Bacterial strains with phage resistance

Abstract

The present invention relates to strains of lactic acid bacterium, such as of the genus Lactococcus having increased resistance to phage infection, methods for the production of a variant strain of a lactic acid bacterium, such as of the genus Lactococcus; compositions comprising such strain, as well as their use as a starter culture, during fermentation, or as host cells in the production of biomolecules.

Description

[0001]BACTERIAL STRAINS WITH PHAGE RESISTANCE FIELD OF THE INVENTION The present invention relates to strains of lactic acid bacteria, such as of the genus Lacto- coccus having increased resistance to phage infection, methods for the production of a variant strain of a lactic acid bacterium, such as of the genus Lactococcus; compositions comprising such strain, as well as their use as a starter culture, during fermentation, or as host cells in the pro- duction of biomolecules. BACKGROUND OF THE INVENTION Lactococcus lactis and Lactococcus cremoris represent a diverse group of strains that are used extensively in starter cultures for fermented dairy products. Due to the nature of commer- cial dairy fermentations, virulent bacterial viruses known as bacteriophages (phages) are com- monly found in the industrial environment (Sturino and Klaenhammer, 2006). If left uncon- trolled, phages will reduce the quality of the final fermented product, and if the infection is se- vere, they can cause complete fermentation failure. Lactococcal phages have been classified into ten groups, from which three types are pre- dominantly problematic in the dairy fermentation industry: c2 (Ceduovirus genus), 936 (Skuna- virus genus), and P335 (Deveau et.al., 2006). Targets for mutagenesis conferring Skunavirus re- sistance often belong to the gene cluster encoding the cell wall polysaccharides (CWPS), which decrease phage adsorption. However, these mutations often have a deleterious effect on the strain physiology, including slower growth rates and cell clumping phenotypes (Romero et al., 2020). L. cremoris DGCC13630 (DSM34734) is a bacteriophage insensitive mutant (BIM) of DGCC12640 that is resistant to skunaviruses. However, phage adsorption was unaffected, and DGCC13630 maintained the milk coagulation properties equivalent to parent DGCC12640, which are both inconsistent with CWPS mutation. Therefore, DGCC13630 potentially harbors a novel, uncharacterized phage-resistance mechanism. The present inventors have identified the uncharacterized phage resistance encoded by DGCC13630. Whole genome sequencing found a mutation in the ynbA gene of DGCC13630. Complementation and deletion experiments confirmed that this mutation in ynbA was responsible for phage resistance. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. pTRK989-ynbA vector map. Gray arrows indicate CDS. Black arrow indicates the P6 promoter region. Figure 2. ynbA targeted deletion strategy example (cremoris species). Gray arrows indicate CDS. White and black arrows indicate regions of homology used to delete ynbA. A. Deletion vector pCremYnbADel. B. ynbA deletion region extracted from DGCC12640. Figure 3: iCinac milk acidification activity test comparison of parent DGCC12640 to DGCC13630 at 32°C. Feature points: Ta indicates the lag time. t5.2 = time to pH 5.2. m6-5 is the slope of the acidification curve. Figure 4: Alignment of DGCC12640 and DGCC13630 ynbA CDS. Dark gray bar indicates identity. Light gray arrows indicate CDS. Zoomed in view of mutation. Figure 5: ynbA deletion gels. A: PCR amplicons of the deletion region in DGCC12640 and DGCC12697 ynbA deletion mutants. Amplicon sizes of mutants are consistent with the desired deletion. B: Plasmid profiles confirm the DGCC12640 and DGCC12697 deletion mutants are de- rived from the parental genotype. SUMMARY OF THE INVENTION It is an object of embodiments of the invention to provide bacterial strains with increased resistance to phage infections. The present invention relates in a broad aspect to strains of a lactic acid bacterium, such as of the genus Lactococcus having increased resistance to phage infection, such as strains func- tionally inactive with respect to phage infection. Accordingly, in a first aspect the present invention relates to a strain of a lactic acid bac- terium, such as of the genus Lactococcus having the ynbA gene or the homologue thereof encod- ing an endogenous YnbA protein or a homologue thereof disrupted, such as deleted or compris- ing a modification to make the YnbA protein or a homologue thereof functionally inactive with re- spect to phage infection. In a second aspect the present invention relates to a strain of a lactic acid bacterium, which is selected from the list consisting of a strain deposited under accession number DSM34734 on 9 August 2023 at the DSMZ or a mutant thereof; and a strain deposited under accession number DSM34735 on 9 August 2023 at the DSMZ or a mu- tant thereof. In a third aspect the present invention relates to a method for the production of a variant strain of a lactic acid bacterium, such as of the genus Lactococcus, resistant to phages, such as phages of the genus Skunavirus, which method comprises the step of either deleting the ynbA gene or homologue thereof or making a suitable modification of the ynbA gene or homologue thereof disrupting or abolishing the expression of a functional YnbA protein. In a further aspect the present invention relates to a composition, such as a food or feed composition comprising a strain of a lactic acid bacterium according to the invention, or a bacte- riophage resistant variant strain produced using the method of the invention. In a further aspect the present invention relates to methods for preparing such food or feed, starter cultures, fer- mentation methods, as well as the use of a lactic acid bacterium according to the invention for use as a host bacterium in the production of an antimicrobial, a biomolecule, or a recombinant proteins including enzymes. DETAILED DESCRIPTION OF THE INVENTION Lactococcus cremoris DGCC13630 (DSM34734) is a bacteriophage insensitive mutant (BIM) of DGCC12640, isolated from challenge with a Skunavirus. DGCC13630 harbors a previ- ously uncharacterized resistance mechanism