Lactic acid bacteria with reduced susceptibility to cos-type bacteriophages

Abstract

The present invention relates to lactic acid bacteria, in particular strains of the genus Streptococcus, which have reduced susceptibility to cos-type bacteriophages. The present invention also relates to a method for engineering said lactic acid bacteria and to the use of said lactic acid bacteria for fermenting milk.

Description

[Technical field]

[0001] The present invention relates to lactic acid bacteria having reduced susceptibility to cos-type bacteriophages. [Background technology]

[0002] Lactic acid bacteria starter cultures are widely used in the food industry for the production of fermented products, including dairy products (e.g., yogurt, butter, and cheese), meat products, bakery products, wine, and vegetable products. Examples of lactic acid bacteria that are widely used in the food industry include the genera Streptococcus, Lactococcus, Lactobacillus, Leuconostoc, Pediococcus, Enterococcus, Oenococcus, and Bifidobacterium. For example, the lactic acid bacteria Streptococcus thermophilus is widely used by the dairy industry. Preparation of the cultures is labor intensive, occupies large amounts of space and equipment, and there is a significant risk of bacteriophage (phage) contamination during milk fermentation.

[0003] Attack of bacterial cultures by infection and proliferation of bacteriophages is considered one of the main problems in industrial applications of bacterial cultures. Many different phage types exist, with different mechanisms of attacking bacteria. Furthermore, new strains of bacteriophages are generated. Many different bacteriophage strains have been found and isolated that are capable of infecting bacterial strains used in industry, including species of lactic acid bacteria, such as Lactococcus lactis and Streptococcus thermophilus.

[0004] Strategies used in the industry to minimize bacteriophage infection and the resulting failure of bacterial cultures are not completely effective. Such strategies include the use of mixed starter cultures to ensure that there is a certain level of resistance to phage attack. Rotation of selected bacterial strains that are sensitive to different bacteriophages is also used. However, rapid replacement of bacterial strains with resistant strains after the emergence of new phages is usually not possible. Therefore, it is not yet possible to eliminate phage contamination in the food industry.

[0005] The lytic infection cycle of bacteriophages specifically involves phage adsorption to the bacterial host cell surface to release newly assembled phage particles, injection of phage DNA into the cell, phage DNA replication, phage protein expression, phage assembly, and host bacterial cell lysis. Bacterial phage resistance mechanisms depend on host factors that are involved in one or more steps of the lytic cycle of phage replication, and are usually classified based on the step of the infection cycle that they interfere with.

[0006] While numerous phage resistance mechanisms have been found in other bacterial species, only a few have been identified in S. thermophilus. The most dominant defense mechanism in this species is the CRISPR-Cas system (Non-Patent Document 1; Non-Patent Document 2). In this defense mechanism, bacterial cells accumulate short sequences from the phage genome, called spacers, which act as cellular memory of previous encounters and serve as guides for specific cleavage of invading phage DNA. Little information is available about other host factors involved in phage infection in this food-grade Streptococcus species. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Barrangou et al., 2007 [Non-Patent Document 2] Horvath et al., 2008 Summary of the Invention [Problem to be solved by the invention]

[0008] There is a continuing need in the art to provide improved bacterial strains for use in the food or feed industry, such as bacterial strains with reduced susceptibility to phages.Therefore, there is a need to identify host factors involved in phage infection in lactic acid bacteria, such as those of the genus Streptococcus. [Means for solving the problem]

[0009] The inventors have surprisingly found that the host gene encoding methionine aminopeptidase (MetAP protein) is involved in phage infection and is essential for the cos-type phage DT1 to complete its lytic cycle. Mutations in the metAP gene provide Streptococcus thermophilus with reduced susceptibility to cos-type phages. Complementation of the mutant strain with the metAP gene of a susceptible strain results in restoration of the phage-susceptible phenotype. Introduction of the same mutation into a Streptococcus mutans strain also results in reduced phage-susceptible phenotype. This suggests the widespread importance of host MetAP in phage infection. The inventors have also shown that the reduced susceptibility phenotype to cos-type phages is stable over time (at least 60 generations). This confirms the stability of metAP mutations and the importance of metAP mutant lactic acid bacteria in industrial fermentation.

[0010] In one aspect, the present invention relates to a method for the production of methionine aminopeptidase protein (MetAP R metAP, which encodes a proteinR Regarding lactic acid bacteria containing the metAP allele, R The allele, when inserted in place of the metAP gene allele of Streptococcus thermophilus SMQ-301 strain, reduces the susceptibility to phage DT1, as determined by the Efficiency of Plaquing (EOP) Assay I. Thus, the present invention provides a method for the detection of methionine aminopeptidase (MetAP R Protein) protein-encoding metAP R Regarding lactic acid bacteria containing the metAP allele, R The allele is defined as a metAP allele that reduces the susceptibility of a Streptococcus thermophilus SMQ-301 derivative to phage DT1, and the SMQ-301 derivative is R The lactic acid bacterium is a Streptococcus thermophilus SMQ-301 strain in which the metAP allele has been replaced by an allele, and susceptibility to phage DT1 is determined by the efficiency of plaque formation (EOP) assay I. In a particular embodiment, the lactic acid bacterium is a strain of the genus Streptococcus selected from the group consisting of Streptococcus thermophilus strains or Streptococcus mutans strains.

[0011] In one aspect, the present invention relates to a bacterial composition comprising the lactic acid bacteria of the present invention.

[0012] In one aspect, the present invention relates to a food or feed product comprising the lactic acid bacteria or bacterial composition of the present invention.

[0013] In one aspect, the present invention relates to a method for producing a fermented product comprising: a) inoculating a substrate with a lactic acid bacteria or bacterial composition of the present invention; and b) fermenting the inoculated substrate obtained from step a) to obtain a fermented product, preferably a fermented dairy product.

[0014] In one aspect the present invention relates to the use of a lactic acid bacteria or bacterial composition of the present invention for producing a food or feed product, preferably a fermented food product, more preferably a fermented dairy product.

[0015] In one aspect, the present invention provides a method for the preparation of a MetAP R The present invention relates to a polynucleotide encoding a protein, which, when inserted in place of an allele of the metAP gene of Streptococcus thermophilus strain SMQ-301, reduces susceptibility to phage DT1, as determined by efficiency of plaque formation (EOP) assay I. Thus, the present invention provides a method for the detection of MetAP R A polynucleotide encoding a protein, the polynucleotide being a metAP allele that reduces the susceptibility of a Streptococcus thermophilus SMQ-301 derivative to phage DT1, the SMQ-301 derivative comprising the metAP allele. R The Streptococcus thermophilus SMQ-301 strain has a metAP allele replaced by the DT1 allele, and its susceptibility to phage DT1 is determined by the efficiency of plaque formation (EOP) assay I.

[0016] In one aspect, the present invention relates to the use of a polynucleotide of the present invention for reducing the susceptibility of a lactic acid bacterium sensitive to cos-type phages to at least one cos-type phage, wherein the susceptibility to at least one cos-type phage is determined by EOP Assay I.

[0017] In one aspect, the present invention relates to a method for preparing a lactic acid bacteria strain with reduced susceptibility to at least one cos-type phage, comprising: a) providing a lactic acid bacteria strain sensitive to cos-type phages; b) substituting an allele of the metAP gene of said lactic acid bacteria strain sensitive to cos-type phages with a polynucleotide of the present invention or modifying the sequence of the metAP gene of said lactic acid bacteria strain sensitive to cos-type phages to have a metAP allele that has the same sequence as the polynucleotide of the present invention; and c) recovering the lactic acid bacteria strain with reduced susceptibility to at least one cos-type phage, wherein the susceptibility to at least one cos-type phage is determined by EOP Assay I.

[0018] In one aspect, the present invention provides a method for the preparation of a MetAP R Protein-encoding metAP R A method for identifying an allele, comprising: a) inserting a metAP allele to be tested in place of an allele of the metAP gene of Streptococcus thermophilus SMQ-301 strain to obtain an SMQ-301 derivative; and b) determining the EOP of phage DT1 against the SMQ-301 derivative of step a) by plaque formation efficiency assay I, wherein an EOP reduction of at least 4 logs, at least 5 logs, or at least 6 logs is found to be indicative of a MetAP allele. R Protein-encoding metAP R The method relates to a method for identifying a metAP allele, DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] General definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0020] The present disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure.

[0021] The headings provided herein are not limitations of the various aspects or embodiments of the disclosure which can be used by reference to the specification as a whole, and accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0022] As used herein, the term "polynucleotide" is synonymous with the term "nucleotide sequence" and / or the term "nucleic acid sequence." Unless otherwise specified, any nucleic acid sequence is written left to right in 5' to 3' orientation.

[0023] The term "protein" as used herein includes proteins, polypeptides, and peptides. The term "amino acid sequence" as used herein is synonymous with the term "protein". In this disclosure and claims, amino acid names, conventional three-letter codes, or conventional one-letter codes are used for amino acid residues. It is also understood that a protein can be coded for by multiple nucleotide sequences due to the degeneracy of the genetic code. Unless otherwise specified, any amino acid sequence is written in amino to carboxy orientation from left to right.

[0024] In the present invention, MetAP R A particular numbering of amino acid residue positions within a protein may be used. By alignment of the amino acid sequence of a sample MetAP protein with the MetAP protein of SEQ ID NO: 2, it is possible to assign to amino acid residue positions within said sample MetAP protein numbers which correspond to the amino acid residue positions or numbering of the amino acid sequence shown in SEQ ID NO: 2 of the present invention.

[0025] Other definitions of terms may appear throughout this specification. Before describing the exemplary embodiments in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, which may of course vary. It should also be understood that the terminology used herein is for the purpose of describing only the specific embodiments, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0026] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0027] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," "containing," and "contains," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. Additionally, the terms "comprising," "comprises," and "comprised of" include the term "consisting of."

[0028] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that any such publication constitutes prior art to the claims appended hereto.

[0029] The present inventors have surprisingly found that the host gene encoding methionine aminopeptidase (MetAP) is a bacterial host factor involved in the propagation of cos-type phages in Streptococcus strains. R Protein-encoding metAP R The allele was found to reduce the susceptibility of Streptococcus strains to at least one cos-type phage.

[0030] In one aspect, the present invention provides a method for the preparation of a MetAP R Protein-encoding metAP R 1. A method for distinguishing between alleles comprising: a) inserting a metAP allele to be tested (candidate metAP allele) in place of an allele of the metAP gene of Streptococcus thermophilus SMQ-301 to obtain a SMQ-301 derivative; b) determining the EOP of phage DT1 against the SMQ-301 derivative of step a) by plaque formation efficiency assay I; At least 4 log reduction in EOP was achieved by the MetAP R Protein-encoding metAP R The present invention provides a method for identifying an allele, the metAP allele.

[0031] As used herein, the phrase "allele of the metAP gene" refers to a version of the metAP gene found in a particular lactic acid bacterium. For most bacterial genes, the nucleotide sequence of the gene can vary, and alleles represent different sequences of the same gene. Thus, an allele of the metAP gene of Streptococcus thermophilus SMQ-301 is as shown in SEQ ID NO:1. This allele defined in SEQ ID NO:1 encodes the MetAP protein shown in SEQ ID NO:2.

[0032] MetAP R Protein-encoding metAP R Alleles The inventors have shown that some of these metAP alleles can reduce the susceptibility of Streptococcus thermophilus SMQ-301 to phage DT1 when inserted in place of the original (i.e., natural) allele of the metAP gene of SMQ-301 (SEQ ID NO: 1). These metAP alleles are referred to as "metAP R These metAP alleles are defined herein as R The protein encoded by the allele is referred to herein as "MetAP R are called "proteins."

[0033] Therefore, any metAP that reduces the susceptibility of the SMQ-301 strain to phage DT1 (as defined herein) R Allele (MetAP R The metAP gene is a gene encoding a metAP protein. R The allele is defined as a metAP allele that reduces the susceptibility of a Streptococcus thermophilus SMQ-301 derivative to phage DT1, the SMQ-301 derivative comprising the metAP allele. R The Streptococcus thermophilus strain SMQ-301 has a metAP allele that replaces the original metAP allele.

[0034] In one embodiment, the metAP R An allele is defined herein (particularly in the context of the lactic acid bacteria or polynucleotides of the invention) as a metAP allele that is identified by the following method: a) inserting a metAP allele to be tested (candidate metAP allele) in place of an allele of the metAP gene of Streptococcus thermophilus SMQ-301 (shown in SEQ ID NO:1) to obtain a SMQ-301 derivative; b) determining the EOP of phage DT1 against the SMQ-301 derivative of step a) by plaque formation efficiency assay I; (At least a 4 log reduction in EOP is required for the MetAP R Protein-encoding metAP R The metAP allele is shown.

[0035] MetAP R Non-limiting examples of proteins are disclosed below.

[0036] The metAP of the present invention R One way to determine the allele-conferred reduction in susceptibility to a bacteriophage is to determine the efficiency of plaque formation (EOP) of a phage against a Streptococcus strain in which a candidate metAP allele of the invention has been inserted in place of the original allele of the metAP gene of the Streptococcus strain.

[0037] In one embodiment, the reduction in susceptibility is determined by calculating the efficiency of plaque formation (EOP) with the Efficiency of Plaque Formation Assay I (EOP Assay I) described herein.

[0038] Plaque formation efficiency assay I One method for determining the reduced susceptibility to cos-type bacteriophages conferred by a candidate metAP allele is to determine the susceptibility of both a lactic acid bacterium that is susceptible to cos-type phages (referred to herein as the "reference strain") and a corresponding derived lactic acid bacterium in which an allele of the metAP gene has been replaced with the candidate metAP allele (referred to herein as the "derived strain").

[0039] The bacteriophage susceptibility of the lactic acid bacteria of the present invention (especially of the genus Streptococcus, in particular of Streptococcus thermophilus strains) can be determined by calculating the plaque-forming efficiency (EOP) using the plaque-forming efficiency assay I (EOP assay I) described herein.

[0040] The enumeration of infectious phage particles is carried out using the double agar overlay plaque assay described by Kropinski et al. 2009, herein named "Plaque-forming efficiency assay I". The method consists of infecting a lawn of bacteria growing on a surface in a soft agar nutrient medium. Infection by phages results in localized clear or translucent zones, called plaques, corresponding to areas where bacteria are dead or not growing. An infectious phage unit is therefore called a plaque-forming unit (pfu). Since the enumeration of phages using the assay requires a minimum of 30 and a maximum of 300 pfu per plate, the phage suspension may require dilution. For this purpose, the primary phage suspension is serially diluted 10-fold in 10 mL of M17 medium containing 5 g / L lactose (v / v).

