 434-450 (1993)]. The characteristics of the R / M mechanisms of types I and II are briefly recalled below:

Type II R / M mechanisms are the most frequent; they consist of two separate enzymes, a methylase and an endonuclease, which are active separately, and which recognize a common target sequence.

R / M type I mechanisms were mainly discovered in enterobacteria, then were also demonstrated in some Grauf bacteria (Bacillus subtilis. And Mycobacterium pulmonis), and in archaebacteria. In these mechanisms, methylase (M subunit, or HsdM) and endonuclease (R subunit, or HsdR) each form a subunit of a multifunctional enzyme complex where they are associated with a third protein (under -S unit, or HsdS), which is responsible for the recognition of specific sequences by the enzyme complex.

These three proteins are the products of genes respectively designated hsdM, hsdR and hsdS. Genetic complementation experiments and the comparison of the sequences of these genes allowed to allocate mechanisms R / M type I in 4 distinct families n ^'having that ^a very low homology among themselves, la, Ib, Ic, and Id .

On the other hand, it was observed that R / M type I mechanisms could acquire a new specificity following recombinations in the hsdS gene [FULLER-PACE and MURRAY, Proc. Natl. Acad. Sci. USA, 83, pp. 9368-9372, (1986); SHARP et al., Proc. Natl. Acad. Sci. USA, 89, pp. 9836-9840 (1992); GUBLER et al. EMBO J., 11, pp. 233-240, (1992)].

The R / M mechanisms so far characterized in lactic acid bacteria, and the use of which has been proposed to make them more resistant to bacteriophages are R / M type II mechanisms [FITZGERALD et al. Nucleic Acids Research, 10, pp. 8171-8179, (1982); NYENGAARD et al., Gene, 136, pp. 371-372 (1993); TOWNEY et al. Gene, 136, pp. 205-209, (1993); DAVIS et al., Appl. About. Microbiol, 59, pp. 777-785, (1995); NYENGAARD et al. Gene, 157, pp. 13-18 (1995); O'SULLIVAN et al., J. Bactériol., 177, pp. 134-143 (1995); MOINEAU et al. Appl. About. Microbiol .. 61, pp. 2193-2202, (1995)].

A major drawback of the use of this type of mecha- nisms is ^the appearance of phage-resistant, in particular due to the fact that the methylase of the R / M recognizes and modifies a certain proportion of the molecules of the phage DNA , which can then escape the action of the endonuclease. This phenomenon appears in particular during prolonged use under industrial fermentation conditions. To limit it, it has been proposed to combine several R / M mechanisms recognizing various target sequences [JOSEPHSEN and KLAENHAMMER, Plasmid, 23, 71-75 (1989)]; it is therefore particularly desirable to isolate new mechanisms, with a specificity different from that of the mechanisms already known.

The inventors have now isolated new R / M mechanisms of the le type, active in lactic acid bacteria, and have expressed the genes coding for their various constituents.

The present invention relates to a polypeptide constituting one of the HsdR subunits. HsdM, or HsdS of a mechanism of resistance to bacteria- phage R / M type le, characterized in that said mechanism is active against the phages of lactic acid bacteria.

According to a preferred embodiment of a polypeptide constituting an HsdR subunit in accordance with the present invention, it comprises the following sequence (I) (represented in code 1 - letter):

KRLARK

According to a preferred embodiment of a polypeptide constituting an HsdM subunit in accordance with the present invention, it comprises at least one of the following sequences (represented in code 1 - letter): - the sequence (II):

GLX ^ YKYLS in which X] represents I or L, - the sequence (III):

ENDX ₂ NLNIPRYVDTFEEEE in which X2 represents Y or F

According to a preferred embodiment of a polypeptide constituting an HsdS subunit in accordance with the present invention, it comprises:

* an N-terminal domain of approximately 150 to 180 amino acids, comprising the following sequence (IV): PX3LRFX ₄ GFTX ₅ DD EERKX ₆ in which X3 represents E or Q, X4 represents E, P, D, or K, X5 represents N or D, Xβ represents L or F;