against skunaviruses. Comparison of whole genome sequences of DGCC13630 and DGCC12640 identified a mutation in the ynbA gene in DGCC13630. Complementation with wild-type ynbA restored phage sensitivity to DGCC13630, and targeted deletion of ynbA in DGCC12640 resulted in phage resistance. This data confirmed ynbA to be responsible for the phage resistance identified in DGCC13630. The term “resistant to phages” refers to a bacterium having a low or no susceptibility to bacterio- phage multiplication or infection when compared to the wild-type bacteria when cultured (e.g., in a dairy medium). In some embodiments, some bacterial cells exhibit "low susceptibility to bacte- riophage multiplication”. This term refers to the level of bacteriophage multiplication in a bacte- rium being below a level that would cause a deleterious effect to a culture in a given period of time. Such deleterious effects on a culture include, but are not limited to, no coagulation of milk during production of fermented milk products (e.g., yogurt or cheese), inadequate or slow lower- ing of the pH during production of fermented milk products (e.g., yogurt or cheese), slow ripen- ing of cheese, and / or deterioration of a food's texture to the point where it is unappetizing or un- suitable for consumption. As used herein, the term "bacteriophage" has its conventional meaning as understood in the art, i.e. a virus that infects bacteria. Many bacteriophages are specific to a particular genus or spe- cies or strain of bacteria. The term "bacteriophage" is synonymous with the term "phage". In- cluded within this definition are bacteriophages selected from a P335-type phage, phages of the Ceduovirus genus (formerly known as c2-type phages), such as7140hy, phages of the Skuna- virus genus (formerly known as 936-type phages), such as any one of GL7, FB3, FB6, RH10, FB10, RH6, FB14, GP14, GP13, GP15, M7671 and D7457. In one embodiment bacteriophage or phage referred to herein is a phage of the Skunavirus genus selected from M7671 and D7457. The term "disrupted," as applied to the gene ynbA, refers to any genetic modification that de- creases or eliminates the expression of the gene and / or the functional activity of the correspond- ing gene product (mRNA and / or protein). Genetic modifications include complete or partial inacti- vation, suppression, deletion, interruption, blockage, or down-regulation of a gene. This can be accomplished, for example, by gene "knockout," inactivation, mutation (e.g., insertion, deletion, point, or frameshift mutations that disrupt the expression or activity of the gene product), or by use of inhibitory RNAs (e.g., sense, antisense, or RNAi technology). A disruption may encompass all or part of a gene's coding sequence. The term “ynbA gene” refers to the gene encoding YnbA protein or homologs, such as a gene identified by any one of SEQ ID Nos 1, 3, 5, 7, 9, 11 or a corresponding homolog or polymorphic sequence thereof. The term “YnbA protein” refers to the protein encoded by the ynbA gene. The protein may also be referred to as site-specific tyrosine recombinase, Tyrosine recombinase XerD-like or just XerD. Reference to the gene name ynbA is not intended to be limiting, but is intended to encompass homologs (i.e., which may be endogenous to a related microbial organism) and polymorphic var- iants. Homologs and variants can be identified based on sequence identity and / or similar biologi- cal (e.g., enzymatic) activity. In certain embodiments, the invention includes a polynucleotide or polypeptide sequence with at least 50%, 60%, 70%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the named gene ynbA or its gene product. "Identity" or "percent identity," in the context of two or more polynucleotide or polypeptide sequences, refers to two or more sequences or sub-sequences that are the same or have a specified percentage of nucleotides or amino acid residues, respectively, that are the same. Percent identity may be de- termined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which may also con- tain gaps to optimize the alignment) for alignment of the two sequences. For example, the se- quence can have a percent identity of at least 50%, 60%, 70%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% over a specified region to a reference sequence when com- pared and aligned for maximum correspondence over a comparison window, or designated re- gion as measured using a sequence comparison algorithm or by manual alignment and visual in- spection. Alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method of Pear- son and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current Protocols, John Wiley & Sons, Inc. (1995 Supplement) (Ausubel)). The term "variant" is used to mean a naturally occurring polypeptide or nucleotide sequences which differs from a wild-type sequence. The lactic acid bacterium used in the present invention may be a bacterium belonging to the ge- nus Lactococcus, for example, Lactococcus allomyrinae, Lactococcus chungangensis, Lactococcus cremoris, Lactococcus formosensis,. Lactococcus fujiensis, Lactococcus garvieae, Lactococcus hir- cilactis, Lactococcus kimchii, Lactococcus lactis, Lactococcus laudensis, Lactococcus nasutiter- mitis, Lactococcus petauri, Lactococcus piscium, Lactococcus plantarum, Lactococcus raffinolac- tis, Lactococcus reticulitermitis, Lactococcus taiwanensis, Lactococcus termiticola, Lactococcus cremoris subsp. tructae, or Lactococcus lactis subsp. hordniae. In some specific embodiment, the lactic acid bacterium is a bacterium selected from the species of Lactococcus lactis or Lactococcus cremoris, and Lactococcus lactis subsp. lactis bv. diacetylac- tis. Lactococcus lactis subsp. lactis bv. diacetylactis may also be referred to as Lactococcus lactis bv. Diacetylactis, and Lactococcus lactis ssp. Diacetylactis. As described above an aspect of the present invention relates to a strain of a lactic acid bacte- rium, such as of the genus Lactococcus having the ynbA gene or the homologue thereof encoding an endogenous YnbA protein or a homologue thereof disrupted, such as deleted or comprising a modification to make the YnbA protein or a homologue thereof functionally inactive with respect to phage infection. In some