[0041] The plaque formation efficiency assay I was as follows: i. Pre-culturing each strain to be tested (reference strain and its derivatives to be tested as defined herein) overnight at 37° C. in M17 medium containing 5 g / L lactose (v / v); ii. inoculating each preculture separately at 1% (v / v) in 5 mL of molten M17-CaCl2 soft agar medium (maintained at 47° C. in a water bath) containing 5 g / L lactose, 10 mM CaCl2, and 5 g / L (w / v) agar; iii. adding 100 μL of the phage dilution to be tested to each inoculated medium; iv. after mixing, pouring each mixture onto the surface of M17-CaCl2 solid agar medium containing 5 g / L lactose, 10 mM CaCl2, and 15 g / L (w / v) agar; v. After the overlay has solidified, incubating the inverted plate at 37° C. for 48 hours; vi. counting the plaques (on plates showing 30-300 plaques); vii. calculating the titer of the virulent phage as: number of plaques x 10 x the reciprocal of the dilution factor, expressed in pfu / mL; viii. calculating the EOP of the phage for the derivative strain as the phage titer for the derivative strain to be tested divided by the phage titer for the reference strain (EOP of the reference strain is 1); has.

[0042] In a particular embodiment, the lactic acid bacteria of the invention are strains of the genus Streptococcus. The susceptibility to bacteriophages conferred by candidate metAP alleles is determined by the plaque formation efficiency assay I described herein using a strain of Streptococcus susceptible to cos-type phages (reference strain) and a corresponding derivative strain of Streptococcus in which an allele of the metAP gene has been replaced by the candidate metAP allele.

[0043] In a particular embodiment, the lactic acid bacteria of the invention are of the species Streptococcus thermophilus. The susceptibility to bacteriophages conferred by candidate metAP alleles is determined by the plaque formation efficiency assay I described herein using Streptococcus thermophilus strains susceptible to cos-type phages (reference strains) and corresponding derived Streptococcus thermophilus strains in which an allele of the metAP gene has been replaced by the candidate metAP allele.

[0044] The candidate metAP allele is a metAP allele according to the present invention. R To determine whether a candidate metAP allele is a candidate for a Streptococcus thermophilus strain, SMQ-301, is used as a "reference strain" and a Streptococcus thermophilus strain obtained by insertion of a candidate metAP allele in place of an allele of the metAP gene of Streptococcus thermophilus strain SMQ-301 is used as a derived strain (referred to herein as an "SMQ-301 derivative").

[0045] Thus, in one embodiment, the plaque formation efficiency assay I defined herein is carried out by: - Streptococcus thermophilus strain SMQ-301 as a "reference strain"; strain SMQ-301 is commercially available, has been used as a model organism to study phage-host interactions (Tremblay and Moineau, 1999; Labrie et al., 2015), and is available at the Felix d'Herelle Reference Centre for Bacterial Viruses (www.phage.ulaval.ca) under reference HER 1368; the genome sequence of this strain is available under the accession number CP011217.1; - Streptococcus thermophilus strain SMQ-301, in which an allele of the metAP gene has been replaced by a candidate metAP allele (an SMQ-301 derivative); thus, the allele of the metAP gene of the SMQ-301 derivative does not encode the MetAP protein shown in SEQ ID NO:2, and in particular is not set out in SEQ ID NO:1; and - Phage DT1, which is commercially available (Tremblay and Moineau 1999; Labrie et al., 2015) and available at the Felix d'Herelle Reference Centre for Bacterial Viruses under reference HER 368; the genome sequence of this phage is available under the accession number NC_002072.2.

[0046] A candidate metAP allele as defined herein is a metAP allele according to the invention if, when inserted in place of an allele of the metAP gene of Streptococcus thermophilus strain SMQ-301, said candidate metAP allele reduces susceptibility to phage DT1. RThe candidate metAP alleles are considered to be alleles, and susceptibility to phage DT1 is determined by the efficiency of plaque formation (EOP) assay I. Thus, a candidate metAP allele is considered to be a metAP allele according to the invention if it reduces the susceptibility of a Streptococcus thermophilus SMQ-301 derivative strain to phage DT1. R The metAP allele is considered to be the SMQ-301 derivative. R The Streptococcus thermophilus SMQ-301 strain has a metAP allele that replaces the original metAP allele, and its susceptibility to phage DT1 is determined by the efficiency of plaque formation (EOP) assay I.

[0047] Candidate metAP alleles (metAP of the present invention) instead of the original allele of the metAP gene of Streptococcus thermophilus SMQ-301 R It should be noted that insertion of a metAP allele (including a metAP mutant allele as defined herein) must result in an SMQ-301 derivative and can be tested by the Efficiency of Plaque Formation (EOP) Assay I. Any metAP allele of a sequence that does not allow for the determination of susceptibility to DT1 using the EOP Assay I is not considered a metAP allele as defined herein.

[0048] "Reduced susceptibility to phage DT1" by EOP Assay I means an EOP reduction of at least 4 logs. In one embodiment, the EOP reduction is at least 5 logs. In one embodiment, the EOP reduction is at least 6 logs. In one embodiment, the EOP reduction is at least 7 logs. In one embodiment, the EOP reduction is at least 8 logs. In one embodiment, a metAP allele is a metAP allele according to the invention if the EOP reduction is selected from the group consisting of at least 4 logs, at least 5 logs, at least 6 logs, at least 7 logs, and at least 8 logs of EOP reduction. R A candidate metAP allele is considered to be an allele and its susceptibility to phage DT1 is determined by the efficiency of plaque formation (EOP) assay I. Thus, a candidate metAP allele is considered to be a metAP allele according to the invention if said candidate metAP allele reduces the susceptibility of a Streptococcus thermophilus SMQ-301 derivative strain to phage DT1 by at least 4 logs. R The metAP allele is considered to be the SMQ-301 derivative. R The Streptococcus thermophilus SMQ-301 strain has a metAP allele replaced by the DT1 allele, and its susceptibility to phage DT1 is determined by the efficiency of plaque formation (EOP) assay I (i.e., compared to the Streptococcus thermophilus SMQ-301 strain).

[0049] In contrast, candidate metAP alleles that do not reduce the EOP by at least 4 logs when inserted in place of an allele of the metAP gene of Streptococcus thermophilus strain SMQ-301 (susceptibility to phage DT1 as determined by the Efficiency of Plaque Formation (EOP) Assay I) are not suitable for use as metAP alleles according to the invention. RThus, a candidate metAP allele that does not reduce the EOP of phage DT1 by at least 4 logs against a Streptococcus thermophilus SMQ-301 derivative, the SMQ-301 derivative being a Streptococcus thermophilus SMQ-301 strain in which the metAP allele has been replaced by the candidate metAP allele, and the susceptibility to phage DT1 is determined by the Efficiency of Plaque Formation (EOP) Assay I, is considered to be a metAP allele according to the present invention. R Not considered to be alleles.

[0050] The expressions "reducing susceptibility" or "reducing EOP" are defined according to assay I, i.e. by determining the EOP of phage DT1 against a Streptococcus thermophilus SMQ-301 derived strain and comparing it with the EOP of phage DT1 against a Streptococcus thermophilus SMQ-301 strain.

[0051] metAP mutant alleles encoding mutant MetAP proteins Candidate metAP alleles that 1) encode a MetAP protein whose sequence has at least 80% identity to SEQ ID NO:2, and 2) do not reduce the EOP or reduce the EOP by less than 3 logs (sensitivity to phage DT1 is determined by plaque-forming efficiency (EOP) assay I) are referred to herein as metAP mutant alleles (encoding MetAP mutant proteins). The phrase "metAP mutant protein" is used interchangeably with the phrase "metAP mutant protein having at least 80% identity to SEQ ID NO:2."

[0052] In one embodiment, the metAP mutant allele reduces the EOP by less than 3 logs when inserted in place of an allele of the metAP gene of Streptococcus thermophilus SMQ-301 strain (susceptibility to phage DT1 is determined by Efficiency of Plaque Formation (EOP) Assay I). Thus, the metAP mutant allele reduces the EOP of phage DT1 by less than 3 logs for a Streptococcus thermophilus SMQ-301 derivative strain, the SMQ-301 derivative strain being a Streptococcus thermophilus SMQ-301 strain in which the metAP allele has been replaced, and susceptibility to phage DT1 is determined by Efficiency of Plaque Formation (EOP) Assay I. In one embodiment, the EOP reduction is less than 3 logs. In one embodiment, the EOP reduction is less than 2 logs. In one embodiment, the EOP reduction is less than 3 logs or less than 2 logs.

[0053] In one embodiment, the metAP mutant allele results in the same EOP (susceptibility to phage DT1 is determined by Efficiency of Plaque Formation (EOP) Assay I) when inserted in place of the allele of the metAP gene of Streptococcus thermophilus strain SMQ-301. Thus, the metAP mutant allele results in the same EOP of phage DT1 for a Streptococcus thermophilus SMQ-301 derived strain as the EOP of phage DT1 for Streptococcus thermophilus strain SMQ-301, said SMQ-301 derived strain being a Streptococcus thermophilus strain SMQ-301 in which the metAP allele has been replaced by said metAP mutant allele, and susceptibility to phage DT1 is determined by Efficiency of Plaque Formation (EOP) Assay I.

[0054] Also in combination with any of the previous EOP reduction embodiments, a metAP mutant allele is defined to encode a MetAP mutant protein whose sequence is at least 80% identical to SEQ ID NO:2. By "at least 80% identical to SEQ ID NO:2" is meant at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%. In one embodiment, a MetAP mutant protein (encoded by a metAP mutant allele) has a sequence that is at least 85% identical to SEQ ID NO:2. In one embodiment, a MetAP mutant protein (encoded by a metAP mutant allele) has a sequence that is at least 85% identical to SEQ ID NO:2. In one embodiment, a MetAP mutant protein (encoded by a metAP mutant allele) has a sequence that is at least 90% identical to SEQ ID NO:2. In one embodiment, a MetAP mutant protein (encoded by a metAP mutant allele) has a sequence that is at least 95% identical to SEQ ID NO:2. In one embodiment, a MetAP mutant protein (encoded by a metAP mutant allele) has a sequence that is at least 96% identical to SEQ ID NO:2. In one embodiment, a MetAP mutant protein (encoded by a metAP mutant allele) has a sequence that is at least 97% identical to SEQ ID NO:2. In one embodiment, a MetAP mutant protein (encoded by a metAP mutant allele) has a sequence that is at least 98% identical to SEQ ID NO:2. In one embodiment, a MetAP mutant protein (encoded by a metAP mutant allele) has a sequence that is at least 99% identical to SEQ ID NO:2.

[0055] In one embodiment, in combination with the percentage of identity, the size of the MetAP mutant protein is the same as the MetAP protein defined in SEQ ID NO:2 (286 amino acid residues); thus, in one embodiment, the metAP mutant allele is additionally defined as encoding a 286 amino acid MetAP mutant protein.

[0056] In one embodiment, the metAP mutant allele is defined herein as: 1) encodes a MetAP mutant protein whose sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2; and 2) When inserted in place of an allele of the metAP gene of Streptococcus thermophilus strain SMQ-301, it reduces the EOP by less than 3 logs or by less than 2 logs (sensitivity to phage DT1 is determined by the Efficiency of Plaque Formation (EOP) Assay I). It is defined as follows.

[0057] Thus, the metAP mutant allele is referred to herein as: 1) encodes a MetAP mutant protein whose sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2; and 2) reduce the EOP of phage DT1 by less than 3 logs against a Streptococcus thermophilus SMQ-301 derivative, the SMQ-301 derivative being a Streptococcus thermophilus SMQ-301 strain in which the metAP allele has been replaced by the metAP mutant allele, and the susceptibility to phage DT1 is determined by the Efficiency of Plaque Formation (EOP) Assay I; It is defined as follows.

[0058] In one embodiment, the metAP mutant allele is defined herein as: 1) encodes a MetAP mutant protein whose sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2; and 2) when inserted in place of an allele of the metAP gene of Streptococcus thermophilus strain SMQ-301, it results in the same EOP (sensitivity to phage DT1 is determined by the efficiency of plaque formation (EOP) assay I) It is defined as follows.

[0059] Thus, the metAP mutant allele is also referred to herein as: 1) encodes a MetAP mutant protein whose sequence is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:2; and 2) yielding the same EOP of phage DT1 against Streptococcus thermophilus SMQ-301 derivative as that against Streptococcus thermophilus SMQ-301 strain, said SMQ-301 derivative being a Streptococcus thermophilus SMQ-301 strain in which the metAP allele has been replaced by said metAP mutant allele, and susceptibility to phage DT1 is determined by the Efficiency of Plaque Formation (EOP) Assay I; It is defined as follows.

[0060] The metAP mutant alleles defined herein can be easily identified by the skilled artisan starting from a Streptococcus thermophilus strain that is known or can be characterized as being sensitive to phage DT1 (susceptibility to phage DT1 is determined by EOP assay I).

[0061] The expressions "same EOP" or "less than 3 logs or less than 2 logs reduced EOP" are defined according to assay I, i.e. by determining the EOP of phage DT1 against Streptococcus thermophilus SMQ-301 derived strains and comparing it with the EOP of phage DT1 against Streptococcus thermophilus SMQ-301 strain.

[0062] Allelic substitution of the metAP gene in lactic acid bacteria (especially strain SMQ-301) The replacement of an allele of the metAP gene of a reference lactic acid bacterium (particularly of a strain of the genus Streptococcus, in particular of a strain of Streptococcus thermophilus) with a metAP allele (e.g. a candidate metAP allele or a metAP mutant allele) is carried out using conventional techniques in molecular biology and is within the capabilities of a person skilled in the art. Generally speaking, suitable routine methods include replacement via homologous recombination.

[0063] The phrase "a metAP allele inserted in place of an allele of the metAP gene" is synonymous with the phrase "an allele of the metAP gene has been replaced by a metAP allele" (e.g., a candidate metAP allele or a metAP mutant allele). R The expression "an allele of the metAP gene is R"Replaced by an allele."

[0064] Substituted (or inserted instead) means that the sequence of the MetAP protein encoded by the metAP allele to be inserted (in particular a candidate metAP allele or a metAP mutant allele) is different from the sequence of the MetAP protein encoded by the allele of the metAP gene of the original strain (e.g. a reference strain). Thus, replaced (or inserted instead) means that the coding sequence of the metAP gene of the original strain (from the first nucleotide of the start codon to the last nucleotide of the stop codon) is replaced by the corresponding coding sequence of a metAP allele (in particular a candidate metAP allele or a metAP mutant allele).

[0065] In the case of the SMQ-301 strain, replaced (or inserted instead) means that the sequence of the MetAP protein encoded by the metAP allele to be inserted (in particular a candidate metAP allele or a metAP mutant allele) is different from the sequence of the MetAP protein encoded by the original metAP gene of the SMQ-301 strain. Thus, replaced (or inserted instead) means that the coding sequence of the metAP gene of the SMQ-301 strain (from the first nucleotide of the start codon to the last nucleotide of the stop codon, i.e. nucleotides 1-861 of SEQ ID NO: 1) is replaced by the corresponding coding sequence of a metAP allele (in particular a candidate metAP allele or a metAP mutant allele).

