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  • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

• 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.
• the phage resistance mechanisms are grouped into 3 main classes, depending on the stage of the bacteriophage reproduction cycle with which they interfere:
• R M mechanisms types I. II, and III
• 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.
• 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.
• 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)].
• 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.
• polypeptide constituting an HsdR subunit comprises the following sequence (I) (represented in code 1 - letter):
• polypeptide constituting an HsdM subunit comprises at least one of the following sequences (represented in code 1 - letter): - the sequence (II):
• polypeptide constituting an HsdS subunit in accordance with the present invention, it comprises:
• 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 X ⁇ 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 ⁇ ⁇ 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):
• the present invention includes in particular:
• 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;
• 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 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.
• 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:
• 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.
• 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.
• 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).
• 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.
• 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.
• 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.
• 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.
• 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)]
• 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.
• plasmid vectors as well as integration vectors usable in lactococci are described in the review of:
• Lactococal plasmid vectors In: K.G.
• 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.
• 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.
• the orfl reading frame codes for a protein with strong homology to RepB proteins already identified in lactococci.
• 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.
• the sequencing of the plasmid ⁇ IL7 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.
• 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.
• 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.
• 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.
• 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 ⁇ M 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 ⁇ l.
• the amplification products obtained from the IL582, IL858, IL910, IL993 strains. MG1363, IL827, IL854, IL964. have been sequenced.
• 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.
• 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 III shows the results obtained with the phage bIL67 (previously propagated on the IL 1403 strain) on:
• 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.

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