embodiments the lactic acid bacterium is of the genus Lactococcus, such as a strain is selected from the species Lactococcus lactis, Lactococcus cremoris, and Lactococcus lactis subsp. lactis bv. diacetylactis. In some embodiments the YnbA protein or a homologue thereof is selected from a YnbA protein and the putative site-specific tyrosine recombinase (XerD). In some embodiments the YnbA protein or a homologue thereof is functionally inactive with re- spect to phage infection due to deletion of the complete ynbA gene. In some embodiments the YnbA protein or a homologue thereof is functionally inactive with re- spect to phage infection due to a modification introduced into the ynbA gene, such as a mutation introducing a stop codon. In some embodiments the YnbA protein or homologue thereof is expressed by a gene comprising or consisting of a nucleotide sequence selected from i) the nucleotide sequence at least 80% identical to a sequence identified by SEQ ID NO:1; ii) the nucleotide sequence at least 80% identical to a sequence identified by SEQ ID NO:3 ; iii) the nucleotide sequence at least 80% identical to a sequence identified by SEQ ID NO:5 ; iv) the nucleotide sequence at least 80% identical to a sequence identified by SEQ ID NO:7; v) the nucleotide sequence at least 80% identical to a sequence identified by SEQ ID NO:9; vi) the nucleotide sequence at least 80% identical to a sequence identified by SEQ ID NO:11; vii) a nucleotide sequence encoding a YnbA protein at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequence of SEQ ID NO:2, 4, 6, 8, 10, or 12; or a truncated version thereof. the YnbA protein or homologue thereof is expressed by a gene comprising or consisting of a nu- cleotide sequence at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to a sequence selected from SEQ ID NO 1, 3, 5, 7, 9, and 11. In some embodiments the modification to make the YnbA protein or a homologue thereof func- tionally inactive with respect to phage infection is selected from the group consisting of a stop codon, a deletion, a mutation, or an insertion, such as an insertion that causes a frame shift, a deletion or a mutation causing a premature stop codon, or any other insertion such as of a gene or transposable element disrupting the ynbA gene. In some embodiments the strain according to the present invention has increased resistance to phages, such as phages of the genus Skunavirus, such as M7671. In some embodiments the strain according to the present invention has increased resistance to phages of the genus Ceduovirus, such as increased resistance to both phages of the genus Skunavirus, such as M7671 and to phages of the genus Ceduovirus. In some embodiments the strain according to the present invention shows a reduction in phage sensitivity as measured by a spot titer assays described herein by a factor of at least 50, such as at least 100, such as at least 500, such as at least 1000, such as at least 10000 for each phage tested. In some embodiments the strain according to the present invention has the endogenous gene ynbA deleted. Sequences: Note that there is potential for the transcription of ynbA to begin at two different start sites. An ATG start codon or a GTG start codon that is 30 nucleotides upstream of the ATG codon. 747-nt (SEQ ID NO:1) Wild-type ynbA gene in DGCC12640 : GTGCCTTCGCTCACGACTTTACAAAATATTATGAAATTACCAAACGAGATTGAGGAG- TATTTAGTAAGTAGAAACTTTTCTGAAAATACTCGCTCAAATTATTATTATGACTTAGTCTATTTGCAGG CTTTTTTTGAAGATAAGTCTGTCACAGAAGAAGCACTAGAACTTTACAAACACCAGCTTAG- TAAACTCTCTCCTGCGGCACAACGGCGAAAAATTTCGAGTGCCAACCAATATTTTCTATTTTTATATGAA AATAAAAAAATAAACCAGTTTTTCAAAATTAAGCAAGTT- GTTCAGAAAAAAACTCAAAGTTCTGAGAGTTATCATCCTATGATAAAAGAATTTCCAGAATTTTATGGAC CTCTAAGTTGTCCAGGGCAATTTTTAGCTTTATTGATATTAGAATTTGGTCTTAATTTT- GCAGAGATTCAAAAATTAAAATGGGAAAATTTTAATTGGAATTTTAAATATCTCACAATCGAAAAAGCTG GAATAAAACGAGTCTTACCCATTCGAGAAAAATTTGCCATCAGAGTTAAGGCCA- TAAAAAATGCCGATGAATTATTTGCAAAATCTCGCCAATTCCTATATACAGAATTAAAGAAATTCACTAA TTATTCTTCTAAAGAGATTAGGGAACAGTATATTTTGCATCAGGTAAAAGCGGGAAAAACAA- TATATGAGTTGGCTAACTTACTTGGCTTAAGCACAATTACTACCTTAGAAAAATATTATAGATAG 248-aa (SEQ ID NO:2) Wild-type YnbA protein encoded by wild-type ynbA gene of DGCC12640: MPSLTTLQNIMKLPNEIEEYLVSRNFSENTRSNYYYDLVYLQAFFEDKSVTEEALE- LYKHQLSKLSPAAQRRKISSANQYFLFLYENKKINQFFKIKQVVQKKTQSSESYHPMIKEFPEFYGPLSC PGQFLALLILEFGLNFAEIQKLKWENFNWNFKYLTIEKAGIKRVLPIREK- FAIRVKAIKNADELFAKSRQFLYTELKKFTNYSSKEIREQYILHQVKAGKTIYELANLLGLSTITTLEKY YR 747-nt (SEQ ID NO:3) Wild-type ynbA gene in DGCC12697 GTGCCTTTGCTCACGACTTTACAAAATATTATGAAATTACCAAACGAGATTGACGAG- TATTTAGCAAGCAGAAATTTTTCAGAGAATACTCGTTCAAATTACCATTATGATTTAGTTTCTTTGCAGG CTTTCTTTGAAGATAAGTCACTCACAACAGAAAACTTAGAACTTTACAAAATTCAAATTAG- TAATCTTTCGCCAGCAGCACAGCGAAGGAAGATTTCGAGTGCAAATCAATATTTGCTTTTCCTGTATCAA AGACAAAAAGTTGACCAG- TACTTTAAAATTAAACAAGTCGTTCAAAAAAAATCACAGACTGCTCAAAGTTATCATCCAATGATTAAGG AATTTCCTGAGTTCTATGGTCCTTTGACCTGTCCAGGACAATTTTTAGCCTTATTAATTTTA- GAATTTGGTTTAAATTTTGCTGAGATACAAAAATTGAAATGGGAAAATTTTAATTGGAACTTTAAATATC TCACGATTGAAAAAGCCGGAATAAAACGAGTTTTACCAATTAGAGAAAAATTTGCCATAA- GAGTTAAAGCCATTAATAATGCTGATGAATTATTCGCAAAATCTCGTCAATTTCTTTATACTGAATTGAA AAAATTTACGAACTATTCTTCCAAAGAAATTAGAGAACAGTATATCTTGCATCAGGTTAAA- GCAGGGAAGTCAATTTATGAACTGGCAACTCTACTTGGTTTAACAACTATTACAACGCTTGAAAAATATT ATAGATAG 248-aa (SEQ ID NO:4) Wild-type YnbA protein encoded by wild-type ynbA gene of DGCC12697: MPLLTTLQNIMKLPNEIDEYLASRNFSENTRSNYHYDLVSLQAFFEDKSLTTENLELYKI- QISNLSPAAQRRKISSANQYLLFLYQRQKVDQYFKIKQVVQKKSQTAQSYHPMIKEFPEFYGPLTCPGQF LALLILEFGLNFAEIQKLKWENFNWNFKYLTIEKAGIKRVLPIREK- FAIRVKAINNADELFAKSRQFLYTELKKFTNYSSKEIREQYILHQVKAGKSIYELATLLGLTTITTLEKY YR Complete sequence of the mutated ynbA gene in DGCC13630 (SEQ ID NO:5) The position of in- serted TC sequence into the wild-type gene is as indicated in bold and underlined: GTGCCTTCGCTCACGACTTTACAAAATATTATGAAATTACCAAACGAGATTGAGGAG- TATTTAGTAAGTAGAAACTTTTCTGAAAATACTCGCTCAAATTATTATTATGACTTAGTCTATTTGCAGG CTTTTTTTGAAGATAAGTCTGTCACAGAAGAAGCACTAGAACTTTACAAACACCAGCTTAG- TAAACTCTCTCTCCTGCGGCACAACGGCGAAAAATTTCGAGTGCCAACCAATATTTTCTATTTTTATATG AAAATAAAAAAATAAACCAGTTTTTCAAAATTAAGCAAGTT- GTTCAGAAAAAAACTCAAAGTTCTGAGAGTTATCATCCTATGATAAAAGAATTTCCAGAATTTTATGGAC CTCTAAGTTGTCCAGGGCAATTTTTAGCTTTATTGATATTAGAATTTGGTCTTAATTTT- GCAGAGATTCAAAAATTAAAATGGGAAAATTTTAATTGGAATTTTAAATATCTCACAATCGAAAAAGCTG