* a central domain, of approximately 50 to 80 amino acids, comprising the following sequence (V): EQX ₇ KI _-8 _Tr X ₉ LDX ₁₀ TIX ₁₁ IJlQRKl_D__LKEQKKGYX ₁ 2Q - ^^ in which Xη represents R or Q, Xg represents S, N, or L, X9 represents E, H, or Q, X \ Q represents A, D, or N, X \ represents A, or V, X \ 2 represents L or F, Xj3 represents A or S, X14 represents I or V, X \ ζ represents E or G, Xi g represents D or E; * a C-terminal domain, of approximately 160 to 200 amino acids, comprising the following sequence (VI):

EQX ₁₅ X _{1 6} IGSFFKQLDX ₁₇ TIX _{1 8} LHQRKLX _{1 9} in which X \ ζ represents K or Q, X \ represents K or Q, Xjγ represents N or D, Xjg represents T, A, or V, Xi 9 represents A, or D; and / or the following sequence (VΙI):

GFL ^Q KMFX _{2 0} in which X20 represents V or H. The present invention includes in particular:

- the HsdR polypeptides corresponding to one of the sequences respectively represented in the sequence list in the appendix under the numbers SEQ ID NO: 2 and SEQ ID NO: 4; - HsdM polypeptides corresponding to one of the sequences respectively represented in the sequence list in the appendix under the numbers SEQ ID NO: 6, and SEQ ID NO: 8;

- the HsdS polypeptides corresponding to one of the sequences respectively represented in the sequence list in the appendix under the numbers SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16.

The association of any of the HsdR polypeptides, with any of the HsdM polypeptides and any of the HsdS polypeptides according to the invention makes it possible to obtain an enzymatic complex constituting an R / M mechanism of the active type. in lactic acid bacteria. The specificity of action of this mechanism is conditioned by the HsdS polypeptide. The sequences (IV), (V), (VI) and (VIT) represent conserved regions of the HsdS polypeptides in lactic acid bacteria (and in particular in lactococci), which are probably involved in the association of these polypeptides with HsdR and HsdM polypeptides; these conserved regions are separated by variable regions involved in the recognition of specific nucleotide sequences.

The present invention also relates to the nucleic acid sequences coding for the polypeptides defined above, as well as their complementary.

DNA sequences in accordance with the invention are for example represented by:

- The hsdR sequences SEQ ID NO: 1, and SEQ ID NO: 3, which code respectively for the polypeptides SEQ ID NO: 2, and SEQ ID NO: 4;

- The hsdM sequences SEQ ID NO: 5, and SEQ ID NO: 7, which code respectively for the polypeptides SEQ ID NO: 6, and SEQ ID NO: 8; - the hsdS sequences SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 15 which code respectively for the polypeptides SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO : 14 and SEQ ID NO: 16.

The present invention also relates to nucleic acid fragments at least 18 bp, homologous or complementary to all or part of a sequence ^of nucleic acid encoding a polypeptide HsdR, HsdM or HsdS according to the invention . These fragments can in particular be used as hybridization probes, and / or amplification primers, for detecting and selecting strains of lactic bacteria, or plasmids hosted by these strains, containing at least one sequence coding for an HsdR polypeptide, HsdM, or HsdS according to the invention, and for isolating and / or cloning said sequence from a strain or a plasmid selected in this way.

Preferred subfragments are those which are located in sequences encoding conserved regions between the HsdR subunits, between the HsdM subunits or between the HsdS subunits of the R / M type II-conforming mechanisms. invention.

For example :

- to select strains of lactic acid bacteria containing a sequence coding for an HsdR subunit, and / or to clone said sequence, advantageously use at least one oligonucleotide homologous or complementary to a sequence coding for at least 6 consecutive amino acids of one of the following peptides (represented in code 1 - letter): TGSGKT KRLARK LLTGFDS which correspond to conserved regions of the HsdR subunits in L. lactis, identified by the inventors from the sequences SEQ ID NO: 2 and SEQ ID NO: 4

- to select strains of lactic acid bacteria containing a sequence coding for an HsdM subunit. and / or to clone said sequence, it is advantageous to use at least one oligonucleotide homologous or complementary to a sequence coding for at least 6 consecutive amino acids of one of the following peptides:

FYKYLS LARMNL LPHGVLFRGAAE

NLNIPRYVDTFEEEE which correspond to conserved regions of HsdM subunits in L. lactis, identified by the Inventors by aligning the sequences SEQ ID NO: 6 and SEQ ID NO: 8; - to select strains of lactic acid bacteria containing a sequence coding for an HsdS subunit. and / or to isolate and clone said sequence, it is advantageous to use at least one homologous or complete oligonucleotide of a sequence coding for at least 6 consecutive amino acids of one of the following peptides:

DD EERK

LHQRKLDLLKEQKKGY QKMFPKNG

PELRFA

FADD E

IGSFFKQLD

GFLQKMF which correspond to conserved regions of HsdS subunits in L. lactis, identified by the Inventors from the sequences SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14 and SEQ ID NO: 16.

The DNA sequences in accordance with the invention can be used to express in a lactic bacterium one or more R / M mechanisms of the le type, in order to increase its resistance to bacteriophages.

In accordance with the invention, to allow the expression in a bacterium, in particular a lactic acid bacterium, of at least one mechanism of resistance to R / M bacteriophages of the lactic acid type le, the transformation of said bacterium is carried out with at least one DNA sequence according to the invention. The method according to the invention can be implemented in different ways. For example, if the host bacteria naturally does not contain any sequence coding for one of the HsdR, HsdM or HsdS subunits, it must be transformed by at least three DNA sequences, each of which codes for one of these polypeptides. If, on the other hand, the host bacterium already contains at least one sequence (carried by the bacterial chromosome or by a plasmid, coding for one of the subunits HsdR. HsdM, or HsdS, it will suffice to transform it with one or coding sequence (s) for the missing subunit (s).

Advantageously, from the same strain of host bacteria which contains at least one sequence coding for an HsdR subunit and at least one sequence coding for an HsdM subunit. it is possible, by transformation with different sequences coding for HsdS subunits of different specificity, to obtain strains exhibiting different resistance characteristics, and to limit the emergence of phages escaping the resistance mechanism.

Advantageously, in order to widen the spectrum of resistance to bacteriophages, it is possible to use simultaneously, in the same host bacterium, sequences coding for HsdS subunits of different specificity; we can also, for the same purpose, express in the transformed bacteria a resistance mechanism to bacteriophages chosen from Abi mechanisms, and R / M type II mechanisms. In this case, it is possible to use a host bacterium already comprising at least one sequence coding for an Abi mechanism and / or at least one sequence coding for an R / M type II mechanism, or to introduce to the times in the host bacterium, the sequence (s) coding for the Abi and / or R / M type II mechanism (s), and the sequence (s) coding for the subunit (s) ) HsdR, HsdM or HsdS.

In transformed bacteria obtained in accordance with the invention, the sequences coding for the mechanisms of resistance to bacteriophages and in particular those coding for the HsdR, HsdM, or HsdS subunits conforming to the invention, can be carried by a same DNA molecule (bacterial chromosome or plasmid), or by different DNA molecules. Several sequences carried by the same DNA molecule can be part of the same transcription unit, or else of different transcription units. They can be placed under the control of their own transcription regulation sequences, or under the control of heterologous sequences. active in the host bacteria.

It is possible, for example, to express the sequences coding for the HsdR, HsdM, or HsdS subunits under the control of promoter sequences permitting constitutive expression, or, advantageously, under the control of promoter sequences permitting inducible expression, for example a promoter inducible by cold such as that described in Application FR 9615731 on behalf of the NATIONAL INSTITUTE FOR AGRONOMIC RESEARCH.

To implement the method according to ^the invention, one can use natural plasmids, previously selected ^by means of nucleic acid probes according to the invention and containing at least one sequence encoding one subunit HsdR, HsdM or HsdS ^a mechanism R / M type of the lactic acid bacterium.

A subject of the present invention is also recombinant vectors, characterized in that they result from the insertion of at least one nucleic acid sequence in accordance with the invention into an appropriate vector, and in particular recombinant vectors usable to transform bacteria according to the invention.