[0066] Generating candidate metAP alleles The candidate metAP allele is metAP RStarting from a non-allelic metAP allele, in particular from a metAP allele encoding the MetAP protein defined in SEQ ID NO:2 (e.g. SEQ ID NO:1), or from a MetAP mutant allele as defined herein, can be generated by random or directed mutagenesis. In one embodiment, candidate metAP alleles are generated by random mutagenesis. In another embodiment, candidate metAP alleles can be generated by directed mutagenesis. Suitable mutagenesis protocols for random or directed mutagenesis are described in the Materials and Methods section below.

[0067] In addition, the candidate metAP allele was identified in the bacterium Streptococcus thermophilus (metAP gene allele is metAP R A strain that is not an allele (e.g., the SMQ-301 strain) can be generated by presenting a strain to challenge with phage DT1. A suitable protocol for challenging a strain with phage is described in the Materials and Methods section below. In one embodiment, to reduce the number of genetic events resulting from the CRISPR-Cas system that result in reduced susceptibility to phage (and thus increase the number of genetic events associated with metAP mutations), it is possible to inhibit or reduce the efficiency of the CRISPR-Cas system by using a phage carrying an anti-CRISPR gene (acr gene) (WO 2018 / 197495).

[0068] The candidate metAP alleles thus generated are known as metAP alleles, as defined herein. R Screening can be done using methods that discriminate between alleles.

[0069] In one aspect, the present invention provides a metAP R Allele (MetAP R and providing a lactic acid bacterium comprising the metAP encoding a protein,R The allele, when inserted in place of the metAP gene allele of Streptococcus thermophilus SMQ-301 strain, reduces the susceptibility to phage DT1, as determined by efficiency of plaque formation (EOP) assay I. Thus, the present invention provides a method for the detection of methionine aminopeptidase (MetAP R Protein) protein-encoding metAP R Regarding lactic acid bacteria containing the metAP allele, R The allele is defined as a metAP allele that reduces the susceptibility of a Streptococcus thermophilus SMQ-301 derivative to phage DT1, and the SMQ-301 derivative is R The Streptococcus thermophilus SMQ-301 strain, in which the metAP allele has been replaced by the metAP allele, and the susceptibility to phage DT1 is determined by the efficiency of plaque formation (EOP) assay I. R An allele is one that has been generated, screened for, and / or is defined herein. The expression "reducing susceptibility to phage DT1" refers to a metAP R For the definition of an allele, it is as defined herein. In particular, and as defined herein, the metAP of the present invention R The allele-associated reduction in susceptibility to phage DT1 is at least a 4 log reduction as determined by the efficiency of plaque formation (EOP) assay I.

[0070] Of note is the expression "metAP R "Contains metAP allele" means that the only allele of the metAP gene contained in the genome of the lactic acid bacteria (LAB) is metAP RThus, an allele of the metAP gene of the LAB of the present invention is a metAP gene as defined herein. R It is not intended that the LAB of the invention contain several alleles of the metAP gene. In one embodiment, the LAB of the invention contains a metAP allele that reduces susceptibility to phage DT1 when inserted in place of an allele of the metAP gene of Streptococcus thermophilus strain SMQ-301 as the only allele of the metAP gene. R The LAB of the present invention contains the metAP allele, which, as the only allele of the metAP gene, reduces the susceptibility of Streptococcus thermophilus SMQ-301 derivatives to phage DT1, as determined by the efficiency of plaque formation (EOP) assay I. R The SMQ-301 derivative comprises the metAP allele. R The Streptococcus thermophilus SMQ-301 strain has a metAP allele replaced by the DT1 allele, and its susceptibility to phage DT1 is determined by the efficiency of plaque formation (EOP) assay I.

[0071] In one aspect, the present invention provides a method for the preparation of a MetAP R The present invention provides a polynucleotide encoding a protein, which when inserted in place of an allele of the metAP gene of Streptococcus thermophilus strain SMQ-301, reduces susceptibility to phage DT1, as determined by efficiency of plaque formation (EOP) assay I. Thus, the present invention provides a method for the detection of MetAP RThe present invention provides a polynucleotide encoding a protein, the polynucleotide being a metAP allele that reduces the susceptibility of a Streptococcus thermophilus SMQ-301 derivative to phage DT1, the SMQ-301 derivative comprising the metAP allele. R and a Streptococcus thermophilus SMQ-301 strain in which the metAP allele has been replaced by an allele, and susceptibility to phage DT1 is determined by efficiency of plaque formation (EOP) assay I. In one embodiment, the polynucleotide is R The polynucleotides may be generated, screened, and / or comprised of metAP alleles as defined herein. R The expression "reducing susceptibility to phage DT1" means R For the definition of an allele, it is as defined herein. In particular, and as defined herein, the metAP of the present invention R The allele-associated reduction in susceptibility to phage DT1 is at least a 4 log reduction as determined by the efficiency of plaque formation (EOP) assay I.

[0072] MetAP R Protein sequence The metAP of the invention (as part of a polynucleotide of the invention or contained in a lactic acid bacterium of the invention) R The allele may be characterized by its nucleotide sequence or by the MetAP gene it encodes, in addition to its ability to reduce the susceptibility to phage DT1, in particular its ability to reduce the susceptibility of Streptococcus thermophilus SMQ-301 derivatives to phage DT1 (as determined herein by EOP assay I). R It can be defined by the amino acid sequence of the protein.

[0073] In one embodiment, the metAP R The allele is a MetAP allele that includes an amino acid suppression, an amino acid addition, an amino acid substitution, or an amino acid suppression and addition to the MetAP protein selected from the group consisting of: R Encode a protein: a) a MetAP protein having the amino acid sequence defined in SEQ ID NO:2; and b) A MetAP mutant protein as defined herein having at least 80% identity to SEQ ID NO: 2. A MetAP mutant protein as defined herein is encoded by a metAP mutant allele that, when inserted in place of an allele of the metAP gene of Streptococcus thermophilus strain SMQ-301, does not reduce the EOP or reduces the EOP by less than 3 logs (sensitivity to phage DT1 is determined by the efficiency of plaque formation (EOP) assay I). As defined herein, a MetAP mutant protein is encoded by a metAP mutant allele that reduces the EOP of phage DT1 by less than 3 logs against a Streptococcus thermophilus SMQ-301 derivative, or that results in the same EOP of phage DT1 against a Streptococcus thermophilus SMQ-301 derivative, the SMQ-301 derivative being a Streptococcus thermophilus SMQ-301 strain in which the metAP allele has been replaced, and susceptibility to phage DT1 is determined by efficiency of plaque formation (EOP) assay I.

[0074] In one embodiment, the metAP R An allele is a MetAP nucleotide sequence having at least 80% identity with (but different from) SEQ ID NO:2. R Encodes proteins.

[0075] In certain embodiments relating to EOP, the identity and size percentages described elsewhere in this application within the context of metAP mutant proteins are R The same is true in the context of proteins.

[0076] In one embodiment, the metAP R The allele comprises a MetAP protein having an amino acid sequence selected from the group consisting of: a) a MetAP protein having the amino acid sequence as defined in SEQ ID NO:2; and b) a MetAP mutant protein as defined herein having at least 80% identity to SEQ ID NO:2. R In certain embodiments, MetAP R The protein is characterized by at least one amino acid restriction, in particular by one, two, three, four, or five amino acid restrictions. R The protein is characterized by a restriction of one amino acid. In a particular embodiment, MetAP R The protein is characterized by a restriction of two, three, four, or five amino acids. R The proteins are characterized by restraints of two, three, four, or five consecutive amino acids.

[0077] In one embodiment, the metAP R The allele is a MetAP protein comprising an amino acid addition to a MetAP protein selected from the group consisting of: a) a MetAP protein having an amino acid sequence as defined in SEQ ID NO:2; and b) a MetAP variant protein as defined herein having at least 80% identity to SEQ ID NO:2. R In certain embodiments, MetAP R The protein is characterized by the addition of at least one amino acid, in particular by the addition of one, two, three, four or five amino acids. RThe protein is characterized by the addition of one amino acid. In a particular embodiment, MetAP R The protein is characterized by the addition of two, three, four, or five amino acids. R Proteins are characterized by the addition of two, three, four, or five consecutive amino acids.

[0078] In one embodiment, the metAP R The allele is a MetAP protein that contains an amino acid substitution relative to a MetAP protein selected from the group consisting of: a) a MetAP protein having an amino acid sequence as defined in SEQ ID NO:2; and b) a MetAP mutant protein as defined herein that has at least 80% identity to SEQ ID NO:2. R In certain embodiments, MetAP R The protein is characterized by at least one amino acid substitution, in particular by one, two, three, four or five amino acid substitutions. R The protein is characterized by a single amino acid substitution. In a particular embodiment, the MetAP R The proteins are characterized by the substitution of two, three, four, or five amino acids.

[0079] In one embodiment, the metAP R The allele is MetAP R MetAP protein encoding R The sequence of the protein does not contain an amino acid selected from the group consisting of glutamine at position 57, leucine at position 153, alanine at position 168, histidine at position 206, valine at position 228, and proline at position 233, and the amino acid sequence shown in SEQ ID NO: 2 is used for numbering.

[0080] In one embodiment, the metAP R The allele is MetAP R MetAP protein encoding RThe sequence of the protein does not include a glutamine at position 57, and the amino acid sequence shown in SEQ ID NO: 2 is used for numbering.

[0081] In one embodiment, the metAP R The allele is MetAP R MetAP protein encoding R The sequence of the protein does not include a leucine at position 153, and the amino acid sequence shown in SEQ ID NO:2 is used for numbering.

[0082] In one embodiment, the metAP R The allele is MetAP R MetAP protein encoding R The sequence of the protein does not include an alanine at position 168, and the amino acid sequence shown in SEQ ID NO:2 is used for numbering.

[0083] In one embodiment, the metAP R The allele is MetAP R MetAP protein encoding R The sequence of the protein does not include a histidine at position 206, and the amino acid sequence shown in SEQ ID NO:2 is used for numbering.

[0084] In one embodiment, the metAP R The allele is MetAP R MetAP protein encoding R The sequence of the protein does not include a valine at position 228, and the amino acid sequence shown in SEQ ID NO:2 is used for numbering.

[0085] In one embodiment, the metAP R The allele is MetAP R MetAP protein encoding R The sequence of the protein does not include a proline at position 233, and the amino acid sequence shown in SEQ ID NO:2 is used for numbering.

[0086] In any of these embodiments, the MetAP R The size of the protein is the same as the MetAP protein defined in SEQ ID NO:2 (286 amino acid residues).

[0087] In one embodiment, the metAP R The allele comprises a MetAP gene that contains an amino acid selected from the group consisting of a lysine or equivalent amino acid at position 57, a proline or equivalent amino acid at position 153, a glutamic acid or equivalent amino acid at position 168, a glutamine or equivalent amino acid at position 206, an aspartic acid or equivalent amino acid at position 228, a glutamine or equivalent amino acid at position 233, and a leucine or equivalent amino acid at position 233. R The amino acid sequence encoding the protein and shown in SEQ ID NO:2 is used for numbering.

[0088] In one embodiment, the metAP R The allele contains a lysine or equivalent amino acid at position 57. R The amino acid sequence encoding the protein and shown in SEQ ID NO:2 is used for numbering.

[0089] In one embodiment, the metAP R The allele contains a proline or equivalent amino acid at position 153, MetAP R The amino acid sequence encoding the protein and shown in SEQ ID NO:2 is used for numbering.

[0090] In one embodiment, the metAP R The allele is MetAP, which contains glutamic acid or an equivalent amino acid at position 168. R The amino acid sequence encoding the protein and shown in SEQ ID NO:2 is used for numbering.

[0091] In one embodiment, the metAP R The allele contains a glutamine or equivalent amino acid at position 206, MetAP RThe amino acid sequence encoding the protein and shown in SEQ ID NO:2 is used for numbering.

[0092] In one embodiment, the metAP R The allele is MetAP, which contains aspartic acid or an equivalent amino acid at position 228. R The amino acid sequence encoding the protein and shown in SEQ ID NO:2 is used for numbering.

[0093] In one embodiment, the metAP R The allele contains a glutamine or equivalent amino acid at position 233, MetAP R The amino acid sequence encoding the protein and shown in SEQ ID NO:2 is used for numbering.

[0094] In one embodiment, the metAP R The allele contains a leucine or equivalent amino acid at position 233. R The amino acid sequence encoding the protein and shown in SEQ ID NO:2 is used for numbering.

[0095] In any of these embodiments, the MetAP R The size of the protein is the same as the MetAP protein defined in SEQ ID NO:2 (286 amino acid residues).

[0096] In one embodiment, the metAP R The allele is a MetAP gene that contains an amino acid selected from the group consisting of a lysine at position 57, a proline at position 153, a glutamic acid at position 168, a glutamine at position 206, an aspartic acid at position 228, a glutamine at position 233, and a leucine at position 233. R The amino acid sequence encoding the protein and shown in SEQ ID NO:2 is used for numbering.

[0097] In one embodiment, the metAP R The allele is MetAP, which contains a lysine at position 57. RThe amino acid sequence encoding the protein and shown in SEQ ID NO:2 is used for numbering.

[0098] In one embodiment, the metAP R The allele is MetAP, which contains a proline at position 153. R The amino acid sequence encoding the protein and shown in SEQ ID NO:2 is used for numbering.

[0099] In one embodiment, the metAP R The allele is MetAP, which contains glutamic acid at position 168. R The amino acid sequence encoding the protein and shown in SEQ ID NO:2 is used for numbering.

[0100] In one embodiment, the metAP R The allele is MetAP, which contains a glutamine at position 206. R The amino acid sequence encoding the protein and shown in SEQ ID NO:2 is used for numbering.

[0101] In one embodiment, the metAP R The allele is MetAP, which contains an aspartic acid at position 228. R The amino acid sequence encoding the protein and shown in SEQ ID NO:2 is used for numbering.

[0102] In one embodiment, the metAP R The allele is MetAP, which contains a glutamine at position 233 R The amino acid sequence encoding the protein and shown in SEQ ID NO:2 is used for numbering.

[0103] In one embodiment, the metAP R The allele is MetAP, which contains a leucine at position 233. R The amino acid sequence encoding the protein and shown in SEQ ID NO:2 is used for numbering.

[0104] In any of these embodiments, the MetAPR The size of the protein is the same as the MetAP protein defined in SEQ ID NO:2 (286 amino acid residues).

[0105] In one embodiment, the metAP R Alleles include MetAP R Encode a protein: a) an amino acid sequence which is otherwise defined as SEQ ID NO:2, but which does not contain an amino acid selected from the group consisting of a glutamine at position 57, a leucine at position 153, an alanine at position 168, a histidine at position 206, a valine at position 228, and a proline at position 233; R The alleles are MetAPs shown in SEQ ID NOs: 5 to 10, respectively. R Encode proteins; b) an amino acid sequence having 80% identity to SEQ ID NO:2 and not including an amino acid selected from the group consisting of a glutamine at position 57, a leucine at position 153, an alanine at position 168, a histidine at position 206, a valine at position 228, and a proline at position 233; or c) an amino acid sequence that is a MetAP mutant protein (as defined herein) that is otherwise at least 80% identical to SEQ ID NO:2, but that does not contain an amino acid selected from the group consisting of a glutamine at position 57, a leucine at position 153, an alanine at position 168, a histidine at position 206, a valine at position 228, and a proline at position 233.