GAATAAAACGAGTCTTACCCATTCGAGAAAAATTTGCCATCAGAGTTAAGGCCA- TAAAAAATGCCGATGAATTATTTGCAAAATCTCGCCAATTCCTATATACAGAATTAAAGAAATTCACTAA TTATTCTTCTAAAGAGATTAGGGAACAGTATATTTTGCATCAGGTAAAAGCGGGAAAAACAA- TATATGAGTTGGCTAACTTACTTGGCTTAAGCACAATTACTACCTTAGAAAAATATTATAGATAG SEQ ID NO:6: The YnbA protein sequence in DGCC13630 resulting from frame shift and prema- ture stop. The truncated YnbA is 90-aa vs 248-aa of DGCC12640: MPSLTTLQNIMKLPNEIEEYLVSRNFSENTRSNYYYDLVYLQAFFEDKSVTEEALE- LYKHQLSKLSLLRHNGEKFRVPTNIFYFYMKIKK 717-nt (SEQ ID NO:7) Wild-type ynbA gene in DGCC12640: ATGAAATTACCAAACGAGATTGAGGAGTATTTAGTAAGTAGAAACTTTTCTGAAAA- TACTCGCTCAAATTATTATTATGACTTAGTCTATTTGCAGGCTTTTTTTGAAGATAAGTCTGTCACAGAA GAAGCACTAGAACTTTACAAACACCAGCTTAGTAAACTCTCTCCTGCGGCACAAC- GGCGAAAAATTTCGAGTGCCAACCAATATTTTCTATTTTTATATGAAAATAAAAAAATAAACCAGTTTTT CAAAATTAAGCAAGTTGTTCAGAAAAAAACTCAAAGTTCTGAGAGTTATCATCCTATGA- TAAAAGAATTTCCAGAATTTTATGGACCTCTAAGTTGTCCAGGGCAATTTTTAGCTTTATTGATATTAGA ATTTGGTCTTAATTTTGCAGAGATTCAAAAATTAAAATGGGAAAATTTTAATT- GGAATTTTAAATATCTCACAATCGAAAAAGCTGGAATAAAACGAGTCTTACCCATTCGAGAAAAATTTGC CATCAGAGTTAAGGCCATAAAAAATGCCGATGAATTATTTGCAAAATCTCGCCAATTCCTA- TATACAGAATTAAAGAAATTCACTAATTATTCTTCTAAAGAGATTAGGGAACAGTATATTTTGCATCAGG TAAAAGCGGGAAAAACAATATATGAGTTGGCTAACTTACTTGGCTTAAGCACAATTACTAC- CTTAGAAAAATATTATAGATAG 238-aa (SEQ ID NO:8) Wild-type YnbA protein encoded by wild-type ynbA gene of DGCC12640: MKLPNEIEEYLVSRNFSENTRSNYYYDLVYLQAFFEDKSVTEEALELYKHQLSKLSPAAQRR- KISSANQYFLFLYENKKINQFFKIKQVVQKKTQSSESYHPMIKEFPEFYGPLSCPGQFLALLILEFGLNF AEIQKLKWENFNWNFKYLTIEKAGIKRVLPIREK- FAIRVKAIKNADELFAKSRQFLYTELKKFTNYSSKEIREQYILHQVKAGKTIYELANLLGLSTITTLEKY YR 717-nt (SEQ ID NO:9) Wild-type ynbA gene in DGCC12697 ATGAAATTACCAAACGAGATTGACGAGTATTTAGCAAGCAGAAATTTTTCAGAGAA- TACTCGTTCAAATTACCATTATGATTTAGTTTCTTTGCAGGCTTTCTTTGAAGATAAGTCACTCACAACA GAAAACTTAGAACTTTACAAAATTCAAATTAGTAATCTTTCGCCAGCAGCACAGCGAAGGAA- GATTTCGAGTGCAAATCAATATTTGCTTTTCCTGTATCAAAGACAAAAAGTTGACCAGTACTTTAAAATT AAACAAGTCGTTCAAAAAAAATCACAGACTGCTCAAAGTTATCATCCAATGAT- TAAGGAATTTCCTGAGTTCTATGGTCCTTTGACCTGTCCAGGACAATTTTTAGCCTTATTAATTTTAGAA TTTGGTTTAAATTTTGCTGAGATACAAAAATTGAAATGGGAAAATTTTAATT- GGAACTTTAAATATCTCACGATTGAAAAAGCCGGAATAAAACGAGTTTTACCAATTAGAGAAAAATTTGC CATAAGAGTTAAAGCCATTAATAATGCTGATGAATTATTCG- CAAAATCTCGTCAATTTCTTTATACTGAATTGAAAAAATTTACGAACTATTCTTCCAAAGAAATTAGAGA ACAGTATATCTTGCATCAGGTTAAAGCAGGGAAGTCAATTTATGAACTGGCAACTCTACTT- GGTTTAACAACTATTACAACGCTTGAAAAATATTATAGATAG 238-aa (SEQ ID NO:10) Wild-type YnbA protein encoded by wild-type ynbA gene of DGCC12697: MKLPNEIDEYLASRNFSENTRSNYHYDLVSLQAFFEDKSLTTENLELYKIQISNLSPAAQRR- KISSANQYLLFLYQRQKVDQYFKIKQVVQKKSQTAQSYHPMIKEFPEFYGPLTCPGQFLALLILEFGLNF AEIQKLKWENFNWNFKYLTIEKAGIKRVLPIREK- FAIRVKAINNADELFAKSRQFLYTELKKFTNYSSKEIREQYILHQVKAGKSIYELATLLGLTTITTLEKY YR Complete sequence of the mutated ynbA gene in DGCC13630 (SEQ ID NO:11) The position of inserted TC sequence into the wild-type gene is as indicated in bold and underlined: ATGAAATTACCAAACGAGATTGAGGAGTATTTAGTAAGTAGAAACTTTTCTGAAAA- TACTCGCTCAAATTATTATTATGACTTAGTCTATTTGCAGGCTTTTTTTGAAGATAAGTCTGTCACAGAA GAAGCACTAGAACTTTACAAACACCAGCTTAGTAAACTCTCTCTCCTGCGGCACAAC- GGCGAAAAATTTCGAGTGCCAACCAATATTTTCTATTTTTATATGAAAATAAAAAAATAAACCAGTTTTT CAAAATTAAGCAAGTTGTTCAGAAAAAAACTCAAAGTTCTGAGAGTTATCATCCTATGA- TAAAAGAATTTCCAGAATTTTATGGACCTCTAAGTTGTCCAGGGCAATTTTTAGCTTTATTGATATTAGA ATTTGGTCTTAATTTTGCAGAGATTCAAAAATTAAAATGGGAAAATTTTAATT- GGAATTTTAAATATCTCACAATCGAAAAAGCTGGAATAAAACGAGTCTTACCCATTCGAGAAAAATTTGC CATCAGAGTTAAGGCCATAAAAAATGCCGATGAATTATTTGCAAAATCTCGCCAATTCCTA- TATACAGAATTAAAGAAATTCACTAATTATTCTTCTAAAGAGATTAGGGAACAGTATATTTTGCATCAGG TAAAAGCGGGAAAAACAATATATGAGTTGGCTAACTTACTTGGCTTAAGCACAATTACTAC- CTTAGAAAAATATTATAGATAG SEQ ID NO:12: The YnbA protein sequence in DGCC13630 resulting from frame shift and prema- ture stop. The truncated YnbA is 90-aa vs 238-aa of DGCC12640: MKLPNEIEEYLVSRNFSENTRSNYYYDLVYLQAFFEDKSVTEEALE- LYKHQLSKLSLLRHNGEKFRVPTNIFYFYMKIKK EXAMPLES EXAMPLE 1 Bacterial Strains and Phages Exemplary bacteria and phages used to illustrate the present invention are listed in Table 1. Table 1: Bacteria and phages used Biological Material Relevant Characteristics Reference Bacteria Lactococcus cremoris DGCC12640 phage-sensitive starter strain This example DGCC13630 Skunavirus-resistant mutant of This example DGCC12640 12640ΔynbA 1-3 DGCC12640 with deletion in ynbA This example 13630-989 DGCC13630 + pTRK989 This example 13630-YnbA 2 and 3 DGCC13630 + pTRK989-ynbA This example DGCC14127 Ceduovirus-resistant mutant of This example (DSM34735) DGCC13630 Lactococcus lactis DGCC12697 Skunavirus-sensitive starter strain This example 12697ΔynbA DGCC12697 with deletion in ynbA This example Escherichia coli TG1 RepA+ supE hsd∆5 thi ∆(kac-proAB) F’ Duwat et al., (traD36 proAB+ lacIq lacZ∆M15) 1997 with chromosomal copy of pWV01 repA Phages M7671 Skunavirus, host DGCC12640 This example 7140hyCeduovirus, host DGCC12640 This example D7457Skunavirus, host DGCC12697 This example Plasmids pTRK989 P6 promoter; CmR Rodrigues da Cunha, 2011 pTRK989-ynbA pTRK989 + wild-type cremoris This example ynbA pG9Min Variant of pGhost9 (Maguin et al., This example 1996) that has been minimized by deletion of regions not required for plasmid maintenance or antibiotic resistance pCremYnbADel pG9Min + regions of homology for This example deletion in cremoris ynbA pLacYnbADel pG9Min + regions of homology for This example deletion in lactis ynbA Lactococcus strains were grown at 30°C in sterile 11% (w / v) non-fat dry milk (NFDM) or in M17 broth (Terzaghi and Sandine, 1975) supplemented with 0.5% (w / v) lactose or glucose. Escherichia coli was propagated aerobically in LB broth (BD Difco, USA) at 37°C. When required, antibiotics were added to the media as follows: chloramphenicol (Cm; 5 µg / mL), erythromycin (Em; 5 µg / mL for lactococci; 150 µg / mL for E. coli). Phage lysates were prepared as described by Terzaghi and Sandine (1975). High titer phage ly- sates were passed through a 0.45 µm sterilized filter and stored at 4°C. Spot titer assays and plaque assays were performed as previously described (Terzaghi and Sandine, 1975) on MRS medium (BD Difco, USA). Phage adsorption tests were performed as previously described (Sand- ers and Klaenhammer, 1980) on MRS medium. Phage Mutagenesis