Depending on the intended use, it is possible to choose either vectors capable of replicating and of maintaining themselves in the host bacterium in the form of a plasmid, such as for example the plasmids pIL252 and pIL253 described by SIMON and CHOPIN, [Biochemistry, 70, p. 559-566, (1988)], or vectors allowing the integration of the inserts which they carry in the chromosomal DNA of the host bacterium, such as for example the vectors described in PCT application WO 94/16086 on behalf of BIOTEKNOLOGISK INSTITUT and CHR. HASSEN'S LABORATORIUM DANMARK A / S, or the plasmid pG ⁺ host5 described by BISWAS et al. [J. Bact., 175, p. 3628-3635, (1995)].

More generally, plasmid vectors as well as integration vectors usable in lactococci are described in the review of:

LEENHOUTS K.J. and VENEMA G. (1993). Lactococal plasmid vectors, In: K.G.

HARDY (ed) Plasmids. A practical approach. Second Edition. IRL Press, Oxford, p.

65-94.

If several vectors are used to introduce into the host bacterium the different HsdR, HsdM, and HsdS subunits, and possibly other mechanisms of resistance to bacteriophage, oh will choose vectors compatible with each other.

The present invention can be advantageously implemented in all areas where industrial fermentation takes place, and in particular in the dairy and cheese industry. Bacterial strains having an increased resistance to bacteriophages can be obtained in accordance with the invention, from strains of lactic acid bacteria of industrial interest, either by selection of bacteria naturally having an R / M system of the Ie type using probes. nucleic acid according to the invention, or by transformation of host bacteria using nucleic acid sequences according to the invention.

The present invention will be better understood with the aid of the additional description which follows, which refers to examples of cloning of sequences coding for sub-units of R / M mechanisms of the Ie type, and of use of these sequences to increase the resistance of host bacteria to attacks by bacteriophages. EXAMPLE 1: OBTAINING DNA FRAGMENTS ENCODING HsdS SUBUNITS OF R / M-TYPE MECHANISMS

The manipulation of DNA, the cloning and the transformation of bacterial cells are, in the absence of contrary details, carried out according to the protocols described by SAMBROOK et al. [(Molecular cloning: a laboratory manual., Cold Spring Harbor Laboratory, Cold Spring Harbor N. Y. (1989)].

GAUTIER and CHOPIN [App. About. Microbiol. 53, 923-927 (1987)], and CHOPIN et al., [Plasmid. 11, pp. 260-263, (1984)] described the existence of mechanisms of resistance to phages of the R / M type, respectively associated with the plasmids pIL 103 and pIL7. The pIL 103 DNA was digested with EcoRI, and the fragments obtained were ligated to the EcoRI site of the vector pIL204 [SIMON and CHOPIN. Biochemistry 70, 559-566, (1988)]. The ligation mixture was used to transform L. Lactis ssp lactis bacteria of the IL 1403 strain. The bacterial colonies were selected on the basis of their resistance to phage bIL67. A recombinant plasmid carrying an insert of about 3.7 kb was obtained from bacterial clones resistant to phage attack. This recombinant plasmid was named pIL261.

Surprisingly, the sequencing of the 3.7 kb insert of the plasmid pIL261 did not reveal any sequence having homology with those of the R / M mechanisms known in lactococci.

However, 2 reading frames orfl and or / 2 were located on this 3.7 kb fragment;

The orfl reading frame codes for a protein with strong homology to RepB proteins already identified in lactococci.

The sequence of the protein encoded by the or / 2 reading frame has no homology with known sequences of lactic acid bacteria proteins; however, the central region and the N-terminal region of this protein contain repeat sequences having some homology with repeat consensus sequences of the HsdS subunits of R / M systems le type of enterobacteria and mycoplasmas.

A nucleic acid sequence containing the orfl reading frame, and the sequence of the corresponding polypeptide are respectively represented in the sequence list in the appendix under the numbers SEQ ID NO: 13 and SEQ ID NO: 14.

Similarly, the sequencing of the plasmid ρIL7 made it possible to highlight a reading frame coding for a protein having a significant homology with that encoded by the orfl reading frame of the plasmid pIL261, and also having repeated sequences recalling those of the sub - HsdS units of type I R / M systems.

A sequence ^of nucleic acid containing the third reading frame, and the sequence of the corresponding polypeptide are respectively represented in the sequence listing in the annex under the numbers SEQ ID NO: 15 and SEQ ID NO: 16.