[0106] In one embodiment, the metAP R The allele is a MetAP variant protein that is normally one of the MetAP variant proteins having at least 80% identity to SEQ ID NO:2 but that does not have a glutamine at position 57 (SEQ ID NO:5), or that has an amino acid sequence that is 80% identical to SEQ ID NO:2 and does not have a glutamine at position 57, or that has an amino acid sequence that is 80% identical to SEQ ID NO:2 but does not have a glutamine at position 57. R Encodes proteins.

[0107] In one embodiment, the metAP R The allele is a MetAP variant protein that is normally one of the MetAP variant proteins having at least 80% identity to SEQ ID NO:2 but that does not include the leucine at position 153 (SEQ ID NO:6), or that has an amino acid sequence that is 80% identical to SEQ ID NO:2 and does not include the leucine at position 153. R Encodes proteins.

[0108] In one embodiment, the metAP R The allele is a MetAP variant that is normally one of the MetAP variant proteins having at least 80% identity to SEQ ID NO:2 but that does not include an alanine at position 168 (SEQ ID NO:7), or that has an amino acid sequence that is 80% identical to SEQ ID NO:2 and does not include an alanine at position 168. R Encodes proteins.

[0109] In one embodiment, the metAP R The allele is a MetAP variant protein that is normally one of the MetAP variant proteins having at least 80% identity to SEQ ID NO:2 but that does not have the histidine at position 206, or an amino acid sequence having 80% identity to SEQ ID NO:2 and that does not have the histidine at position 206 (SEQ ID NO:8). R Encodes proteins.

[0110] In one embodiment, the metAP RThe allele is a MetAP variant that contains an amino acid sequence, normally defined as SEQ ID NO:2, but that does not contain a valine at position 228 (SEQ ID NO:9), or contains an amino acid sequence that has 80% identity to SEQ ID NO:2 and does not contain a valine at position 228, or contains an amino acid sequence that is normally one of the MetAP variant proteins that has at least 80% identity to SEQ ID NO:2, but that does not contain a valine at position 228. R Encodes proteins.

[0111] In one embodiment, the metAP R The allele is a MetAP variant that is normally defined as SEQ ID NO:2 but contains an amino acid sequence (SEQ ID NO:10) that does not contain the proline at position 233, or contains an amino acid sequence that has 80% identity to SEQ ID NO:2 and does not contain the proline at position 233, or contains an amino acid sequence that is normally one of the MetAP variant proteins that has at least 80% identity to SEQ ID NO:2 but does not contain the proline at position 233. R Encodes proteins.

[0112] In any of these embodiments, the MetAP R The size of the protein is the same as the MetAP protein defined in SEQ ID NO:2 (286 amino acid residues).

[0113] In one embodiment, the metAP R Alleles include MetAP R Encode a protein: a) an amino acid sequence which is otherwise defined in SEQ ID NO:2 but which includes an amino acid substitution selected from the group consisting of a lysine or equivalent amino acid at position 57, a proline or equivalent amino acid at position 153, a glutamic acid or equivalent amino acid at position 168, a glutamine or equivalent amino acid at position 206, an aspartic acid or equivalent amino acid at position 228, a glutamine or equivalent amino acid at position 233, and a leucine or equivalent amino acid at position 233; b) an amino acid sequence having 80% identity with SEQ ID NO:2 and comprising an amino acid residue selected from the group consisting of a lysine or equivalent amino acid at position 57, a proline or equivalent amino acid at position 153, a glutamic acid or equivalent amino acid at position 168, a glutamine or equivalent amino acid at position 206, an aspartic acid or equivalent amino acid at position 228, a glutamine or equivalent amino acid at position 233, and a leucine or equivalent amino acid at position 233; or c) an amino acid sequence which is normally one of the MetAP mutant proteins having at least 80% identity to SEQ ID NO:2 (MetAP mutant proteins as defined herein), but which comprises an amino acid substitution selected from the group consisting of a lysine or equivalent amino acid at position 57, a proline or equivalent amino acid at position 153, a glutamic acid or equivalent amino acid at position 168, a glutamine or equivalent amino acid at position 206, an aspartic acid or equivalent amino acid at position 228, a glutamine or equivalent amino acid at position 233, and a leucine or equivalent amino acid at position 233.

[0114] In one embodiment, the metAP R The allele is a MetAP variant protein that is normally defined as SEQ ID NO:2 but includes an amino acid sequence that includes a lysine at position 57 or an equivalent amino acid, or that has 80% identity to SEQ ID NO:2 and includes a lysine at position 57 or an equivalent amino acid, or that is normally a MetAP variant protein that has at least 80% identity to SEQ ID NO:2 but includes an amino acid sequence that includes a lysine at position 57 or an equivalent amino acid. R Encodes proteins.

[0115] In one embodiment, the metAP RThe allele is a MetAP variant protein that is normally defined as SEQ ID NO:2 but includes an amino acid sequence that includes a proline at position 153 or an equivalent amino acid, or that has 80% identity with SEQ ID NO:2 and includes a proline at position 153 or an equivalent amino acid, or that is normally a MetAP variant protein that has at least 80% identity with SEQ ID NO:2 but includes an amino acid sequence that includes a proline at position 153 or an equivalent amino acid. R Encodes proteins.

[0116] In one embodiment, the metAP R The allele is a MetAP variant protein that is normally defined as SEQ ID NO:2 but includes an amino acid sequence that includes a glutamic acid or an equivalent amino acid at position 168, or that has 80% identity with SEQ ID NO:2 and includes a glutamic acid or an equivalent amino acid at position 168, or that is normally a MetAP variant protein that has at least 80% identity with SEQ ID NO:2 but includes an amino acid sequence that includes a glutamic acid or an equivalent amino acid at position 168. R Encodes proteins.

[0117] In one embodiment, the metAP R The allele is a MetAP variant protein that is normally defined as SEQ ID NO:2 but includes an amino acid sequence that includes a glutamine or an equivalent amino acid at position 206, or that has 80% identity with SEQ ID NO:2 and includes a glutamine or an equivalent amino acid at position 206, or that is normally a MetAP variant protein that has at least 80% identity with SEQ ID NO:2 but includes an amino acid sequence that includes a glutamine or an equivalent amino acid at position 206. R Encodes proteins.

[0118] In one embodiment, the metAP RThe allele is a MetAP variant protein that is normally defined as SEQ ID NO:2 but includes an amino acid sequence that includes an aspartic acid at position 228 or an equivalent thereof, or that has 80% identity to SEQ ID NO:2 and includes an aspartic acid at position 228 or an equivalent thereof, or that is normally a MetAP variant protein that has at least 80% identity to SEQ ID NO:2 but includes an amino acid sequence that includes an aspartic acid at position 228 or an equivalent thereof. R Encodes proteins.

[0119] In one embodiment, the metAP R The allele is a MetAP variant protein that is normally defined as SEQ ID NO:2 but includes an amino acid sequence that includes a glutamine or an equivalent amino acid at position 233, or that has 80% identity with SEQ ID NO:2 and includes a glutamine or an equivalent amino acid at position 233, or that is normally a MetAP variant protein that has at least 80% identity with SEQ ID NO:2 but includes an amino acid sequence that includes a glutamine or an equivalent amino acid at position 233. R Encodes proteins.

[0120] In one embodiment, the metAP R The allele is a MetAP variant protein that is normally defined as SEQ ID NO:2 but includes an amino acid sequence that includes a leucine or an equivalent amino acid at position 233, or that has 80% identity with SEQ ID NO:2 and includes a leucine or an equivalent amino acid at position 233, or that is normally a MetAP variant protein that has at least 80% identity with SEQ ID NO:2 but includes an amino acid sequence that includes a leucine or an equivalent amino acid at position 233. R Encodes proteins.

[0121] In any of these embodiments, the MetAP R The size of the protein is the same as the MetAP protein defined in SEQ ID NO:2 (286 amino acid residues).

[0122] In one embodiment, the metAP R Alleles include MetAP R Encode a protein: a) an amino acid sequence that is otherwise defined in SEQ ID NO:2, but that contains an amino acid substitution selected from the group consisting of a glutamine at position 206, a lysine at position 57, a proline at position 153, a glutamic acid at position 168, an aspartic acid at position 228, a glutamine at position 233, and a leucine at position 233; these metAPs R The alleles are MetAP as shown in SEQ ID NOs: 4 and 11 to 16, respectively. R Encode proteins; b) an amino acid sequence having 80% identity with SEQ ID NO:2 and comprising an amino acid residue selected from the group consisting of a glutamine at position 206, a lysine at position 57, a proline at position 153, a glutamic acid at position 168, an aspartic acid at position 228, a glutamine at position 233, and a leucine at position 233; or c) an amino acid sequence that is a MetAP mutant protein (as defined herein) that would normally have at least 80% identity to SEQ ID NO:2, but that contains an amino acid substitution selected from the group consisting of a lysine at position 57, a proline at position 153, a glutamic acid at position 168, a glutamine at position 206, an aspartic acid at position 228, a glutamine at position 233, and a leucine at position 233.

[0123] In one embodiment, the metAP R The allele is a MetAP variant protein that is normally defined as SEQ ID NO:2 but includes an amino acid sequence (SEQ ID NO:11) that includes a lysine at position 57, or that has 80% identity to SEQ ID NO:2 and includes a lysine at position 57, or that is normally a MetAP variant protein that has at least 80% identity to SEQ ID NO:2 but includes an amino acid sequence that includes a lysine at position 57. R Encodes proteins.

[0124] In one embodiment, the metAP RThe allele is a MetAP variant protein that is normally defined as SEQ ID NO:2 but includes an amino acid sequence (SEQ ID NO:12) that includes a proline at position 153, or that has 80% identity to SEQ ID NO:2 and includes an amino acid sequence that includes a proline at position 153, or that is normally a MetAP variant protein that has at least 80% identity to SEQ ID NO:2 but includes an amino acid sequence that includes a proline at position 153. R Encodes proteins.

[0125] In one embodiment, the metAP R The allele is a MetAP variant protein that is normally defined as SEQ ID NO:2 but includes an amino acid sequence (SEQ ID NO:13) that includes a glutamic acid at position 168, or that has 80% identity to SEQ ID NO:2 and includes a glutamic acid at position 168, or that is normally a MetAP variant protein that has at least 80% identity to SEQ ID NO:2 but includes an amino acid sequence that includes a glutamic acid at position 168. R Encodes proteins.

[0126] In one embodiment, the metAP R The allele is a MetAP variant protein that is normally defined as SEQ ID NO:2 but includes an amino acid sequence (SEQ ID NO:4) that includes a glutamine at position 206, or that has 80% identity to SEQ ID NO:2 and includes a glutamine at position 206, or that is normally a MetAP variant protein that has at least 80% identity to SEQ ID NO:2 but includes an amino acid sequence that includes a glutamine at position 206. R Encodes proteins.

[0127] In one embodiment, the metAP RThe allele is a MetAP variant protein that is normally defined as SEQ ID NO:2 but includes an amino acid sequence (SEQ ID NO:14) that includes an aspartic acid at position 228, or that has 80% identity to SEQ ID NO:2 and includes an amino acid sequence that includes an aspartic acid at position 228, or that is normally a MetAP variant protein that has at least 80% identity to SEQ ID NO:2 but includes an amino acid sequence that includes an aspartic acid at position 228. R Encodes proteins.

[0128] In one embodiment, the metAP R The allele may be a MetAP variant protein that is normally defined as SEQ ID NO:2 but includes an amino acid sequence (SEQ ID NO:15) that includes a glutamine at position 233, or that has 80% identity to SEQ ID NO:2 and includes a glutamine at position 233, or that is normally a MetAP variant protein that has at least 80% identity to SEQ ID NO:2 but includes an amino acid sequence that includes a glutamine at position 233. R Encodes proteins.

[0129] In one embodiment, the metAP R The allele is a MetAP variant protein that is normally defined as SEQ ID NO:2 but includes an amino acid sequence (SEQ ID NO:16) that includes a leucine at position 233, or that has 80% identity to SEQ ID NO:2 and includes a leucine at position 233, or that is normally a MetAP variant protein that has at least 80% identity to SEQ ID NO:2 but includes an amino acid sequence that includes a leucine at position 233. R Encodes proteins.

[0130] In any of these embodiments, the MetAP R The size of the protein is the same as the MetAP protein defined in SEQ ID NO:2 (286 amino acid residues).

[0131] In one embodiment, the metAP RThe allele is a MetAP protein having the sequence shown in SEQ ID NO:2, which is obtained by an amino acid substitution selected from the group consisting of Q57K, L153P, A168E, H206Q, V228D, P233Q, and P233L. R Encodes proteins.

[0132] In one embodiment, the metAP R The allele is a MetAP protein variant having at least 80% identity to SEQ ID NO: 2, which is obtained by an amino acid substitution selected from the group consisting of Q57K, L153P, A168E, H206Q, V228D, P233Q, and P233L. R Encodes proteins.

[0133] In one embodiment, the metAP R The allele is a MetAP protein having the sequence shown in SEQ ID NO:2, or a MetAP mutant protein as defined herein having at least 80% identity with SEQ ID NO:2, derived by a Q57K substitution. R Encodes proteins.

[0134] In one embodiment, the metAP R The allele is a MetAP protein having the sequence shown in SEQ ID NO:2, or a MetAP mutant protein as defined herein having at least 80% identity with SEQ ID NO:2, derived by the L153P substitution. R Encodes proteins.

[0135] In one embodiment, the metAP R The allele is a MetAP protein having the sequence shown in SEQ ID NO:2, or a MetAP mutant protein as defined herein having at least 80% identity with SEQ ID NO:2, derived by the A168E substitution. R Encodes proteins.

[0136] In one embodiment, the metAPR The allele may be a MetAP protein having the sequence shown in SEQ ID NO:2, or a MetAP mutant protein as defined herein having at least 80% identity with SEQ ID NO:2, derived by the H206Q substitution. R Encodes proteins.

[0137] In one embodiment, the metAP R The allele may be a MetAP protein having the sequence shown in SEQ ID NO:2, or a MetAP mutant protein as defined herein having at least 80% identity with SEQ ID NO:2, derived by a V228D substitution. R Encodes proteins.

[0138] In one embodiment, the metAP R The allele may be a MetAP protein having the sequence shown in SEQ ID NO:2, or a MetAP mutant protein as defined herein having at least 80% identity with SEQ ID NO:2, derived by a P233Q substitution. R Encodes proteins.

[0139] In one embodiment, the metAP R The allele may be a MetAP protein having the sequence shown in SEQ ID NO:2, or a MetAP mutant protein as defined herein having at least 80% identity with SEQ ID NO:2, derived by a P233L substitution. R Encodes proteins.