A single-step phage challenge was performed by exposing DGCC12640 to M7671 and DGCC13630 to phage 7140hy at varying multiplicities of infection followed by plating in MRS overlay. Colonies that grew in the presence of phage were randomly selected and streaked onto FSDA II medium (Sandine 1985), with the trimagnesium phosphate substituted with β-glycer- ophosphate to evaluate their ability to acidify and then subsequently inoculated into milk over- night to evaluate their ability to clot milk. Acidification Testing For a more thorough assessment of acidification properties, overnight 11% NFDM cul- tures were transferred into M17-Lac and grown. After the cultures reached an OD600 ~ 0.50, they were inoculated at 0.75% into activity milk (commercial 2% fat Kemp’s). Samples were kept in a 32˚C water bath overnight with pH probes and iCinac software monitoring acidification of each strain every 2 minutes. Each strain was run in triplicate. DNA preparation, PCR, and sequencing Plasmid DNA was isolated from lactococci as previously described (Anderson and McKay, 1983). PCRs were performed with GoTaq® Colorless Master Mix (Promega Corp., USA) or Phusion HF Mastermix (Thermo Scientific, USA) according to manufacturer’s instructions. Primer sequences are listed in Table 2. Primers were synthesized by Integrated DNA Technologies (USA). PCR products were purified using Wizard SV Gel and PCR Clean-Up System (Promega Corp., USA). Whole genome sequencing of DGCC12640 and DGCC13630 was performed using Illumina technology. Sanger sequencing was performed by Eurofins Genomics (USA), and results were analyzed using Geneious Prime 2021.0.3 (https: / / www.geneious.com). Protein analysis was performed using HHpred (Homology detection & structure prediction by HMM-HMM comparison) (Zimmermann et al., 2018). Table 2: Primer and gBlock Sequences Primer name Sequence (5’ – 3’) ynbA 2-F ATGTAAGAGAAGATCAAGCG ynbA 2-R GCAGGAATTTATGGGAATCG 989ynbA-R CCGAAAATATGCACTCGAGAAGAGAAGATCAAGCGGACCCTC new 989ynbA-F GATCCTCTAGAGAGCTCAAGCCGTAGTTCGTCAAGAAATATTG 989F GCTTGAGCTCTCTAGAGGATC 989R TTCTCGAGTGCATATTTTCGG new pG9+Cm F2 CCTCTTTAGCTCCTTGGAAG new pG9+Cm R2 TCCCTAGCGGACTCTAGAGG ynbaDelDownF CTTCCAAGGAGCTAAAGAGGAAGCACAATTACTACCTTAG ynbaDelDownR CCTCTAGAGTCCGCTAGGGATTTGTTGCCTTTGTAATTCC ynbA down DelF AAGCACAATTACTACCTTAG minus hom new ynbA del upR CTAAGGTAGTAATTGTGCTTCTAAATACTCCTCAATCTCG new ynbA del upF CTTCCAAGGAGCTAAAGAGGACTCTGAGTCTACAAAGCTC check ynbA del-F GAAGAGCCATAAATGTTGTGACG (cremoris) check ynbA del-R CTTATCGAAATCTCCAACATG (cremoris) check ynbA del-F CTTATCAGCGATTGTAAATTTC (lactis) check ynbA del-R CATATCGAAATCTGCAACATG (lactis) lactis ynbA gBlock CTTCCAAGGAGCTAAAGAGGACTCTAAGTCTACAAAGCTCTTTAAGGAGTAATTT- GCATGATTGATAAAAAGATTGAGGATTTTATCTTGAATCAAAGTGAAACCTTTATG ATTCCTGCCTCAAATGTTGCTGTACTTTATGCTGAGCATAA- TATTGCTCATGCCAAACTGATGCTTAGTCAGTATAAGTATTCACGAGTTCCAGTTT TAAATGAAAAAAAAGAATACGTTGGTGTTTTGGGTTTAACAGAAATCGTT- GAATTT- GAAATGGAACAGAATTTCTTTTTTGAAAAATTACATAATACATCAATTTCGGAAAT AGTTAATAAAGATGTTCAAACAGTTGGACCATCAGTAACCTTGGAAGA- GATGATGCACAAATTAATTAAGGAGCCATTTCTCCCAGTAGTCAAAAGACAGGAA TTTATGGGGATTGTTGTTAGGCAAGAAATATTAAAGGCCTTTAGTGCCTTT- GCTCAC- GACTTTACAAAATATTATGAAATTACCAAACGAGATTGACGAGTATTTAAACAACT ATTACAACGCTTGAAAAATATTATAGATAGGAATTTACAC- TATTTTTATTATGTAAAC- TATGATTAAAGAAATCAATATAAAAATTAAAAACTTCGAGGGTCCTCTCGACCTTT TATTGCATTTGGTAAGTCAGTATGAAATGGATATTTTTGAAGTTCCATTGGTAC- CAG- TTATTGAGCAATATCTTATCTATATTCAAACCATGAAAGAACTTGAATTGGAAGTA GCTGGAGAGTACATGTTAATGGCAAGTCAACTGATGTTAATTAAATCAAGAA- GACTTTTAC- CGACAGTTACCGAAACTTTTATTGAAGATACAGAACAACTTGAGTATGATTTATTG GCTCAAATTGATGAATATCGGAAATATAAAATGTTGTCACAAGATTTA- GATGAACTTCATCAGGAACGTAGTCATTTTTATTCAAAAGCTAAGACAGAAATTAT CACTGATGAAACTGTACTTTTGCAGGATAAATCTGCTTTAGAT- TTATTTCTAGCTTTTACTA- AAATTCTTGAGTTGCAAAGACAACATTTCCCTAGCGGACTCTAGAGG Molecular cloning and deletion of ynbA Cloning vectors were constructed in TG1 RepA+ using NEBuilder HiFi DNA Assembly. Fig- ure 1 shows the vector map for pTRK989-YnbA (A). Figure 2 shows an example of the strategy used for deletion in ynbA using homologous recombination. While Figure 2 shows only the vector map for pCremYnbADel (A) and ynbA deletion region in L. cremoris DGCC12640 (B), a similar vector and strategy were used for deletion of ynbA in L. lactis DGCC12697. Fragments used for each assembly are listed in Table 3. Fragments were assembled using NEBuilder HiFi DNA As- sembly MasterMix (New England Biolabs, USA), and assemblies were electroporated into TG1 RepA+according to manufacturer’s instructions. Vectors were purified from TG1 RepA+trans- formants using the GeneJET Plasmid Miniprep Kit (Thermo Scientific, USA). Electroporations of lactococci and E. coli were performed as previously described (Holo and Nes, 1989; Dower et al., 1988). Table 3: Hi-Fi assembly fragments Construct Fragment 1 (PCR amplicon) Fragment 2 (PCR amplicon or gBlock) F Pri- R primer Template F Pri- R primer Template mer mer pTRK989- 989F 989R pTRK989 new 989ynbA-R DGCC12640 ynbA 989ynbA -F pCremYn- new new pG9Min yn- yn- DGCC12640 bADel inter- pG9+C pG9+Cm baDelDo baDelDownR mediate m F2 R2 wnF pCremYn- new ynbA down pCremYn- new new ynbA DGCC12640 bADel pG9+C DelF minus bADel in- ynbA del del upF m F2 hom termediate upR pLacYnbADel new new pG9Min lactis ynbA gBlock pG9+C pG9+Cm m F2 R2 Deletion of ynbA was performed by homologous recombination between the respective deletion vector and the host chromosome. DGCC12640 containing pCremYnbADel and DGCC12697 containing pLacYnbADel were propagated first at 37°C in M17 + Em broth. Because the origin of replication of the vectors is temperature-sensitive, growth at 37°C while keeping antibiotic pressure should force integration of each element into the host chromosome. Cultures were plated after subculturing up to five times at 37°C. Single colonies were checked for integra- tion by PCR using primers check ynbA del-F and check ynbA del-R of their respective species. Colonies giving amplicons consistent with integration of the full vector or the deletion event were propagated in M17 without Em at 30°C with subculturing over 3 days. Cultures were plated M17 without Em and incubated at 30°C overnight. Single colonies were evaluated for the deletion by PCR, and deletions were confirmed by Sanger sequencing