Oligonucleotide probes derived from the sequences coding for regions conserved between the sequences SEQ ID NO: 14 and SEQ ID NO: 16 were used to search for the existence of other homologous sequences of the hsdS type. These probes made it possible to demonstrate sequences of this type, on the chromosomal DNA of the strain IL 1403. as well as on a plasmid derived from the strain of L. lactis ssp. lactis IL420, hosted by IL403. The hsdS type sequence obtained from the chromosomal DNA of the IL 1403 strain, and the sequence of the corresponding polypeptide are respectively represented in the sequence list in the appendix under the numbers SEQ ID NO: 9 and SEQ ID NO: 10 La hsdS type sequence obtained from the plasmid derived from the strain of L. lactis ssp. lactis IL420, and the sequence of the corresponding polypeptide are respectively represented in the sequence list in the appendix under the numbers SEQ ID NO: 11 and SEQ ID NO: 12.

EXAMPLE 2: OBTAINING DNA FRAGMENTS ENCODING FOR HsdR AND HsdM SUBUNITS OF R / M-TYPE MECHANISMS

The regions of the chromosome of the strain IL 1403, and of the plasmid derived from the strain IL420 on which the hsdS type sequences have been located have been entirely sequenced.

This sequencing made it possible to demonstrate, in the 2 cases, directly upstream of the hsdS sequence, 2 open reading frames, coding for proteins respectively containing regions having a significant homology with conserved domains of subunits R and M of R / M systems of the type le of enterobacteria and mycoplasmas. These 2 open reading frames were respectively named, because of this homology, hsdR and hsdM. On the chromosome of the IL 1403 strain, as on the plasmid derived from the IL420 strain, the order of the open reading frames is, from upstream to downstream: hsdR, hsdMet hsdS.

The hsdR type sequence obtained from the chromosomal DNA of the IL 1403 strain and the sequence of the corresponding polypeptide are respectively represented in the sequence list in the appendix under the numbers SEQ ID NO: 1 and SEQ ID NO: 2.

The hsdM type sequence obtained from the chromosomal DNA of the strain IL 1403, and the sequence of the corresponding polypeptide are respectively represented in the sequence list in the appendix under the numbers SEQ ID NO: 5 and SEQ ID NO: 6.

The hsdR type sequence obtained from the plasmid derived from the strain of L. lactis ssp. lactis IL420, and the sequence of the corresponding polypeptide are respectively represented in the annexed sequence list under the numbers SEQ ID NO: 3 and SEQ ID NO: 4 The hsdM type sequence obtained from the plasmid derived from the strain of L. lactis ssp. lactis IL420, and the sequence of the corresponding polypeptide are respectively represented in the sequence list in the appendix under the numbers SEQ ID NO: 7 and SEQ ID NO: 8.

EXAMPLE 3: USE OF OLIGONUCLEOTIDES DERIVED FROM THE HSDR, HSDM AND HSDS SEQUENCES FOR THE DETECTION OF BACTERIAL STRAINS HAVING R / M-TYPE SYSTEM SUB-UNITS.

Regions of the hsdM sequences of the plasmid derived from the IL420 strain, and of the chromosome of the IL 1403 strain, coding for the same peptide sequences have been identified. The following oligonucleotides:

* oligo 66: TTA GCA CGT ATG AAC TTA

* oligo 67: TTG GCT CGA ATG AAT TTA represent sequences which code respectively in the plasmid of the strain IL420, and in the chromosome of the strain IL 1403. for the peptide sequence (code 1 letter) LARMNL.

The following oligonucleotides:

* oligo 68: CTC TTC AAA GGT ATC CAC

* oligo 69: TTC CTC AAA GGT ATC TAC represent sequences complementary to the sequences coding respectively, in the chromosome of the strain IL 1403, and in the plasmid of the strain IL420, for the peptide sequence (code 1 letter) VDTFEE.

These oligonucleotides are used as primers ^for PCR amplification (66/69 and 67/68 pairs) of 16 different strains of Lactococcus.