[0140] In any of these embodiments, the MetAP R The size of the protein is the same as the MetAP protein (286 amino acid residues) defined in SEQ ID NO:2.

[0141] In one embodiment, the metAP R The allele contains a truncated amino acid sequence of MetAP RBy "last quarter" is meant that the C-terminal portion of the MetAP protein consists of residues 215 to 286, and the amino acid sequence shown in SEQ ID NO: 2 is used for numbering. Thus, in one embodiment, metAP R The allele is MetAP, which contains a stop codon at residues 215 to 286. R The amino acid sequence encoding the protein and shown in SEQ ID NO: 2 is used for numbering. R The allele is MetAP, which contains a stop codon at residues 226 to 286. R The amino acid sequence encoding the protein and shown in SEQ ID NO:2 is used for numbering.

[0142] In one embodiment, the metAP R The allele contains a truncated amino acid sequence of MetAP R MetAP protein encoding R The sequence of the protein has at least 80% identity with the corresponding portion of SEQ ID NO: 2. By "corresponding portion" it is meant that the percentage identity is calculated based on the alignment of the two sequences for the same amino acid range, starting from residue 1 to the position of the stop codon (as an example, if the protein is truncated at position 226, the MetAP codon with SEQ ID NO: 2 is not identical). R (80% protein identity is calculated based on alignment of the two sequences for residues 1-226). "At least 80% identity with the corresponding portion of SEQ ID NO:2" means at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity with the corresponding portion of SEQ ID NO:2. In one embodiment, metAP is R The allele is MetAP, which contains an amino acid sequence normally as defined in SEQ ID NO:2, but which contains a stop codon within the last quarter. R Encodes proteins.

[0143] In one embodiment, the stop codon results from the insertion and / or deletion of nucleotides.

[0144] In one embodiment, the stop codon is derived from a single nucleotide substitution; in one embodiment, the metAP R The allele is MetAP truncated at position 226 (i.e., exhibits a stop codon at position 226). R The amino acid sequence encoding the protein and shown in SEQ ID NO: 2 is used for numbering. R Codon 226 of the allele is TGA.

[0145] In one embodiment, the metAP R The allele may be a MetAP variant that is ordinarily as defined in SEQ ID NO:2 but includes an amino acid sequence (SEQ ID NO:17) that includes a stop codon at position 226, or that includes an amino acid sequence that has 80% identity to SEQ ID NO:2 and includes a stop codon at position 226, or that is ordinarily a MetAP variant protein (as defined herein) that has at least 80% identity to SEQ ID NO:2 but includes a stop codon at position 226. R Encodes proteins.

[0146] In one embodiment, the metAP R The allele is derived from the MetAP protein having the sequence shown in SEQ ID NO:2. R In one embodiment, the metAP protein encodes a nucleotide sequence similar to that of the nucleotide sequence 144 of the present invention, wherein the glycine at position 226 is replaced with a stop codon. R The allele is a MetAP variant derived from a MetAP variant protein as defined herein that has at least 80% identity to SEQ ID NO:2. R It encodes a protein in which the glycine at position 226 has been replaced with a stop codon.

[0147] Amino acid numbering In the present invention, specific numbering of amino acid residue positions is used in characterizing the MetAP proteins of the present invention. R Alignment of the amino acid sequence of the candidate MetAP protein, or of the MetAP mutant protein, with the MetAP protein defined in SEQ ID NO:2, respectively, identifies the candidate MetAP protein, the MetAP mutant protein, R Numbers can be assigned to amino acid residue positions within the protein, or said MetAP mutant protein, which correspond to the amino acid residue positions or numbering of the amino acid sequence shown in SEQ ID NO:2.

[0148] Another way to describe the amino acid numbering used in this application is to say that an amino acid position is identified by an amino acid position that "corresponds" to a particular position in the amino acid sequence shown in SEQ ID NO:2. This should not be interpreted as meaning that the sequences of the present invention must contain the amino acid sequence shown in SEQ ID NO:2. One of skill in the art will readily recognize that MetAP protein sequences vary among various bacterial strains. Reference to the amino acid sequence shown in SEQ ID NO:2 is used simply to allow identification of a particular amino acid position in any particular MetAP protein. Such amino acid positions can be routinely identified using sequence alignment programs, the use of which is well known in the art.

[0149] lactic acid bacteria The term "lactic acid bacteria" or "LAB" as used herein refers to gram-positive bacteria that ferment sugars to produce exclusively or predominantly lactic acid.

[0150] In one aspect, the present invention provides a metAP of the present invention. R A lactic acid bacterium comprising the allele is provided. In another aspect, the present invention provides a lactic acid bacterium comprising a polynucleotide of the present invention.

[0151] The most industrially useful lactic acid bacteria are found in the genera Streptococcus, Lactococcus, Lactobacillus, Leuconostoc, Pediococcus, Enterococcus, Oenococcus and Bifidobacterium, and therefore in one embodiment the lactic acid bacteria is preferably selected from this group of genera.

[0152] In one embodiment, the lactic acid bacteria of the present invention are of the genus Streptococcus. In one embodiment, the lactic acid bacteria of the present invention are of the species Streptococcus thermophilus or Streptococcus mutans. In a particular embodiment, the lactic acid bacteria of the present invention are of the species Streptococcus thermophilus.

[0153] In one embodiment, the lactic acid bacteria of the present invention are R As previously described, the metAP R "Contains metAP allele" means that the only allele of the metAP gene contained in the genome of the LAB is the metAP R In one embodiment, the lactic acid bacterium of the present invention has the metAP gene of the present invention as the only allele of the metAP gene. R Includes alleles.

[0154] Such lactic acid bacteria can be used to express the original allele of the metAP gene as a R It may be engineered by substitution with an allele; substitutions can be made using conventional techniques as defined herein.

[0155] In another embodiment, such lactic acid bacteria include: a) challenging a parent LAB strain (reference strain) susceptible to cos-type phages with said cos-type phages; b) selecting strains with reduced susceptibility to said cos-type phages (susceptibility to said cos-type phages is determined by efficiency of plaque formation (EOP) assay I); c) selecting from the strains with reduced susceptibility to said cos-type phages identified in step b) a strain having a metAP allele whose sequence differs from the metAP allele of the parent LAB strain; d) determining whether the metAP allele of the strain identified in step c) is a metAP allele as defined herein; R Checking for alleles The method may be performed by a method including:

[0156] A suitable protocol for challenging the strains with the phages of step a) is described in the Materials and Methods section. The reduction in susceptibility (step b) is determined by the Efficiency of Plaque Formation (EOP) Assay I defined herein using the parental LAB strain as a reference strain. In a particular embodiment, the reduction in susceptibility is characterized by an EOP reduction of at least 4 logs, at least 5 logs, at least 6 logs, at least 7 logs, or at least 8 logs as determined by EOP Assay I. The sequence difference between the metAP gene of the strains identified in step b) and the metAP gene of the parental strains can be carried out by any conventional method, for example DNA sequencing. In a particular embodiment, the checking step d) is performed by checking the MetAP gene as defined herein. R Protein-encoding metAP R This is carried out using methods that discriminate between alleles.

[0157] In one embodiment, the method also comprises an additional step [step b1] between steps b) and c), which consists of comparing the CRISPR loci of the strains identified in step b) with the CRISPR loci of the parental LAB strains, and selecting strains whose CRISPR loci are not modified compared to the corresponding CRISPR loci of the parental LAB strains. The comparison of the CRISPR loci can be carried out by comparing the length of the CRISPR loci or by sequencing the CRISPR loci. Step c) is then carried out on the strains selected by step b1).

[0158] Bacteriophage The term "bacteriophage" as used herein has its conventional meaning as understood in the art, i.e., a virus that selectively infects bacteria. Many bacteriophages are specific for a particular genus, species, or strain of bacteria. The term "bacteriophage" is synonymous with the term "phage."

[0159] The definition of "cos-type phages" refers to the presence of specific sequences (cos sites) required for packaging of phage DNA according to the art. Cos-type phages thus incorporate "sticky" ends into their genome (cos sites), in contrast to pac-type phages that use a so-called headful DNA packaging system and thus may incorporate additional redundant DNA into their genome (pac sites). Cos-type phages are known that infect species of bacteria, including lactic acid bacteria, and can be identified based on their DNA packaging strategy or on genetic or genomic analysis.

[0160] In S. thermophilus, cos- and pac-type phages (family Siphoviridae) have been described in detail by Le Marrec et al. (1997). A representative cos-type phage that infects Streptococcus thermophilus strains is phage DT1 (described herein).

[0161] A representative cos-type phage infecting Streptococcus mutans strains is the phage M102AD, which has been described by Delisle et al. (2012). The M102AD phage is available at the Felix d'Herelle Reference Centre for Bacterial Viruses under reference HER503. The host strain for this phage is Streptococcus mutans HER 1503, available at the Felix d'Herelle Reference Centre for Bacterial Viruses under reference HER1503.

[0162] Polynucleotides In a further aspect, the present invention provides a metAP of the present invention. R Allele [MetAP R In one embodiment, the polynucleotide comprises or consists of a metAP protein of the invention. R Allele [MetAP R [coding for proteins].

[0163] In one embodiment, the size of the polynucleotide of the invention is at least 843 nucleotides, at least 846 nucleotides, at least 849 nucleotides, at least 852 nucleotides, at least 855 nucleotides, at least 858 nucleotides, or at least 861 nucleotides. In one embodiment, the size of the polynucleotide of the invention is less than 3 kb, less than 2 kb, or less than 1 kb. In one embodiment, the size of the polynucleotide ranges from a minimum size selected from the group consisting of at least 843 nucleotides, at least 846 nucleotides, at least 849 nucleotides, at least 852 nucleotides, at least 855 nucleotides, at least 858 nucleotides, and at least 861 nucleotides to a maximum size selected from the group consisting of 1 kb, 2 kb, and 3 kb. In one embodiment, the size of the polynucleotide is 858 nucleotides or 861 nucleotides.

[0164] In one embodiment, the polynucleotide of the invention comprises a metAP sequence, as defined herein, flanked independently on one side (5' and 3') or both sides of a nucleotide region spanning 500 bp to 1 kb. R It consists of alleles.

[0165] Typically, polynucleotides encompassed by the scope of the present invention are prepared using recombinant DNA techniques (i.e., recombinant DNA) as described herein. However, in alternative embodiments of the present invention, the polynucleotides can be synthesized in whole or in part using chemical methods well known in the art (see Caruthers MH et al., (1980) Nuc Acids Res Symp Ser 215-23 and Horn T et al., (1980) Nuc Acids Res Symp Ser 225-232).

[0166] MetAP as defined herein RThe polynucleotides encoding the proteins can be identified and / or isolated and / or purified from any lactic acid bacteria. Various methods are known in the art for identifying and / or isolating and / or purifying polynucleotides.

[0167] As an example, once a suitable polynucleotide has been identified and / or isolated and / or purified, PCR amplification techniques can be used to prepare more copies of the polynucleotide.

[0168] As a further example, MetAP R A genomic DNA library can be constructed using chromosomal DNA from protein-producing lactic acid bacteria. R Based on the protein sequence, oligonucleotide probes can be synthesized and used to identify protein-coding clones from a genomic library prepared from lactic acid bacteria.

[0169] In addition, the MetAP of the present invention R Polynucleotides encoding proteins can be prepared synthetically by established standard methods, such as the phosphoramidite method described by Beucage Slet al., 1981, Tetrahedron Letters 22:1859-1869, or the method described by Matthes et al., 1984, EMBO J., 3:801-805. In the phosphoramidite method, oligonucleotides are synthesized, for example, in an automatic DNA synthesizer, purified, annealed, ligated, and cloned into appropriate vectors.

[0170] Polynucleotides can be prepared by polymerase chain reaction (PCR) using specific primers, for example as described in US Pat. No. 4,683,202 or Saiki RK et al., 1988, Science, 239:487-491.

[0171] Polynucleotides and nucleic acids encompassed by the present invention may be isolated or substantially purified. By "isolated" or "purified" it is intended that the polynucleotide is substantially or essentially free from components that are normally found associated with the polynucleotide in the natural state. Such components include other cellular material, media from recombinant production, and various chemicals used in chemically synthesizing the nucleic acid.

[0172] An "isolated" polynucleotide or nucleic acid is typically free from nucleic acid sequences (such as coding sequences present at the 5' or 3' end) that flank the nucleic acid of interest in the genomic DNA of the organism from which the nucleic acid is derived. However, the molecule may contain some additional bases or moieties that do not adversely affect the essential characteristics of the composition.

[0173] vector The present invention also relates to a vector comprising the polynucleotide of the present invention. In one embodiment, the vector is a plasmid.

[0174] In one embodiment, the vector contains one or more selectable marker genes, such as genes that confer antibiotic resistance, such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance. In one embodiment, the vector includes a nucleotide sequence that allows the vector to replicate in the host cell in question. Examples of such sequences include the origins of replication of plasmids pUC19, pACYC177, pUB1 10, pE194, pAMBl, and pIJ702.

[0175] The vectors of the invention can be used to engineer the lactic acid bacteria of the invention.

[0176] Uses and methods based on the polynucleotides or vectors of the invention In one embodiment, the present invention relates to the use of a polynucleotide or vector of the present invention for reducing the susceptibility of a lactic acid bacterium susceptible to cos-type phages to at least one cos-type phage. It is not intended that the LAB of the present invention contain several alleles of the metAP gene. Thus, the polynucleotide or vector is such that the resulting lactic acid bacterium does not contain one allele of the metAP gene in its genome. R In one embodiment, a polynucleotide or vector is used such that an allele of the metAP gene of the original lactic acid bacterium is replaced by a polynucleotide of the invention; the replacement can be carried out using conventional techniques as defined herein.

[0177] In one aspect, the present invention relates to a method for preparing a lactic acid bacteria strain having reduced susceptibility to at least one cos-type phage, comprising: a) preparing a lactic acid bacteria strain sensitive to cos-type phages (reference strain); b) replacing the metAP gene of said lactic acid bacteria strain sensitive to cos-type phages with the polynucleotide of the present invention; c) recovering a lactic acid bacteria strain having reduced susceptibility to at least one cos-type phage; The present invention relates to a method comprising the steps of:

[0178] In one embodiment, step b) comprises translating the metAP gene of said lactic acid bacteria strain sensitive to cos-type phages into the metAP gene of the present invention. R It involves replacing a polynucleotide with one that comprises the allele.

[0179] In one aspect, the present invention relates to a method for preparing a lactic acid bacteria strain having reduced susceptibility to at least one cos-type phage, comprising: a) preparing a lactic acid bacteria strain sensitive to cos-type phages (reference strain); b) The metAP gene of a lactic acid bacteria strain sensitive to cos-type phages is transformed into the metAP gene of the present invention. Rmodifying the allele to have the same sequence; c) recovering a lactic acid bacteria strain having reduced susceptibility to at least one cos-type phage; The present invention relates to a method comprising the steps of:

[0180] In these two method embodiments, the lactic acid bacteria are of the genus Streptococcus. In a particular embodiment, the lactic acid bacteria are of the species Streptococcus thermophilus or Streptococcus mutans. In one embodiment, the lactic acid bacteria are of the species Streptococcus thermophilus.