with respective species primers check ynbA del-F and check ynbA del-R. RESULTS Development and characterization of DGCC13630 and DGCC14127 BIM DGCC13630 (DSM34734) was isolated from a mutagenesis of DGCC12640 using phage M7671. Spot titer assays found DGCC13630 to be fully resistant to M7671 (Table 4). Phage adsorption assays found no difference in level of phage adsorption between parent and BIM (Table 4). iCinac activity curves showed that, despite an increase in lag time, DGCC13630 was slightly faster to pH 5.2 than parent strain at 32°C (Figure 3). Subsequently, DGCC14127 (DSM34735) was isolated from mutagenesis of DGCC13630 with Ceduovirus7140hy. DGCC14127 was confirmed to be fully resistant to 7140hy (Table 4). DGCC13630 encodes a mutation in ynbA Comparison of Illumina draft genomes of DGCC12640 and DGCC13630 identified a muta- tion in ynbA in DGCC13630 (Fig. 4). This mutation consists of a TC insert at nucleotide 194, re- sulting in a frame shift and a premature stop codon. Mutation was confirmed by Sanger sequenc- ing using primers ynbA 2-F and ynbA 2-R (data not shown). Complementation with wild-type ynbA restores phage sensitivity Wild-type ynbA was successfully assembled on pTRK989 in TG1 RepA+. Resultant vector pTRK989-ynbA and empty vector pTRK989 were successfully electroporated into DGCC13630. Representative pTRK989-ynbA+transformants 13630-YnbA 2 and 13630-YnbA 3 were found to be sensitive to M7671 in spot titer assays, while representative pTRK989 transformant 13630- 989 remained phage resistant (Table 4). PCR and sequencing using primers ynbA 2-F and ynbA 2-R confirmed that the chromosomal copy of ynbA in 13630-YnbA 2 and 13630-YnbA 3 remained mutated (data not shown). Targeted deletion of ynbA results in phage resistance Targeted deletion of ynbA in DGCC12640 was performed using homologous recombina- tion. Deletion vector pCremYnbADel was successfully assembled in TG1 RepA+ and electro- porated into DGCC12640. Colonies containing the desired deletion were isolated from propaga- tions at 37°C in the presence of Em followed by propagations at 30°C without Em. Three colonies that contained the desired ynbA deletion were analyzed (Fig. 5). Spot titer assays found that the ynbA deletion provided complete resistance against M7671 in DGCC12640 mutants (Table 4). L. lactis encodes a homolog of ynbA that is ~83% identical at the nucleotide level to the L. cremoris ynbA. To determine if the L. lactis ynbA is also involved in phage sensitivity, targeted deletion was performed in L. lactis DGCC12697. pLacYnbADel was successfully assembled in TG1 RepA+ and electroporated into DGCC12697. Colonies containing the desired deletion were iso- lated from propagations at 37°C in the presence of Em followed by propagations at 30°C without Em. A representative colony that contained the desired ynbA deletion was analyzed (Fig. 5). Spot titer assays found that the ynbA deletion provided complete resistance against D7457 in the DGCC12697 mutant (Table 4). Table 4: Phage assays. EOP = efficiency of plaquing; NT = not tested M7671 7140hy D7457 EOP Adsorption EOP EOP DGCC12640 1 99.7% 1 NT DGCC13630 <4 x 10-6 99.8% 1 NT 13630-989 <9.1 x 10-7 NT NT NT 13630-YnbA 2 0.27 NT NT NT 13630-YnbA 3 1 NT NT NT 12640ΔynbA1 < 3.13 x 10-6 NT NT NT 12640ΔynbA2 < 3.13 x 10-6NT NT NT 12640ΔynbA3 < 3.13 x 10-6 NT NT NT DGCC14127 < 3.33 x 10-6 NT < 5.0 x 10-5 NT DGCC12697 NT NT NT 1 12697ΔynbA NT NT NT < 5.0 x 10-6 YnbA HHPred analysis The predicted protein sequence encoded by the L. cremoris ynbA was analysed using HHPred to identify any conserved domains within the protein that may give insight into its func- tion. Table 5 shows the top ten hits from the HHPred analysis. Table 5: HHPred YnbA analysis. Top 10 domain hits No Hit name Prob E- P- Score SS aligned Query Template value value cols HMM HMM 1 5C6K_B Integrase; Inte- 98.98 8.90E- 1.40E- 186.1 22.9 238 5- 8-261 (292) grase, tyrosine 30 34 248 recombinase, in- tegration, site- specific recombi- nation, hydrolase; 1.9A {Enterobacteria phage P2} SCOP: d.163.1.1 5VFZ_A Gp33; Bacterio- 98.97 2.20E- 3.40E- 186.2 23.3 240 6- 12-271 phage, Brujita, 29 34 248 (318) DNA-binding, In- tegrase, DNA BINDING PRO- TEIN; HET: ACT, GOL; 1.847A {Mycobacterium phage Brujita} 2A3V_C site-specific re- 98.97 6.00E- 9.50E- 183.2 22.3 234 11- 3-304 combinase 29 34 248 (320) IntI4; Protein- DNA complex, RECOMBINA- TION; 2.8A {Vibrio cholerae O1 biovar eltor str. N16961} 1A0P_A SITE-SPECIFIC 98.97 1.30E- 2.00E- 179.1 23.7 235 11- 4-279 RECOMBINASE 28 33 248 (290) XERD; XERD, RECOMBINASE, DNA BINDING, DNA RECOMBI- NATION; 2.5A {Escherichia coli} SCOP: d.163.1.1, a.60.9.1 6EMY_A Int protein; 98.97 2.10E- 3.30E- 180.1 23.3 236 10- 1-302 transposase pro- 28 33 248 (317) tein-DNA com- plex, tyrosine recombinase, Y- transposase, Tn916-like con- jugative trans- poson, antibiotic resistance trans- fer, RECOMBI- NATION; 2.5A {Enterococcus faecalis} 1Z19_B Integrase; PRO- 98.96 2.20E- 3.50E- 172.4 22.4 233 11- 1-271 (283) TEIN-DNA COM- 27 32 248 PLEX, DNA BINDING PRO- TEIN-DNA COM- PLEX; HET: PTR, MSE; 2.8A {En- terobacteria phage lambda} SCOP:d.163.1.1 4A8E_A PROBABLE TY- 98.96 4.60E- 7.20E- 172 23.2 236 5- 1-263 (292) ROSINE RECOM- 27 32 248 BINASE XERC- LIKE; CELL CY- CLE, CHROMO- SOME DIMER RESOLUTION, PAB0255; HET: SO4; 2.99A {PY- ROCOCCUS AB- YSSI} 5HXY_A Tyrosine recom- 98.96 7.30E- 1.20E- 172.9 22.9 238 4- 30-300 binase XerA; re- 27 31 248 (317) combinase, XerA, RECOMBI- NATION; HET: PO4, MSE; 2.5A {Thermoplasma acidophilum DSM 1728} 1Z1B_B Integrase; PRO- 98.96 1.20E- 1.90E- 174.2 20.6 235 9- 72-344 TEIN-DNA COM- 26 31 248 (356) PLEX, DNA BINDING PRO- TEIN-DNA COM- PLEX; HET: PTR; 3.8A {Entero- bacteria phage lambda} SCOP: d.163.1.1, d.10.1.4 1XO0_B Recombinase 98.95 1.30E- 2.10E- 171.1 19.2 233 10- 1-307 (324) CRE; CRE RE- 26 31 248 COMBINASE, HOLLIDAY JUNC- TION, RECOMBI- NATION, COM- PLEX (RECOM- BINASE-DNA), HYDROLASE, LIGASE-DNA COMPLEX; 2.0A {Enterobacteria phage P1} SCOP: d.163.1.1, a.60.9.1 DISCUSSION Three predominant phage groups, c2 (Ceduovirus genus), P335, and 936 (Skunavirus ge- nus), are commonly problematic for lactococcal starter strains used in industrial dairy fermenta- tions (Deveau et.al., 2006). L. cremoris DGCC12640 mutant DGCC13630 