The amplification conditions used are as follows: the DNA extracted from each of the strains is placed in the presence of triphosphate deoxyribonucleotides (0.2 mM of each), 0.1 μM of each primer, and 2.5 Taq polymerase units (PROMEGA) in a standard Taq polymerase buffer (PROMEGA).

The reaction is carried out in a final volume of 100 μl.

After denaturation (94 ° C, 5 minutes), 30 cycles are carried out alternating a step of fixing the primers at 50 ° C for 30 seconds, an extension step at 72 ° C for 30 seconds, and a denaturation step at 94 ° C for 30 seconds.

As shown in Table I below, for 8 of the 16 strains tested (IL582, IL858, IL910. IL993. IL964. IL827, IL854. MG1363) we observe, with one or the other of the 2 pairs of primers the presence of an amplification product of the expected length (approximately 670 bp). TABLE I

(L): ssp. lactis

(D) ssp. lactis biovar. diacetylactis (C) ssp. cremoris ++ clearly detectable amplification product; + detectable amplification product; +/- weakly detectable amplification product; - no detectable amplification product.

The amplification products obtained from the IL582, IL858, IL910, IL993 strains. MG1363, IL827, IL854, IL964. have been sequenced. In IL582 strains. IL858, IL993, IL827, IL854. IL964. the sequence is very similar to the hsdM sequence of the chromosome of the strain IL 1403. and in the strains IL910 and MG1363 the sequence is very similar to the hsdM sequence of the plasmid of the strain IL420. EXAMPLE 4: RESISTANCE TO THE PHAGIC ATTACK CONFERRED BY THE HsdR, HsdM AND HsdS SUBUNITS.

The strains used are: the strains MG1363, and IL582 which each have a sequence coding for the M subunit of an R / M system of the type, and the strain IL 1403, which has on its chromosome sequences coding for the 3 R, M and S subunits of an R / M system of type le.

The bacteria are cultured and infected with phages under the conditions described by TERZAGHI and SANDINE, [Appl. Microbiol., 29, 807-815, (1975)] The results of the infection are expressed in PFU / ml, and the propagation efficiency (eop) is determined, for each test, by the ratio between the number of PFU / ml for the bacterial strain tested, and the number of PFU / ml for a bacterial strain chosen as a control.

Table II below shows the results obtained with phage c2 on the strain MG1363, chosen as a control, and the strain IL 1403.

TABLE II

Table III below shows the results obtained with the phage bIL67 (previously propagated on the IL 1403 strain) on:

- IL 1403 strain without plasmids (1), and

- strain IL582 (5) chosen as controls,

- the IL 1403 strain transformed with the plasmid derived from the IL420 strain, carrying sequences hsdR, hsdM and hsdS (2).

- the IL 1403 strain transformed with the plasmid piL7, carrying an hsdS sequence (3)

- the IL 1403 strain transformed with the plasmid pIL261 carrying an hsdS sequence (4)

- the IL582 strain transformed with the plasmid pIL261 carrying an hsdS sequence (6)

TABLE III

These results show that:

* The HsdS subunits of the different resistance mechanisms do indeed have a different specificity: the mechanism carried by the chromosome of the IL 1430 strain is weakly effective against the phage bIL67, while on the other hand the HsdS subunits carried by the plasmids increase, to a greater or lesser degree, resistance to this phage. * The HsdS subunits coded by the plasmids pIL7 and pIL261 can associate with the HsdR and HsdM subunits coded by the chromosome of IL1430 to reconstitute an R / M complex of the functional type.