[0181] Within the use or method of the present invention, the susceptibility of lactic acid bacteria to bacteriophages can be determined by calculating the plaque-forming efficiency (EOP) in the plaque-forming efficiency assay I described herein.

[0182] "Reduction in susceptibility to cos-type phages" means an EOP reduction of at least 4 log as determined by EOP Assay I. In one embodiment, the EOP reduction is at least a 5 log reduction. In one embodiment, the EOP reduction is at least a 6 log reduction. In one embodiment, the EOP reduction is at least a 7 log reduction. In one embodiment, the EOP reduction is at least an 8 log reduction.

[0183] Lactic acid bacteria comprising the polynucleotide of the present invention The present invention relates to a lactic acid bacterium comprising a polynucleotide of the present invention.

[0184] In a further aspect the present invention relates to a lactic acid bacterium obtained by the use or method of the present invention.

[0185] In yet another aspect, the present invention provides a lactic acid bacterium according to the invention produced by the method of the present invention.

[0186] The definitions given above under the section "Lactic acid bacteria" apply here as well.

[0187] In one embodiment, the lactic acid bacteria are of the genus Streptococcus. In one embodiment, the lactic acid bacteria are of the species Streptococcus thermophilus or Streptococcus mutans. In one embodiment, the lactic acid bacteria are of the species Streptococcus thermophilus.

[0188] In one embodiment, the lactic acid bacteria of the present invention may be engineered to contain the polynucleotide of the present invention. It is not intended that the LAB of the present invention contain several alleles of the metAP gene. In one embodiment, the lactic acid bacteria of the present invention contain the polynucleotide of the present invention as the only allele of the metAP gene. Such lactic acid bacteria may be engineered by replacing the original allele of the metAP gene with the polynucleotide of the present invention; the replacement can be carried out using conventional techniques as defined herein. In one embodiment, the lactic acid bacteria of the present invention are engineered by the method of preparing a strain of lactic acid bacteria described herein.

[0189] bacterial composition The present invention also relates to a bacterial composition comprising or consisting of at least one, preferably one, lactic acid bacteria strain of the invention. In a particular embodiment, the lactic acid bacteria is Streptococcus thermophilus. In one embodiment, the bacterial composition is a pure culture, i.e. comprises or consists of a single lactic acid bacteria strain of the invention. In another embodiment, the bacterial composition is a mixed culture, i.e. comprises or consists of a lactic acid bacteria strain of the invention and at least one other bacterial strain. In one embodiment, the bacterial composition is a pure culture, i.e. comprises or consists of a single Streptococcus thermophilus strain of the invention. In another embodiment, the bacterial composition is a mixed culture, i.e. comprises or consists of a Streptococcus thermophilus strain of the invention and at least one other bacterial strain. "At least" one other bacterial strain means one or more, in particular one, two, three, four or five strains.

[0190] Thus, in one embodiment, the bacterial composition of the invention comprises or consists of a lactic acid bacteria strain of the invention, such as a Streptococcus thermophilus strain, as well as one or more further lactic acid bacteria of species selected from the group consisting of Lactococcus spp., Streptococcus spp., Lactobacillus spp. including Lactobacillus acidophilus, Enterococcus spp., Pediococcus spp., Leuconostoc spp., Bifidobacterium spp. and Oenococcus spp., or any combination thereof. Lactococcus species include Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, and Lactococcus lactis subsp. biovar diacetylactis. Bifidobacterium species include Bifidobacterium animalis, especially Bifidobacterium animalis subsp. lactis.Other lactic acid bacteria species include Leuconostoc species, Streptococcus thermophilus, Lactobacillus delbrueckii subsp. bulgaricus, and Lactobacillus helveticus.

[0191] In one embodiment, the bacterial composition comprises or consists of a Streptococcus thermophilus strain of the invention, and at least one Streptococcus thermophilus strain different from the Streptococcus thermophilus strain of the invention, and / or at least one strain of Lactobacillus spp., and / or any combination thereof. In certain embodiments, the bacterial composition comprises or consists of a Streptococcus thermophilus strain of the invention, one or several strains of the species Lactobacillus delbrueckii subsp. bulgaricus, and / or one or several strains of the species Lactobacillus helveticus, and / or any combination thereof, and optionally at least one Streptococcus thermophilus strain different from the Streptococcus thermophilus strain of the invention. In certain embodiments, the bacterial composition comprises or consists of a Streptococcus thermophilus strain of the invention, at least one strain of the Streptococcus thermophilus species different from a Streptococcus thermophilus strain of the invention, and a strain of the Lactobacillus delbrueckii subsp. bulgaricus species.In another specific embodiment, the bacterial composition comprises or consists of a Streptococcus thermophilus strain of the invention and a strain of the species Lactobacillus delbrueckii subsp. bulgaricus.

[0192] In one embodiment, the bacterial composition comprises or consists of a lactic acid bacteria strain of the invention, Lactococcus lactis subsp. lactis, and / or Lactococcus lactis subsp. cremoris. In one embodiment, the bacterial composition comprises or consists of a Streptococcus thermophilus strain of the invention, Lactococcus lactis subsp. lactis, and / or Lactococcus lactis subsp. cremoris.

[0193] In certain embodiments of any bacterial composition defined herein, either as a pure culture or a mixed culture, the bacterial composition further comprises at least one probiotic strain, such as Bifidobacterium animalis subsp. lactis, Lactobacillus acidophilus, Lactobacillus paracasei, or Lactobacillus casei.

[0194] In a particular embodiment, the bacterial composition is in a frozen, dried, lyophilized, liquid or solid format, in the form of a pellet or frozen pellet, or in a powder or dry powder, either as a pure culture or mixed culture as defined above. In a particular embodiment, the bacterial composition of the invention is in a frozen format, or in the form of a pellet or frozen pellet, in particular contained in one or more boxes or sachets. In another embodiment, the bacterial composition defined herein is in powder form, for example a dry powder or lyophilized powder, in particular contained in one or more boxes or sachets.

[0195] In a particular embodiment, the bacterial composition of the invention is prepared either as a pure culture or as a mixed culture as defined above, and whatever the format (frozen, dried, lyophilized, liquid or solid format, in pellet or frozen pellet form, or powder or dry powder), containing a lactic acid bacteria strain of the invention at a concentration of 10 per gram of bacterial composition. 5 ~10 12 In a particular embodiment, the concentration of the lactic acid bacteria strain in the bacterial composition of the present invention is within the range of 10 per gram of bacterial composition. 7 ~10 12 in the range of cfu, particularly at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , or at least 10 11 cfu / g of bacterial composition. In a particular embodiment, the concentration of the lactic acid bacteria strain of the present invention (as a pure culture or as a mixed culture) in the bacterial composition, when in the form of a frozen concentrate or a dry concentrate, is 10 8 ~10 12 cfu / g of frozen concentrate or dried concentrate, more preferably at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , or at least 10 12cfu / g of frozen concentrate or dry concentrate.

[0196] In a particular embodiment, the bacterial composition of the invention, either as a pure culture or a mixed culture as defined above, and whatever the format (frozen, dried, lyophilized, liquid or solid format, in pellet or frozen pellet form, or powder or dry powder), contains 10 Streptococcus thermophilus strains of the invention per gram of bacterial composition. 5 ~10 12 In certain embodiments, the concentration of Streptococcus thermophilus strains in the bacterial composition of the present invention is in the range of 10 per gram of bacterial composition. 7 ~10 12 in the range of cfu, particularly at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , or at least 10 11 cfu / g of bacterial composition. In a particular embodiment, the concentration of Streptococcus thermophilus strains (as pure cultures or mixed cultures) in the bacterial composition, when in the form of a frozen concentrate or dry concentrate, is 10 8 ~10 12 cfu / g of frozen concentrate or dried concentrate, more preferably at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , or at least 10 12 cfu / g of frozen concentrate or dry concentrate.

[0197] In one embodiment, whatever the number and nature of the strains, the composition format and concentration of the strains, the bacterial composition further comprises food acceptable components.

[0198] In a further aspect, a method for producing a fermented product is provided, comprising a) inoculating a substrate with a lactic acid bacteria or bacterial composition according to the invention and b) fermenting the inoculated substrate to obtain a fermented product. In a particular embodiment, the lactic acid bacteria strain of the invention is inoculated as a bacterial composition as defined herein, such as a pure culture or a mixed culture. Preferably, the substrate is a dairy substrate, more preferably milk. By "dairy substrate" is meant milk of animal and / or plant origin. In a particular embodiment, the dairy substrate is of animal origin, in particular of any mammal, such as cow, goat, sheep, buffalo, zebra, horse, donkey or camel. The milk may be in the natural state, reconstituted milk, skimmed milk or milk supplemented with compounds essential for the growth of the bacteria or for the subsequent processing of the fermented milk. Preferably, the dairy substrate comprises a solid item. Preferably, the solid item comprises or consists of fruit, chocolate product or cereals. Preferably, the fermented product is a fermented dairy product. Thus, in certain embodiments, the present invention also provides a method of producing a fermented milk product, the method comprising: a) inoculating a milk substrate with a Streptococcus thermophilus strain or bacterial composition of the present invention; and b) fermenting the inoculated milk substrate to obtain a fermented milk product.

[0199] The present invention also provides in a further aspect the use of a lactic acid bacteria or bacterial composition according to the invention for producing a food or feed product, preferably a fermented dairy product.

[0200] The present invention also relates to a fermented milk product obtained with the lactic acid bacteria strain or bacterial composition of the invention, in particular obtained or obtainable by the method of the invention. Thus, the present invention relates to a fermented milk product comprising the lactic acid bacteria strain of the invention. Preferably, the fermented milk product comprises the Streptococcus thermophilus strain of the invention. In a particular embodiment, the fermented milk food product of the invention is fresh fermented milk.

[0201] product Any product prepared from, containing or including the lactic acid bacteria or bacterial compositions of the present invention is contemplated according to the present invention.

[0202] Suitable products include, but are not limited to, food or feed products.

[0203] These include, but are not limited to, fruits, legumes, forage crops, and vegetables, including derived products, grains and grain-derived products, dairy and dairy-derived products, meat, poultry, and seafood. Preferably, the food or feed product is a dairy product, a meat product, or a cereal product.

[0204] The term "food" is used in a broad sense and includes feed, foodstuff, food ingredient, food supplement, and functional food. In this specification, the term "food" is used in a broad sense and includes human food and animal food (i.e., feed). In a preferred embodiment, the food is for human consumption.

[0205] The term "dietary ingredient" as used herein includes formulations that are or can be added to foods, including formulations that can be used at low levels in a wide variety of products, for example those requiring acidification or emulsification.

[0206] The term "functional food" as used herein means a food that is not only capable of providing a nutritional effect and / or taste satisfaction, but also capable of delivering additional beneficial effects to the consumer. Although there is no legal definition of functional food, most parties interested in this field agree that there are foods marketed as having certain health benefits.

[0207] The lactic acid bacteria of the present invention may be or may be added to a food ingredient, a food supplement, or a functional food.

[0208] The food product may be in the form of a solution or as a solid, depending on the application and / or the mode of use and / or the mode of administration.

[0209] The lactic acid bacteria of the invention can be used in the preparation of food products, such as confectionery products, dairy products, meat products, poultry products, fish products or bakery products.

[0210] By way of example, the bacteria can be used as ingredients to prepare soft drinks, fruit juices or beverages containing whey protein, tea, cocoa drinks, dairy drinks, and lactobacillus drinks, yoghurt, drinking yoghurt, and wine.

[0211] Preferably, the food product described herein is a dairy product. More preferably, the dairy product described herein is one or more of the following: yoghurt, cheese (e.g. sour curd cheese, hard cheese, semi-hard cheese, cottage cheese), buttermilk, quark, sour cream, kefir, fermented whey-based beverage, koumiss, milk beverage, yoghurt beverage, fermented milk, mature cream, cheese, fromage frais, milk, dairy retentate, processed cheese, creamy dessert, or infant milk.

[0212] Preferably, the food product described herein is a fermented food product. More preferably, the food product described herein is a fermented dairy product, such as fermented milk, yogurt, cream, mature cream, cheese, fromage frais, dairy drink, processed cheese, creamy dessert, cottage cheese, yogurt drink, dairy retentate, or infant milk.

[0213] Preferably, the dairy product according to the invention comprises milk of animal and / or plant origin.

[0214] Milk is understood to mean anything of animal origin, in particular any mammal, such as cow, goat, sheep, buffalo, zebra, horse, donkey or camel. The term milk is also used for what is commonly called vegetable milk, i.e. processed plant material or otherwise extracts, for example of legumes (soybean, chickpea, lentil, etc.) or oilseeds (rapeseed, soybean, sesame, cotton, etc.), which contain proteins in solution or in colloidal suspension, which can be coagulated by chemical action, by acid fermentation and / or by heat. Finally, the word milk also denotes a mixture of animal milk and of vegetable milk.

[0215] In one embodiment, the term "milk" refers to commercially available UHT milk supplemented with 3% (w / w) semi-skimmed milk powder pasteurized by heating at 90°C±0.2°C for 10 minutes±1 minute.

[0216] MetAP of the present invention R Amino acid equivalents in a protein sequence In one embodiment, the MetAP of the present invention R The protein is characterized by comprising an amino acid selected from the group consisting of a lysine or an equivalent amino acid at position 57, a proline or an equivalent amino acid at position 153, a glutamic acid or an equivalent amino acid at position 168, a glutamine or an equivalent amino acid at position 206, an aspartic acid or an equivalent amino acid at position 228, a glutamine or an equivalent amino acid at position 233, and a leucine or an equivalent amino acid at position 233.

[0217] "The equivalent amino acid" is MetAP R Protein-encoding metAP Rmeans any amino acid having similarity in the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residue, so long as the allele, when inserted in place of an allele of the metAP gene of Streptococcus thermophilus strain SMQ-301, reduces susceptibility to phage DT1 (as defined herein), as determined by the efficiency of plaque formation (EOP) assay I.

[0218] In one embodiment, amino acids equivalent to lysine are selected from the group consisting of glutamic acid, aspartic acid, and arginine.

[0219] In one embodiment, the amino acid equivalent to proline is selected from the group consisting of alanine and glycine.

[0220] In one embodiment, the equivalent amino acid to glutamic acid is selected from the group consisting of aspartic acid and lysine.

[0221] In one embodiment, amino acids equivalent to glutamine are selected from the group consisting of asparagine and serine.

[0222] In one embodiment, the equivalent amino acids to aspartic acid are selected from the group consisting of glutamic acid and lysine.

[0223] In one embodiment, amino acids equivalent to leucine are selected from the group consisting of methionine, valine, isoleucine, and phenylalanine.

