Evolved distal tail protein of skunaviruses facilitates adsorption to exopolysaccharide-encoding lactococci.

Aim: Lactococcal skunaviruses are diverse and problematic in the industrial dairy environment. Host recognition involves the specific interaction of phage-encoded proteins with saccharidic host cell surface structures. Lactococcal plasmid pEPS6073 encodes genes required for the biosynthesis of a cell surface-associated exopolysaccharide (EPS), designated 6073-like. Here, the impact of this EPS on Skunavirus sensitivity was assessed. Methods: Conjugal transfer of pEPS6073 into two model strains followed by phage plaque assays and adsorption assays were performed to assess its effect on phage sensitivity. Phage distal tail proteins were analyzed bioinformatically using HHpred and modeling with AlphaFold. Construction of recombinant phages carrying evolved Dits was performed by supplying a plasmid-encoded template for homologous recombination. Results: pEPS6073 confers resistance against a subset of skunaviruses via adsorption inhibition. IFF collection skunaviruses that infect strains encoding the 6073-like eps gene cluster carry insertions in their distal tail protein-encoding (dit) genes that result in longer Dit proteins (so-called evolved Dits), which encode carbohydrate-binding domains. Three skunaviruses with classical Dits (no insertion) were unable to fully infect their hosts following the conjugal introduction of pEPS6073, showing reductions in both adsorption and efficiency of plaquing. Cloning the evolved Dit into these phages enabled full infectivity on their host strains, both wild type and transconjugant carrying pEPS6073, with recombinant phages adsorbing slightly better to the EPS+ host than wild type. Conclusion: The 6073-like EPS potentially occludes the phage receptor for skunaviruses that encode a classical Dit protein. Skunaviruses that infect strains encoding the 6073-like EPS harbor evolved Dits, which likely help promote phage adsorption rather than just allow the phage to circumvent the putative EPS barrier. This work furthers our knowledge of phage-host interactions in Lactococcus and proposes a role for insertions in the Dit proteins of a subset of skunaviruses.

Novel P335-like Phage Resistance Arises from Deletion within Putative Autolysin yccB in Lactococcus lactis.

Lactococcus lactis and Lactococcus cremoris are broadly utilized as starter cultures for fermented dairy products and are inherently impacted by bacteriophage (phage) attacks in the industrial environment. Consequently, the generation of bacteriophage-insensitive mutants (BIMs) is a standard approach for addressing phage susceptibility in dairy starter strains. In this study, we characterized spontaneous BIMs of L. lactis DGCC12699 that gained resistance against homologous P335-like phages. Phage resistance was found to result from mutations in the YjdB domain of yccB, a putative autolysin gene. We further observed that alteration of a fused tail-associated lysin-receptor binding protein (Tal-RBP) in the phage restored infectivity on the yccB BIMs. Additional investigation found yccB homologs to be widespread in L. lactis and L. cremoris and that different yccB homologs are highly correlated with cell wall polysaccharide (CWPS) type/subtype. CWPS are known lactococcal phage receptors, and we found that truncation of a glycosyltransferase in the cwps operon also resulted in resistance to these P335-like phages. However, characterization of the CWPS mutant identified notable differences from the yccB mutants, suggesting the two resistance mechanisms are distinct. As phage resistance correlated with yccB mutation has not been previously described in L. lactis, this study offers insight into a novel gene involved in lactococcal phage sensitivity.

Host-encoded, cell surface-associated exopolysaccharide required for adsorption and infection by lactococcal P335 phage subtypes.

Lactococcus lactis and Lactococcus cremoris compose commercial starter cultures widely used for industrial dairy fermentations. Some lactococcal strains may produce exopolysaccharides (EPS), which have technological applications, including texture production and phage resistance. Two distinct gene clusters associated with EPS production, designated 6073-like and 7127-like, were identified on plasmids in lactococcal strains. Infectivity of two subsets of P335 group phages, distinguished based on their single-component baseplate/receptor-binding protein nucleotide sequences, was correlated to the presence of a host-encoded 6073-like or 7127-like eps gene cluster. Furthermore, phages belonging to these subsets differentially adsorbed to lactococcal strains harboring the respective eps gene cluster. Loss of the respective EPS-encoding plasmid from a fully phage-sensitive strain resulted in loss of phage adsorption and resistance to the phage. Transmission electron microscopy (TEM) showed that the EPS produced by strains encoding the 6073-like or 7127-like eps gene clusters are cell-surface associated, which, coupled with phage plaquing and adsorption data, shows that specific capsular EPS are involved in host recognition by certain P335 phage subgroups. To our knowledge, this is the first description of the involvement of EPS produced via the Wzx/Wzy-dependent pathway in phage sensitivity of L. lactis or L. cremoris. This study also shows strains that do not appear to be phage-related based on plaque formation may still be related by phage adsorption and indicates that optimal formulation of phage-robust cultures should take into account the EPS type of individual strains.

Dairy lactococcal and streptococcal phage-host interactions: an industrial perspective in an evolving phage landscape.