Claims

CLAIMS 1) Polypeptide, constituting one of the HsdR, HsdM, or HsdS subunits of a mechanism of resistance to R / M bacteriophages of type le, characterized in that said mechanism is active against the phages of lactic acid bacteria. 2) Polypeptide according to claim 1, characterized in that it is chosen from the group consisting of: - the polypeptides constituting an HsdR subunit comprising the following sequence (I): KRLARK - the polypeptides constituting an HsdM subunit comprising at least one of the following sequences: - the sequence (II): GLX ^ YKYLS in which X \ represents I or L. - the sequence (III): ENDX ₂ NLNIPRYVDTFEEEE in which X2 represents Y or F - the polypeptides constituting an HsdS subunit comprising: * an N-terminal domain of approximately 150 to 180 amino acids, comprising the following sequence (IV): PX3LRFX ₄ GFTX ₅ DDWEERKX ₆ in which X3 represents E or Q, X4 represents E, P, D. or K, X5 represents N or D, Xg represents L or F; * a central domain, of approximately 50 to 80 amino acids, comprising the following sequence (V): E ^ ₇ ra <_X ₈ F_T <9l_I_X ₁₀ TIX ₁₁ __H ^^ in which X7 represents R or Q, Xg represents S, N, or L, X9 represents E, H, or Q, X \ Q represents A, D, or N, X \ \ represents A, or V, X γι represents L or F, X13 represents A or S, X \ __ represents I or V, X15 represents E or G, X \ β represents D or E; * a C-terminal domain, of approximately 160 to 200 amino acids, comprising the following sequence (VI): EQX ₁₅ X _{1 6} IGSFFKQLDX ₁₇ TIX ₁₈ LHQRKLX _{1 9} in which X15 represents K or Q, X \ represents K or Q, X17 represents N or D, X \ g represents T, A, or V, Xi 9 represents A, or D; and / or the following sequence (VΙI): GFLQKMFX ₂ o in which X20 represents V or H. 3) Polypeptide according to claim 2, characterized in that it is chosen from the group consisting of: - an HsdR polypeptide corresponding to one of the sequences respectively represented in the sequence list in the appendix under the numbers SEQ ID NO: 2 and SEQ ID NO: 4; - an HsdM polypeptide corresponding to one of the sequences respectively represented in the sequence list in the appendix under the numbers SEQ ID NO: 6, and SEQ ID NO: 8; - an HsdS polypeptide consisting of: * an N-terminal domain of approximately 150 to 180 amino acids whose sequence is that of the N-terminal domain of any of the sequences represented in the sequence list in the appendix under the numbers SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16; * a central domain, of approximately 50 to 80 amino acids, the sequence of which is that of the central domain of any of the sequences represented in the sequence list in the annex under the numbers SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16; * a C-terminal domain, of approximately 160 to 200 amino acids, the sequence of which is that of the C-terminal domain of any of the sequences represented in the sequence list in the appendix under the numbers SEQ ID NO: 10. SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16. 4) Nucleic acid sequence characterized in that it codes for a polypeptide according to any one of claims 1 to 3. 5) Nucleic acid sequence according to claim 4 characterized in that it is chosen from the group consisting of: - the hsdR sequences SEQ ID NO: 1, and SEQ ID NO: 3; - the hsdM sequences SEQ ID NO: 5, and SEQ ID NO: 7; - the hsdS sequences SEQ ID NO: 9, SEQ ID NO: 1 1, SEQ ID NO: 13, and SEQ ID NO: 15. 6) A fragment of nucleic acid ^of at least 18 bp homologous or complementary to all or part ^of a nucleic acid sequence according to any of claims 4 or 5. 7) A nucleic acid fragment according to claim 6, chosen from the group consisting of oligonucleotides homologous or complementary to a sequence coding for at least 6 consecutive amino acids of one of the following peptides; TGSGKT KRLARK LLTGFDS FYKYLS LAR NL LPHGVLFRGAAE NLNIPRYVDTFEEEE DD EERK LHQRKLDLLKEQKKGY QK FPKNG PELRFA FADD E IGSFFKQLD GFLQKMF. 8) Use of at least one nucleic acid fragment according to any one of claims 6 or 7 to select lactic acid bacteria containing a nucleic acid sequence coding for at least one subunit of an R / M mechanism of the type the. 9) Use of at least one fragment ^of nucleic acid according to any one of claims 6 or 7 to isolate and / or clone a nucleic acid com- taking a sequence according to any one of claims 4 or 5. 10) Use of at least one nucleic acid sequence according to any one of claims 4 or 5 to allow the expression in a lactic acid bacterium of at least one mechanism of resistance to bacteriophages R / M of the le type. 11) A bacterium transformed with at least one nucleic acid sequence according to any one of claims 4 or 5. 12) Bacterium transformed according to Claim 11, characterized in that it is also capable of expressing a mechanism of resistance to bacteriophages chosen from Abi mechanisms, and R / M type II mechanisms.