[0224] Percentage of MetAP protein identity A metAP allele is defined herein as a metAP mutant allele (encoding a MetAP mutant protein) that 1) encodes a MetAP protein whose sequence has at least 80% identity to SEQ ID NO:2, and 2) does not reduce EOP or reduces EOP by less than 3 logs (sensitivity to phage DT1 is determined by plaque formation efficiency (EOP) assay I). A percentage of at least 80% identity to SEQ ID NO:2 means a percentage of identity selected from the group consisting of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%. In one embodiment, the sequence of the MetAP mutant protein differs from SEQ ID NO:2, but the size of the MetAP mutant protein is the same as the MetAP protein (286 amino acid residues) defined in SEQ ID NO:2.

[0225] 1) encodes a MetAP protein, the sequence of which has at least 80% identity to SEQ ID NO:2; and 2) reduces the susceptibility of a Streptococcus thermophilus SMQ-301 derivative to phage DT1 by at least 4 logs, the SMQ-301 derivative being a Streptococcus thermophilus SMQ-301 strain in which the metAP allele has been replaced by a metAP allele, as determined by efficiency of plaque formation (EOP) assay I. R Allele (MetAP R A percentage of identity of at least 80% to SEQ ID NO:2 means a percentage of identity selected from the group consisting of at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%. R The protein sequence differs from SEQ ID NO:2, but isR The size of the protein is the same as the MetAP protein defined in SEQ ID NO:2 (286 amino acid residues).

[0226] Comparison of sequences can be performed by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially or freely available computer programs can calculate similarity or identity scores between two or more sequences.

[0227] The percentage of homology can be calculated over aligned contiguous sequences, i.e., one sequence is aligned to another and each amino acid in one sequence is directly compared to the corresponding amino acid in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively small number of residues.

[0228] This is a very simple and consistent method, but it can greatly reduce identity when performing a global alignment, since it fails to take into account that, for example, in an otherwise identical pair of sequences, an insertion or deletion will cause subsequent amino acid residues to be removed from the alignment. As a result, most sequence comparison methods are designed to produce an optimal alignment that takes into account possible insertions and deletions without unduly penalizing the overall identity score. This is achieved by inserting "gaps" in the sequence alignment to attempt to maximize local identity. These more complex methods assign a "gap penalty" to each gap that occurs in the alignment, such that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible (reflecting a higher relatedness between the two compared sequences) will achieve a higher score than one with more gaps. Typically, an "affine gap cost" is used that imposes a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap (gap extension penalty). This is the most commonly used gap scoring system. A high gap penalty will of course produce an optimized alignment with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, when using such software for sequence comparison, it is possible to use the default values, since they have been adjusted to give reasonable results in most cases. Thus, calculation of the maximum percentage of identity first requires that an optimal alignment is created, taking into account gap penalties. A suitable computer program for performing such an alignment is Vector NTI (Invitrogen Corp.). Examples of software that can perform sequence comparison include, but are not limited to, the BLAST package (Ausubel et al., 1999, Short Protocols in Molecular Biology, 4th Ed-Chapter 18).

[0229] Although alignment quality can be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​commonly used that assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. One example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). Vector NTI programs generally use the public default values, or a custom comparison table if provided (see user manual for further details). Alternatively, the percentage of similarity can be calculated using the multiple alignment feature in Vector NTI (Invitrogen Corp.), which is based on an algorithm similar to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244).

[0230] Once the software has produced an optimal alignment, it is possible to calculate the percentage of sequence similarity, and preferably the percentage of sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0231] In one embodiment, the degree of identity with respect to protein (amino acid) sequences is determined over at least 50 contiguous amino acids, over at least 100 contiguous amino acids, over at least 150 contiguous amino acids, over at least 200 contiguous amino acids, or over at least 250 contiguous amino acids.

[0232] In one embodiment, the degree of identity for amino acid or protein sequences may be determined over the entire sequence of SEQ ID NO:2.

[0233] In one embodiment, the sequence [MetAP RThe candidate mutant protein sequence and SEQ ID NO:2 are aligned by a global alignment program and sequence identity is calculated by identifying the number of exact matches identified by the program divided by the length of the candidate sequence.

[0234] In one embodiment, MetAP R The degree of sequence identity between a candidate mutant protein sequence and SEQ ID NO:2 is determined by: 1) aligning the two sequences with any appropriate alignment program using a default scoring matrix and default gap penalties; 2) identifying the number of exact matches, where an exact match is when the alignment program identifies an identical amino acid in the two aligned sequences at a given position in the alignment; and 3) dividing the number of exact matches by the length of the candidate sequence.

[0235] In one embodiment, the global alignment program is selected from the group consisting of CLUSTAL and BLAST, in particular CLUSTAL, using default parameters, and sequence identity is calculated by identifying the number of exact matches identified by the program divided by the length of the subject sequence.

[0236] In one embodiment, the global alignment program is CLUSTAL using default parameters, and sequence identity is determined in the BioEdit software (http: / / www.mbio.ncsu.edu / BioEdit / bioedit.html) [select the "Sequence" drop-down menu, then the "Pairwise alignment" submenu, and then select the "Calculate identity / similarity for two sequences" menu item].

[0237] General recombinant DNA methodology techniques The present invention employs, unless otherwise indicated, conventional techniques of biochemistry, molecular biology, microbiology, and recombinant DNA, which are within the capabilities of one of ordinary skill in the art, and such techniques are explained in the literature. For example, J. Sambrook, EFFritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3, Cold Spring Harbor Laboratory Press; Ausubel, FMet al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and, 16, John Wiley&Sons, New, York, NY); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; MJ Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, Irl Press; and DMJ Lilley and JEDahlberg, 1992, Methods of See Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press. Each of these texts is generally incorporated herein by reference.

[0238] The present invention will now be further illustrated by examples, which should serve to assist those skilled in the art in practicing the present invention and are not intended to limit the scope of the present invention in any way. EXAMPLES

[0239] Materials and Methods Cell and phage proliferation Bacterial strains, phages, and plasmids are listed in Table 1. S. thermophilus was grown in M17 medium (Oxoid, Nepean) supplemented with 0.5% lactose (LM17) at 37°C or 42°C. Chloramphenicol (Sigma, Oakville) was added to a final concentration of 5 μg / mL, if necessary, for growth and selection of S. thermophilus strains containing pNZ123 (De Vos et al. 1987) and derivatives. For solid media, agar (LabMat, Quebec) was added to a final concentration of 1%. Escherichia coli was grown with agitation at 37°C in Luria Broth (LB) supplemented with 30 μg / mL kanamycin and 20 μg / mL chloramphenicol for selection. Streptococcus mutans HER1503 was grown at 37°C and 5% CO in Brain Heart Infusion (BHI, Difco) supplemented with 10 μg / mL chloramphenicol for selection of pNZ123+ cells. Phages were propagated as previously described (Hynes et al., 2017).

[0240] Isolation of bacteriophage-insensitive mutants Bacteriophage insensitive mutants (BIMs) were isolated using a soft agar overlay assay as previously described (Hynes et al., 2017). Briefly, wild-type host S. thermophilus SMQ-301 was challenged with phage DT1. The bacterial host was incubated in LM17 at 42 °C for 1 h at 600 nm optical density (OD 600nm) reached 0.6. 300 μL of the culture was then added to 3 mL of molten LM17 soft agar supplemented with calcium chloride (10 mM) and phage DT1 was added to achieve a multiplicity of infection (MOI) of 0.1. The plates were incubated at 42°C for 16 h. The resulting colonies were streaked onto LM17+1% agar and screened for the presence or absence of spacer gain at the CR1 and CR3 loci using primers CR1-fwd and CR1-revLong for the CR1 locus and primers CR3-fwd and CR3-rev for the CR3 locus (Hynes et al., 2017). If no spacer gain was detected within the CRISPR array, the BIM was saved for genome sequencing.

[0241] [Table 1]

[0242] DNA isolation, sequencing, and bioinformatics analysis Genomic DNA of BIM was extracted as described previously (Bissonnette et al., 2000). Genomes were sequenced using an Illumina MiSeq platform. Libraries were prepared with the Nextera XT DNA Sample Preparation Kit according to the manufacturer's instructions and sequenced using MiSeq reagents (2 × 250 nt paired ends). Average coverage ranged from 6.8 to 80.1-fold. Reads obtained for the nine BIM genomes were aligned against the S. thermophilus SMQ-301 genome (Labrie et al., 2015) using Novoalign (http: / / www.novocraft.com) with default settings. Mutations were extracted from the alignment files using SAMTools (Li et al., 2009). Mutations in the bacterial genome were mapped and translated into protein sequences using an in-house Python script. The mutation with the highest score provided by SAMTools was considered as the first.

[0243] Complementation assays S. thermophilus SMQ-301:MetAP H206QThe strain was complemented with the metAP allele shown in SEQ ID NO:1 (metAP allele of SMQ-301). First, the metAP allele shown in SEQ ID NO:1 was cloned into pNZ123 using Gibson assembly. The vector pNZ123 was linearized with XbaI (Roche) according to the manufacturer's instructions. The insert was amplified with primers SJL154 and SJL155 with Q5 high fidelity DNA polymerase (NEB). Both primers had a 30 nt extension that complemented the 3' and 5' ends of the linearized vector, respectively. A 3:1 insert / vector ratio was used for the assembly. A master mix for Gibson assembly was prepared as previously described (Gibson et al., 2009). The resulting construct was transformed into E. coli NEB5α according to the manufacturer's instructions and clones were selected on LB medium supplemented with 1% agar and 25 μg / mL chloramphenicol. One clone was confirmed by Sanger sequencing (ABI 3730xl) at the Plateforme de Sequencage et de Genotypage des Genomes of the CHUL Center. Plasmid DNA was extracted using the QIAprep Spin Miniprep kit (Qiagen) and transformed into S. thermophilus (Hynes et al., 2017). Clones were selected by spreading the transformation mixture on LM17 supplemented with 1% agar and 5 μg / mL chloramphenicol.

[0244] Directed and Random Mutagenesis The metAP allele carrying the desired mutation was amplified by PCR using primers SJL128 and SJL130 (Table 2). At least 10 PCR reactions of 50 μL were performed per assay to obtain enough DNA for natural transformation. All reactions were pooled and precipitated by adding 1.5 mL of 95% ethanol and 75 μL of 3 M sodium acetate (pH 5.2). The tubes were centrifuged at 25,000×g for 20 min at 4° C. The DNA pellet was washed twice with 1 mL of 70% ethanol and dried at room temperature for 10 min to remove traces of ethanol. Finally, the pellet was dissolved in 150 μL of water. The linear DNA fragment was introduced into S. thermophilus by natural transformation (Gardan et al., 2009). Briefly, S. thermophilus was grown overnight at 37° C. in 1 mL of LM17. The culture was centrifuged at 17,000 x g for 1 min, and the bacterial cells were washed twice with synthetic medium (CDM) and harvested in 1 mL of CDM. The final culture was diluted 30-fold to an OD of 0.05. 600nm was obtained and incubated at 37°C for 75 min before storing at -20°C. For transformation, 1 μM peptide ComS and 1 μg linear DNA were added to 300 μL of naturally competent cells. The mixture was incubated at 37°C for 3 h. The cells were serially diluted and spread on non-selective LM17 agar to obtain isolated colonies. Colonies were screened using a PCR approach specifically designed to detect clones carrying the desired mutation. One of the primers used in this protocol contained the desired mutation at its 3' end. Thus, PCR amplification was only positive if the mutation was present in the screened clone. The sequences of primers SJL154 and SJL155 used to screen transformants are listed in Table 2. For random mutagenesis, the same protocol as for directed mutagenesis was used with the only exception that MnSO4 was added (10 μM final) and phage DT1 was used to select resistant clones.

[0245] [Table 2]

[0246] Directed metAP mutagenesis in Streptococcus mutans The metAP gene allele was then analyzed for the desired mutation (MetAP H206Q The fragments were amplified from S. mutans HER 1503 with two sets of primers containing the nucleotide sequence SJL160 and SJL161, and the nucleotide sequence SJL162 and SJL163. The two sets of primers (SJL160 and SJL161, and SJL162 and SJL163) generated two overlapping amplicons with mutations in the overlapping regions. The two fragments were purified using a QIAquick PCR Purification Kit (Qiagen) and fused by Gibson Assembly (Gibson et al., 2009). PCR was then performed on the Gibson Assembly sample with primers SJL160 and SJL163 to amplify the fusion fragment. After purification using a QIAquick PCR purification kit, the mutated metAP amplicon was transformed into S. mutans HER 1503 by natural transformation; competence stimulating peptide (CSP) ordered from Biomatik was added at a concentration of 1 μM to 500 μl of an exponentially growing culture of S. mutans (OD of 0.1). 600nm ) along with 1 μg of purified mutant metAP amplicon (Dufour et al., 2011). Cultures were incubated overnight at 37°C with 5% CO2 before being mixed with virulent phage M102AD in BHI supplemented with 0.7% agar and plated on BHI supplemented with 1% agar. The metAP allele in surviving derivatives was amplified by PCR using primers SJL160 and SJL163 and sequenced to confirm the presence of the mutation.

[0247] BIM S.mutans HER 1503:MetAP H206QFor complementation of, the metAP allele of HER 1503 S. mutans was cloned into pNZ123. pNZ123 was linearized with XhoI and EcoRI to remove 24 base pairs between the two sites, and the resulting plasmid was purified using a QIAquick Gel Extraction kit (Qiagen). The metAP allele was amplified from S. mutans HER 1503 using primers CM_145 and CM_146 to generate a gene flanked by XhoI and EcoRI restriction sites. The PCR sample was purified using a QIAgen PCR purification kit, cut with XhoI and EcoRI, and ligated into linearized pNZ123 using T4 DNA ligase. The insert / vector molar ratio used during ligation was 3:1. The ligated plasmids were transformed into E. coli NEB5α according to the manufacturer's instructions and plated on solid LB medium supplemented with 25 μg / mL chloramphenicol. The sequences cloned into the complementing plasmid were confirmed by amplification of the metAP allele with primers CM_145 and CM_146 and sequencing. Plasmid DNA was extracted using a QIAprep Spin Miniprep kit and used to identify the S. mutans HER 1503:MetAP allele. H206Q The strain was transformed into BHI at an OD of 0.1. 600nm The cultures were grown to reach a total volume of 100 μL, and 500 μL aliquots were exposed to 1 μM CSP and transformed with 1 μg of the complementing plasmid. After a 2.5-h incubation period at 37° C. with 5% CO2, the cultures were plated onto BHI+1% agar supplemented with 10 μg / mL chloramphenicol.