was isolated from a phage challenge with Skunavirus M7671. Skunaviruses have been shown to require the host CWPS as their receptor, and mutants against these phages often have mutations in the cwps gene cluster and display decreased phage adsorption. However, these cwps mutations often have a deleterious effect on the strain physiology, including slower growth rates and cell clumping phenotypes (Romero et al., 2020). Although phage resistant, DGCC13630 did not show a de- crease in phage adsorption, nor did it display impaired acidification, which is inconsistent with what would be expected from a CWPS mutation. Subsequently, DGCC14127 (DSM34735) was isolated from mutagenesis of DGCC13630 with a Ceduovirus to further increase the phage ro- bustness of this genotype. Due to the phage resistance phenotype and satisfactory acidification profile of DGCC13630, characterization of its mutation that results in Skunavirus resistance was prioritized. Genome comparisons identified a mutation in the ynbA gene of DGCC13630, a putative tyrosine recombinase. Complementation with the wild-type ynbA gene restored phage sensitivity in DGCC13630. Furthermore, targeted deletion of ynbA in DGCC12640 resulted in phage re- sistance. This confirmed the role of the ynbA mutation in the phage resistance phenotype. The ynbA gene in L. cremoris shares ~83% nucleotide identity with its homolog in L. lactis. There- fore, ynbA was deleted in a L. lactis strain (DGCC12697), which confirmed that deletion of ynbA also provides phage resistance in L. lactis. CONCLUSION Lactococcus cremoris DGCC13630 and DGCC14127 (DSM34735) are Skunavirus-resistant mutants of the DGCC12640 genotype. This resistance was found to result from truncation of a single gene, ynbA. Targeted deletion of the ynbA homolog in L. lactis DGCC12697 also resulted in phage resistance, indicating that ynbA is involved in Skunavirus infection of both lactococcal species, thereby identifying a new target for the construction of phage-resistant lactococcal starter strains. Rapidly constructing a phage-resistant variant from an existing starter strain is essential in commercial dairy industry settings to prevent fermentation failure and will further help us gain a deeper understanding of phage-host relationships. STRAINS AND DEPOSITS DGCC numbers are internal references to DuPont Danisco collection; DSM numbers are the numbers assigned by the the Leibniz-Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstrasse 7B, D-38124 Braunschweig, Germany, following deposit under the Budapest Treaty. Strain DSM34734 and DSM34735 disclosed herein have been deposited under the Budapest Treaty in the name of DuPont Nutrition Biosciences ApS on 9 August 2023 at the Leibniz-Insti- tute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstrasse 7B, D-38124 Braunschweig, Germany. The applicant requests that a sample of the deposited micro-organisms stated herein may only be made available to an expert, until the date on which the patent is granted. In respect to those designations in which a European Patent is sought, a sample of these de- posited microorganisms will be made available until the publication of the mention of the grant of the European patent or until the date on which application has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a sample to an expert nominated by the per- son requesting the sample, and approved either i) by the Applicant and / or ii) by the European Patent Office, whichever applies. REFERENCES Anderson, D. G., & McKay, L. L. (1983). Simple and rapid method for isolating large plasmid DNA from lactic Streptococci. Applied Environmental Microbiology, 46, 549–552. doi: 10.1128 / aem.46.3.549-552.1983 Deveau, H., Labrie, S. J., Chopin, M.-C., & Moineau, S. (2006). Biodiversity and classification of lactococcal phages. Applied Environmental Microbiology, 72, 4338-4346. doi: 10.1128 / AEM.02517-05 Dower, W. J., Miller, J. F., & Ragsdale, C. W. (1988). High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Research, 16, 6127–6145. doi: 10.1093 / nar / 16.13.6127 Duwat, P., Cochu, A., Ehrlich, S. D., & Gruss, A. (1997). Characterization of Lactococcus lactis UV-sensitive mutants obtained by ISS1 transposition. Journal of Bacteriology, 179, 4473–4479. doi: 10.1128 / jb.179.14.4473-4479.1997 Holo, H., & Nes, I. F. (1989). High-frequency transformation, by electroporation, of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Applied Environmental Microbiology, 55, 3119–3123. doi: 10.1128 / aem.55.12.3119-3123.1989 Maguin, E., Prévost, H., Ehrlich, S. D., & Gruss, A. (1996). Efficient insertional mutagenesis in lactococci and other Gram-positive bacteria. Journal of Bacteriology, 178, 931–935. doi: 10.1128 / jb.178.3.931-935.1996 Rodrigues da Cunha, L. (2011). Genotyic and phenotypic characterization of Lactobacillus gasseri isolated from a newborn infant. Universidade Federal de Vicosa. PhD dissertation. Romero, D. A., Magill, D., Millen, A., Horvath, P. & Fremaux, C. (2020). Dairy lactococcal and streptococcal phage–host interactions: an industrial perspective in an evolving phage landscape. FEMS Microbiology Reviews, 44, 909–932. doi: 10.1093 / femsre / fuaa048 Sandine, W. E. (1985). The Streptococci: Milk Products, p. 5 - 23. In S.E. Gilliland (ed), Bacterial Starter Cultures for Food, CRC Press, Inc. Sanders, M. E., & Klaenhammer, T. R. (1980). Restriction and modification in Group N strepto- cocci: effect of heat on development of modified lytic bacteriophage. Applied Environmental Mi- crobiology, 40, 500-506. doi: 10.1128 / aem.40.3.500-506.1980 Sturino, J. M., & Klaenhammer, T. R. (2006). Engineered bacteriophage-defense systems in bio- processing. Nature Reviews Microbiology, 4(5), 395. doi: 10.1038 / nrmicro1393 Terzaghi, B. E., & Sandine, W. E. (1975). Improved medium for lactic streptococci and their bac- teriophages. Applied Environmental Microbiology, 29, 807-813. doi: 10.1128 / am.29.6.807- 813.1975 Zimmermann, L., Stephens, A., Nam, S-Z., Rau, D., Kübler, J., Lozajic, M., Gabler, F., Söding, J., Lupas, A. N., & Alva, V. (2018). A completely reimplemented MPI bioinformatics toolkit with a new HHpred server at its core. Journal of Molecular Biology, 430, 2237-2243. doi: 10.1016 / j.jmb.2017.12.007