Almost a century has elapsed since the discovery of bacteriophages (phages), and 85 years have passed since the emergence of evidence that phages can infect starter cultures, thereby impacting dairy fermentations. Soon afterward, research efforts were undertaken to investigate phage interactions regarding starter strains. Investigations into phage biology and morphology and phage-host relationships have been aimed at mitigating the negative impact phages have on the fermented dairy industry. From the viewpoint of a supplier of dairy starter cultures, this review examines the composition of an industrial phage collection, providing insight into the development of starter strains and cultures and the evolution of phages in the industry. Research advances in the diversity of phages and structural bases for phage-host recognition and an overview of the perpetual arms race between phage virulence and host defense are presented, with a perspective toward the development of improved phage-resistant starter culture systems.

Lactococcus lactis type III-A CRISPR-Cas system cleaves bacteriophage RNA.

CRISPR-Cas defends microbial cells against invading nucleic acids including viral genomes. Recent studies have shown that type III-A CRISPR-Cas systems target both RNA and DNA in a transcription-dependent manner. We previously found a type III-A system on a conjugative plasmid in Lactococcus lactis which provided resistance against virulent phages of the Siphoviridae family. Its naturally occurring spacers are oriented to generate crRNAs complementary to target phage mRNA, suggesting transcription-dependent targeting. Here, we show that only constructs whose spacers produce crRNAs complementary to the phage mRNA confer phage resistance in L. lactis. In vivo nucleic acid cleavage assays showed that cleavage of phage dsDNA genome was not detected within phage-infected L. lactis cells. On the other hand, Northern blots indicated that the lactococcal CRISPR-Cas cleaves phage mRNA in vivo. These results cannot exclude that single-stranded phage DNA is not being targeted, but phage DNA replication has been shown to be impaired.

Genetic determinants of lactococcal C2viruses for host infection and their role in phage evolution.

Lactococcus lactis is an industrial starter culture used for the production of fermented dairy products. Pip (phage infection protein) bacteriophage-insensitive mutant (BIM) L. lactis DGCC11032 was isolated following challenge of parental strain DGCC7271 with C2viruses. Over a period of industrial use, phages infecting DGCC11032 were isolated from industrial whey samples and identified as C2viruses. Although Pip is reported to be the receptor for many C2viruses including species type phage c2, a similar cell-membrane-associated protein, YjaE, was recently reported as the receptor for C2virus bIL67. Characterization of DGCC7271 BIMs following challenge with phage capable of infecting DGCC11032 identified mutations in yjaE, confirming YjaE to be necessary for infection. DGCC7271 YjaE mutants remained sensitive to the phages used to generate pip variant DGCC11032, indicating a distinction in host phage determinants. We will refer to C2viruses requiring Pip as c2-type andC2viruses that require YjaE as bIL67-type. Genomic comparisons of two c2-type phages unable to infect pip mutant DGCC11032 and four bIL67-type phages isolated on DGCC11032 confirmed the segregation of each group based on resemblance to prototypical phages c2 and bIL67, respectively. The distinguishing feature is linked to three contiguous late-expressed genes: l14-15-16 (c2) and ORF34-35-36 (bIL67). Phage recombinants in which the c2-like l14-15-16 homologue gene set was exchanged with corresponding bIL67 genes ORF34-35-36 were capable of infecting a pip mutated host. Together, these results correlate the phage genes corresponding to l14-15-16 (c2) and ORF34-35-36 (bIL67) to host lactococcal phage determinants Pip and YjaE, respectively.

Mobile CRISPR/Cas-mediated bacteriophage resistance in Lactococcus lactis.

Lactococcus lactis is a biotechnological workhorse for food fermentations and potentially therapeutic products and is therefore widely consumed by humans. It is predominantly used as a starter microbe for fermented dairy products, and specialized strains have adapted from a plant environment through reductive evolution and horizontal gene transfer as evidenced by the association of adventitious traits with mobile elements. Specifically, L. lactis has armed itself with a myriad of plasmid-encoded bacteriophage defensive systems to protect against viral predation. This known arsenal had not included CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins), which forms a remarkable microbial immunity system against invading DNA. Although CRISPR/Cas systems are common in the genomes of closely related lactic acid bacteria (LAB), none was identified within the eight published lactococcal genomes. Furthermore, a PCR-based search of the common LAB CRISPR/Cas systems (Types I and II) in 383 industrial L. lactis strains proved unsuccessful. Here we describe a novel, Type III, self-transmissible, plasmid-encoded, phage-interfering CRISPR/Cas discovered in L. lactis. The native CRISPR spacers confer resistance based on sequence identity to corresponding lactococcal phage. The interference is directed at phages problematic to the dairy industry, indicative of a responsive system. Moreover, targeting could be modified by engineering the spacer content. The 62.8-kb plasmid was shown to be conjugally transferrable to various strains. Its mobility should facilitate dissemination within microbial communities and provide a readily applicable system to naturally introduce CRISPR/Cas to industrially relevant strains for enhanced phage resistance and prevention against acquisition of undesirable genes.