[0248] Mutation stability test To test the stability of the mutation H206Q, strains S. thermophilus SMQ-301 and DGCC7796, and their respective mutants [SMQ-301:MetAP H206Q and DGCC7796:MetAP H206Q

[00116] was inoculated (1%) with 1% of the 1% inoculum. The strains were incubated at 42°C during the day. The 1% inoculum was then transferred to fresh medium or milk and incubated at 37°C overnight. After 9 transfers (5 days, 4 nights), corresponding to approximately 60 generations, 10 colonies were randomly selected from each of the four strains to identify S. thermophilus SMQ-301 and S. thermophilus SMQ-301:MetAP H206Q Phage DT1(cos), S. thermophilus DGCC7796 and S. thermophilus DGCC7796:MetAP H206Q The susceptibility of SMQ-301 and SMQ-301:MetAP to phages D4090 (cos) and D4274 (pac) was tested. Sequencing of the metAP gene and CR1 was performed after transfer and strain differentiation. H206Q Colonies were run with primers SJL128 / SJL130 and CR1-fwd / CR1-rev to confirm the presence of the mutation.

[0249] Example 1 Mutations in the gene encoding methionine aminopeptidase reduce susceptibility to phages Several BIMs of S. thermophilus SMQ-301 with reduced susceptibility to phage DT1 were isolated according to the protocol described above in the section entitled "Bacteriophage-insensitive mutant isolation". More than 95% of the BIMs had acquired a new spacer in their CRISPR locus that targets the phage DT1 genome. Nine SMQ-301 BIMs that did not acquire a new spacer were selected to speculate that they had a mutation elsewhere in the bacterial genome that achieved reduced susceptibility to phage DT1. The genomes of these nine non-CRISPR BIMs were sequenced. By comparing these sequences with the genome of S. thermophilus SMQ-301 (Labrie et al., 2015), several mutations were found. Many of these mutations could potentially be involved in phage infection, but the same mutation (T->G) occurred at position 618 of the metAP gene in three of the nine BIMs. This gene encodes methionine aminopeptidase (MetAP) and the mutation induces an amino acid substitution of histidine to glutamine at position 206 in the MetAP protein (H206Q). BLAST searches showed that this histidine is conserved in all analyzed MetAP protein sequences. The S. thermophilus MetAP protein has not been studied previously but shares 89% identity and 96% similarity with the MetAP protein of Streptococcus pneumoniae TIGR4, which has also been characterized at the structural level (PDB 4KM3) (Arya et al. 2013).

[0250] Example 2 Mutations in the metAP gene reduce susceptibility to phages in S. thermophilus Reduced susceptibility to phages is associated with MetAP H206QTo verify that the mutation is due to the MetAP mutation (and not due to other mutations in the genome), H206Q Protein-encoding alleles were amplified and transformed into S. thermophilus SMQ-301 using natural competence. Phage selection is fairly effective, but it greatly increases the probability of selecting for CRISPR BIM and other mutations that confer reduced susceptibility to phages. Instead, a PCR strategy was designed to detect transformants that integrated the PCR product carrying the mutation into the genome in the absence of phage selection. Primer SJL150 in combination with SJL151, which carries the desired mutation at its 3' end, was used to specifically detect the mutation in the bacterial genome. To avoid false positives due to the potential presence of residual linear DNA from the transformation in the bacterial cytoplasm, primers were designed that matched the flanking genomic regions of the transformed DNA fragment. A total of 282 clones were screened for the presence or absence of integration of the H206Q metAP allele to detect the desired MetAP allele. H206Q Four positive clones carrying the mutation were obtained. One of these clones, S. thermophilus SMQ-301:MetAP H206Q were randomly selected and their phage susceptibility was determined by EOP assay I. No plaque formation was visible on bacterial cultures with different dilutions of phage lysate, indicating a fairly high phage resistance phenotype (EOP10 -8 Various experimental conditions were used to try to isolate phage mutants that would overcome the effect of H206Q MetAP, but to no avail. Changing the incubation temperature, using anaerobic conditions, adding different concentrations of glycine to the medium to weaken the bacterial cell wall, replacing the agar with different concentrations of agarose, and attempting rescue experiments in liquid medium all failed to isolate phage mutants that would overcome the effect of H206Q MetAP. H206Q No phage DT1 derivatives capable of growing on .

[0251] [Table 3]

[0252] Example 3 Complementation with an allele of the metAP gene of SMQ-301 restores phage sensitivity. An allele of the metAP gene of SMQ-301 was cloned into the shuttle expression vector pNZ123. The resulting construct was designated S. thermophilus SMQ-301:MetAP H206Q The mutation was complemented in trans by transformation into pNZ123:MetAP. Sensitivity to phage DT1 was restored to two of the three BIMs by complementation with the plasmid pNZ123:MetAP (Table 4). H206Q It was confirmed that the substitutions were responsible for the phage phenotype. Of note, for one BIM (#3), the sensitivity to phage DT1 was not fully restored (EOP=0.02), suggesting that mutations in other genes are likely responsible for the phage DT1 resistance (Table 4). The mutated metAP gene was also cloned into pNZ123 and transformed into S. thermophilus SMQ-301. pNZ123:MetAP H206Q There was no difference in EOP between strains carrying pNZ123 compared to strains carrying pNZ123, suggesting that the mutation does not result in a dominant phenotype.

[0253] [Table 4]

[0254] Example 4 The MetAPH206Q substitution has broad coverage against cos-type phages MetAP H206QTo determine whether the resistance conferred by the substitution was phage dependent, we tested the cos-type phage MD2, which can also infect SMQ-301, and was stringently inhibited by H206Q MetAP.

[0255] To demonstrate that this mutation is not strain dependent, MetAP H206Q was introduced into several S. thermophilus strains using directed mutagenesis. Mutations were introduced into S. thermophilus strain DGCC782, which is sensitive to cos-type phages N1032, N1117, N1119, N1169, N1358, and N3782. Mutations were also introduced into S. thermophilus strain DGCC7710, which is sensitive to cos-type phages, namely D5691, D5913, D6037, and D6215, and several pac-type phages, namely 858, 2972, D3288, 4259, D4752, D4754, and D939. Finally, the mutation was introduced into S. thermophilus DGCC7796, which is sensitive to several cos-type phages, namely D4090, D5821, D4807, and D6179, and several pac-type phages, namely D2765, D4274, D5787, and D5876. The mutation was less effective against cos-type phages infecting S. thermophilus DGCC7710 (EOP10 -5 ), which conferred resistance to all cos-type phages tested. H206Q The introduction of the allele requires at least 10 -6 This led to a reduction in the EOP of 10. -1The MetAP mutants corresponded to the lysis zone of 100 ng / ml of phage (the lysis zone of 100 ng / ml of phage in the phage genome). Furthermore, no phage mutants could be recovered from the assay. On the other hand, none of the S. thermophilus pac-type phages tested were affected by the mutations, suggesting that MetAP is not important for the replication of this group of phages.

[0256] To determine whether MetAP mutations confer reduced susceptibility to other bacterial species, H206Q The protein-encoding allele was introduced into S. mutans strain HER 1503, which is sensitive to the cos-type phage M102AD (Delisle et al., 2012). The metAP gene of S. mutans HER 1503 has the same length (861 bp) as that of S. thermophilus SMQ-301. The two metAP genes share 74% identity, while the two methionine aminopeptidases (MetAP proteins) share 85% identity. The histidine residue found at position 206 is present in S. mutans. Introducing MetAP (in place of the original metAP allele) into the S. mutans genome H206Q Insertion of the protein-encoding allele resulted in reduced susceptibility to phage M102AD (EOP<1×10 -7 , Table 5). Complementation of the mutant strain with the original metAP allele restored the phage-susceptible phenotype (Table 5). Overall, these data suggest a relatively widespread role for MetAP in phage replication.

[0257] [Table 5]

[0258] Example 5 Other mutations in MetAP reduce susceptibility to phage DT1 To determine whether other mutations in the metAP gene could reduce susceptibility to phage DT1, an error-prone PCR amplification approach was used. The resulting PCR products were transformed into S. thermophilus SMQ-301 using natural competence. Since no antibiotic resistance marker was introduced, clones were selected using phage DT1. A positive control (MetAP H206Q ) resulted in more BIM than the negative control (no DNA). The gene encoding the MetAP protein was sequenced. Seven mutants had mutations in the metAP gene that resulted in amino acid substitutions. For six of these mutants, the original amino acid was replaced by another amino acid (Q57K, L153P, A168E, V228D, P233Q, and P233L). For another mutant, the original amino acid was replaced by a stop codon (G226*) (Table 6).

[0259] These seven mutants were retested for susceptibility to phage DT1. All seven mutants had reduced susceptibility to phage DT1, with an EOP of 1×10 -6 When the resistant strain was complemented with pNZ123:MetAP, MetAP shown in SEQ ID NO:2, restored the phage susceptibility phenotype as observed above, confirming that all seven of these mutations in the MetAP protein reduce susceptibility to cos-type phages.

[0260] [Table 6]

[0261] Example 6 MetAP H206Q The mutation is stable One of the important features for industrial fermentation is the stability of the phage resistance phenotype. Thus, the mutant MetAP mutants of S. thermophilus SMQ-301 and DGCC7796, and their mutants, in milk and LM17 over 60 generations were H206Q The stability of the mutant MetAP was confirmed. All selected colonies of the wild-type strain remained phage sensitive, while all 10 colonies from each mutant remained phage resistant. H206Q This shows that it is stable.

[0262] conclusion Here, it is demonstrated that reduced susceptibility to cos-type phages can be obtained by mutations in the metAP gene of S. thermophilus and S. mutans. However, none of the mutations identified in the metAP gene reduce susceptibility to pac-type phages. Finally, the inability to isolate phage mutants that overcome the MetAP H206Q substitution confirmed that such a mutation can confer a robust resistance phenotype.

[0263] All publications mentioned herein above are incorporated herein by reference. Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with certain preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

Claims

1. Methionine aminopeptidase (MetAP) Ｒ Protein) metAP encoding a protein Ｒ A strain of the genus Streptococcus selected from the group consisting of a Streptococcus thermophilus strain or a Streptococcus mutans strain, comprising the metAP allele, Ｒ The allele is defined as a metAP allele that reduces the susceptibility of a Streptococcus thermophilus SMQ-301 derivative to phage DT1, the SMQ-301 derivative being Ｒ Streptococcus thermophilus strain SMQ-301, in which the metAP allele has been replaced by an allele, the susceptibility to phage DT1 being determined by the efficiency of plaque formation (EOP) assay I, R The protein has an amino acid sequence that has at least 90% identity with SEQ ID NO:

2. comprising or consisting of the sequence of said MetAP R protein comprises an amino acid selected from the group consisting of a lysine at position 57, a proline at position 153, a glutamic acid at position 168, a glutamine at position 206, an aspartic acid at position 228, a glutamine at position 233, and a leucine at position 233, where the amino acid sequence shown in SEQ ID NO:2 is used for numbering; or The MetAP R protein is: a) an amino acid sequence comprising a stop codon at position 226, and otherwise as defined in SEQ ID NO:2; b) an amino acid sequence having at least 90% identity to SEQ ID NO:2 and including a stop codon at position 226; c) an amino acid sequence defined as the amino acid sequence of a MetAP mutant protein having at least 90% identity to SEQ ID NO:2, including a stop codon at position 226; A strain comprising or consisting of an amino acid sequence selected from the group consisting of:

2. The metAP gene is the only allele of the metAP gene. Ｒ Alleles included, claims Item 1. The strain described in item 1.

3. MetAP Ｒ A polynucleotide encoding a protein, the polynucleotide being a metAP allele that reduces the susceptibility of a Streptococcus thermophilus SMQ-301 derivative to phage DT1, the SMQ-301 derivative comprising the metAP allele Ｒ Streptococcus thermophilus strain SMQ-301, in which the metAP allele has been replaced by an allele, the susceptibility to phage DT1 being determined by the efficiency of plaque formation (EOP) assay I, R The protein has at least 90% identity with SEQ ID NO:

2. comprising or consisting of the amino acid sequence the sequence of said MetAP R protein comprises an amino acid selected from the group consisting of a lysine at position 57, a proline at position 153, a glutamic acid at position 168, a glutamine at position 206, an aspartic acid at position 228, a glutamine at position 233, and a leucine at position 233, where the amino acid sequence shown in SEQ ID NO:2 is used for numbering; or The MetAP R protein is: a) an amino acid sequence comprising a stop codon at position 226, and otherwise as defined in SEQ ID NO:2; b) an amino acid sequence having at least 90% identity to SEQ ID NO:2 and including a stop codon at position 226; c) an amino acid sequence defined as the amino acid sequence of a MetAP mutant protein having at least 90% identity to SEQ ID NO:2, including a stop codon at position 226; A polynucleotide comprising or consisting of an amino acid sequence selected from the group consisting of:

4. 4. The strain of claim 1 or 2, or the polynucleotide of claim 3, wherein the reduction in susceptibility to phage DT1 is characterized by an EOP reduction of at least 4 logs, at least 5 logs, or at least 6 logs.

5. 5. A bacterial composition comprising a strain according to any one of claims 1, 2 and 4 and, optionally, one or more further lactic acid bacteria selected from the group consisting of the genera Streptococcus, Lactococcus, Lactobacillus, Leuconostoc, Pediococcus, Enterococcus, Oenococcus and Bifidobacterium.

6. 8. A food or feed product, in particular a dairy product, a meat product or a cereal food or feed product, more particularly a fermented dairy product, comprising a strain according to any one of claims 1, 2 and 4 or a bacterial composition according to claim 5.

7. 1. A method for producing a fermented product comprising: a) inoculating a substrate, preferably a milk substrate, with a strain according to any one of claims 1, 2 and 4, or with a bacterial composition according to claim 5; b) fermenting the inoculated substrate from step a) to obtain a fermented product, preferably a fermented milk product. The method includes:

8. A method for producing a food or feed product, preferably a fermented food product, more preferably a fermented dairy product, comprising the steps of: use.

9. Use of a polynucleotide according to claim 3 or a vector comprising a polynucleotide according to claim 3, which reduces the susceptibility of a bacterium of the genus Streptococcus, in particular a Streptococcus thermophilus strain or a Streptococcus mutans strain, which is susceptible to cos-type phages, to at least one cos-type phage, the susceptibility to at least one cos-type phage being determined by EOP assay I.

10. A method for preparing a bacterium of the genus Streptococcus, in particular a Streptococcus thermophilus or Streptococcus mutans strain, having reduced susceptibility to at least one cos-type phage, comprising the steps of: a) providing a bacterium of the genus Streptococcus that is sensitive to a cos-type phage; b) replacing an allele of the metAP gene of said bacterium of the genus Streptococcus that is sensitive to cos-type phages with the polynucleotide of claim 3, or modifying the sequence of the metAP gene of said bacterium of the genus Streptococcus that is sensitive to cos-type phages so that it has a metAP allele that is the same in sequence as the polynucleotide of claim 3; c) recovering a bacterium of the genus Streptococcus having reduced susceptibility to at least one cos-type phage; wherein susceptibility to at least one cos-type phage is determined by EOP Assay I.

11. 11. The use according to claim 9 or the method according to claim 10, wherein the reduction in susceptibility is characterized by an EOP reduction of at least 4 logs, at least 5 logs, or at least 6 logs.

12. A lactic acid bacterium obtained by the method according to claim 10 or 11.

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