Claims

CLAIMS 1. A strain of a lactic acid bacterium, such as of the genus Lactococcus having the ynbA gene or the homologue thereof encoding an endogenous YnbA protein or a homologue thereof dis- rupted, such as deleted or comprising a modification to make the YnbA protein or a homo- logue thereof functionally inactive with respect to phage infection.

2. The strain of a lactic acid bacterium according to claim 1, which is of the genus Lactococcus, such as a strain selected from the species Lactococcus lactis, Lactococcus cremoris, and Lac- tococcus lactis subsp. lactis bv. diacetylactis.

3. The strain of a lactic acid bacterium according to claims 1 or 2, wherein the YnbA protein or a homologue thereof is selected from a YnbA protein and the putative site-specific tyrosine re- combinase (XerD).

4. The strain of a lactic acid bacterium according to any one of claims 1-3, wherein the YnbA protein or a homologue thereof is functionally inactive with respect to phage infection due to deletion of the complete ynbA gene.

5. The strain of a lactic acid bacterium according to any one of claims 1-3, wherein the YnbA protein or a homologue thereof is functionally inactive with respect to phage infection due to a modification introduced into the ynbA gene, such as a mutation introducing a stop codon.

6. The strain of a lactic acid bacterium according to any one of claims 1-5, wherein the YnbA protein or homologue thereof is expressed by a gene comprising or consisting of a nucleotide sequence selected from i) the nucleotide sequence at least 80% identical to a sequence identified by SEQ ID NO:1; ii) the nucleotide sequence at least 80% identical to a sequence identified by SEQ ID NO:3 ; iii) the nucleotide sequence at least 80% identical to a sequence identified by SEQ ID NO:5 ; iv) the nucleotide sequence at least 80% identical to a sequence identified by SEQ ID NO:7; v) the nucleotide sequence at least 80% identical to a sequence identified by SEQ ID NO:9; vi) the nucleotide sequence at least 80% identical to a sequence identified by SEQ ID NO:11; vii) a nucleotide sequence encoding a YnbA protein at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptide sequence of SEQ ID NO:2, 4, 6, 8, 10, or 12; or a truncated version thereof.

7. The strain of a lactic acid bacterium according to claim 6, wherein the YnbA protein or homo- logue thereof is expressed by a gene comprising or consisting of a nucleotide sequence at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identical to a sequence selected from SEQ ID NO 1, 3, 5, 7, 9, and 11.

8. The strain of a lactic acid bacterium according to any one of claims 1-7, wherein said modifi- cation is selected from the group consisting of a stop codon, a deletion, a mutation, or an in- sertion, such as an insertion that causes a frame shift, a deletion or a mutation causing a premature stop codon, or any other insertion such as of a gene or transposable element dis- rupting the ynbA gene.

9. The strain of a lactic acid bacterium according to any one of claims 1-8, which strain has in- creased resistance to phages, such as phages of the genus Skunavirus, such as M7671.

10. The strain of a lactic acid bacterium according to any one of claims 1-9, which strain has in- creased resistance to phages of the genus Ceduovirus, such as increased resistance to both phages of the genus Skunavirus, such as M7671 and to phages of the genus Ceduovirus.

11. The strain of a lactic acid bacterium according to claims 1-10, which strain shows a reduction in phage sensitivity as measured by a spot titer assays described herein by a factor of at least 50, such as at least 100, such as at least 500, such as at least 1000, such as at least 10000 for each phage tested.

12. The strain of a lactic acid bacterium according to claims 1-11, in which the endogenous gene ynbA is deleted.

13. A strain of a lactic acid bacterium, which is selected from the list consisting of a strain depos- ited under accession number DSM34734 on 9 August 2023 at the DSMZ or a mutant thereof; and a strain deposited under accession number DSM34735 on 9 August 2023 at the DSMZ or a mutant thereof.

14. A method for the production of a variant strain of a lactic acid bacterium, such as of the ge- nus Lactococcus, resistant to phages, such as phages of the genus Skunavirus, which method comprises the step of either deleting the ynbA gene or homologue thereof or making a suita- ble modification of the ynbA gene or homologue thereof disrupting or abolishing the expres- sion of a functional YnbA protein.

15. The method according to claim 14, which strain of a lactic acid bacterium is as defined in any one of claims 2-13.

16. A composition comprising a strain of a lactic acid bacterium as defined in any one of claims 1-13, or a bacteriophage resistant variant strain produced using the method set forth in claim 14.

17. A food or feed comprising the composition of claims 16.

18. A method for preparing a food or feed comprising adding the composition of claim 16 to said food or feed.

19. A starter culture, probiotic culture, or dietary supplement culture comprising the composition of claim 16.

20. A fermentation method comprising adding the composition of claim 16 to a starter culture.

21. A fermentation method comprising adding the composition of claims 16 to a fermentation medium, under conditions such that fermentation of the components of said fermentation medium occur.

22. A strain of a lactic acid bacterium, such as of the genus Lactococcus having the ynbA gene or the homologue thereof encoding an endogenous YnbA protein or a homologue thereof either deleted or comprising a modification to make the YnbA protein or a homologue thereof func- tionally inactive with respect to phage infection; for use as a host bacterium in the production of an antimicrobial, a biomolecule, or a recombinant protein including enzymes.

23. A strain of a lactic acid bacterium for use according to claim 22, wherein the strain of a lactic acid bacterium is as defined in any one of claims 1-13, or a bacteriophage resistant variant strain produced using the method set forth in claim 14.

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