Identification of genes involved in the DNA injection process by lactococcal P335 phages

Bacteriophages (phages) belonging to the P335 lactococcal group are one of the most frequently isolated phages in fermentation processes using Lactococcus lactis strains as the primary starter culture. Despite efforts to prevent phage attack, problems caused due to phage infection during large scale milk fermentations are still regularly reported. Lactococcus lactis subsp. cremoris (L. lactis) 3107 is a dairy starter strain and host for the model lactococcal P335 phage TP901-1. Three derivatives of this strain that are resistant to TP901-1, while remaining sensitive to infection by another P335 phage called LC3 were employed to define molecular players involved in the phage infection pathway. Comparative genomic analysis of L. lactis 3107 and its phageresistant derivatives identified several mutations that may cause the observed resistance. Complementation assays with one specific construct, termed pNZgtfA, restored phage sensitivity and lysogenization frequency in two of the derivative strains. pNZgtfA expresses the L3107_1442 gene (renamed here as gtfA) which encodes a predicted membrane-associated glycosyltransferase (GTF), GtfA. Interestingly, the mutant strains also exhibited reduced sensitivity to the rare lactococcal 949 group phages, 949 and WRP3, while complementation assays fully restored infectivity by these phages, thus suggesting that gtfA is also involved in phage-host interactions of these phages. TP901-1 escape mutants capable of infecting these phage-resistant derivatives possess mutations in the gene encoding the structural and/or lytic domain of the Tail-associated lysin in order to adapt to cells which have acquired phage resistance. Interestingly, the gene gtfA is ubiquitous among Lactococcus genomes, L. lactis subsp. cremoris NZ9000 harbours a 99 % identical homologue of gtfA3107 on its genome, referred to here as gtfANZ9000 (corresponding to locus tag LLNZ_08920). Therefore, a mutation (stop codon) was introduced into the coding sequence of gtfANZ9000 by targeted mutagenesis in order to expose the mutant strain generated to NZ9000-infecting 936 group phages. Results showed that a non-functional GtfA protein has no effect on phages belonging to the lactococcal 936 group. GtfA3107 possess similar characteristics to proteins belonging to the GT-C family of GTFs which are known to be involved in glycosylation of substrates at the out-facing side of the cytoplasmic membrane. Therefore, GtfA is presumed to glycosylate a substrate, most likely a cell envelope-associated glycopolymer, such as a cell wall polysaccharide (CWPS) or teichoic acid (TA). If gtfANZ9000 is involved in the modification of CWP components, the inactivation of this gene may have generated a mutant strain deficient in the glycosylation of PSP or rhamnan. In an attempt to identify the GtfA substrate, the biochemical composition and structure of CWPS components (rhamnan and PSP) of L. lactisstrains 3107 and NZ9000, and a number of derivative strains were analysed. Results showed that derivatives possessing a non-functional gtfA or over-expressing gtfA do not possess any structural modifications in either the rhamnan or PSP components of the CWPS as compared to the 3107 or NZ9000 parent strains. In addition, the biochemical composition and structure of lipoteichoic acids (LTAs) in L. lactis 3107 and a representative phage-resistant derivative was also analysed, results revealed that gtfA is not responsible for terminal galactose substitution of LTA. Currently, further biochemical analysis of TA composition in L. lactis 3107 is being performed in order to identify the GtfA substrate and its relevance to phage infection. Attempts to generate LC3-resistant derivatives of L. lactis 3107 resulted in the isolation of lysogens of this phage that displayed resistance to multiple P335 phages. The LC3-prophage encoded superinfection exclusion (Sie) protein Sie2009/LC3, which is an identical (100 % identity) homologue of the previously identified Sie system Sie2009 was shown to be responsible for the phage resistance observed. Furthermore, superinfection immunity (Sii) against certain phages belonging to the P335 group was also exhibited in the presence of the LC3-prophage encoded repressor protein RepLC3. Overall this study has identified bacterial and phage-encoded protein(s) required for, or interfering with, the infection process of the dairy starter strain L. lactis 3107 by phages belonging to the lactococcal P335 group, as well as 936 and 949 group phages. This study forms the basis for future studies aimed to limit the proliferation of phages in the industrial contex

Complete genome sequence of Lactococcus lactis subsp. Cremoris 3107, host for the model Lactococcal P335 Bacteriophage TP901-1

The complete genome sequence of Lactococcus lactis subsp. cremoris 3107, a dairy starter strain and a host for the model lactococcal P335 bacteriophage TP901-1, is reported here. The circular chromosome of L. lactis subsp. cremoris 3107 is among the smallest genomes of currently sequenced lactococcal strains. L. lactis subsp. cremoris 3107 harbors a complement of six plasmids, which appears to be a reflection of its adaptation to the nutrient-rich dairy environment

Lysogenization of a Lactococcal Host with Three Distinct Temperate Phages Provides Homologous and Heterologous Phage Resistance

Lactococcus lactis is the most widely exploited microorganism in global dairy fermentations. Lactococcal strains are described as typically harboring a number of prophages in their chromosomes. The presence of such prophages may provide both advantages and disadvantages to the carrying host. Here, we describe the deliberate generation of three distinct lysogens of the model lactococcal strain 3107 and the impact of additional prophage carriage on phage-resistance and anti-microbial susceptibility. Lysogen-specific responses were observed, highlighting the unique relationship and impact of each lysogenic phage on its host. Both homologous and heterologous phage-resistance profiles were observed, highlighting the presence of possible prophage-encoded phage-resistance factors. Superinfection exclusion was among the most notable causes of heterologous phage-resistance profiles with resistance observed against members of the Skunavirus, P335, P087, and 949 lactococcal phage groups. Through these analyses, it is now possible to identify phages that may pursue similar DNA injection pathways. The generated lysogenic strains exhibited increased sensitivity to the antimicrobial compounds, nisin and lysozyme, relative to the parent strain, although it is noteworthy that the degree of sensitivity was specific to the individual (pro)phages. Overall, the findings highlight the unique impact of each prophage on a given strain and the requirement for strain-level analysis when considering the implications of lysogeny

Host genetic requirements for DNA release of lactococcal phage TP901‐1

Abstract The first step in phage infection is the recognition of, and adsorption to, a receptor located on the host cell surface. This reversible host adsorption step is commonly followed by an irreversible event, which involves phage DNA delivery or release into the bacterial cytoplasm. The molecular components that trigger this latter event are unknown for most phages of Gram‐positive bacteria. In the current study, we present a comparative genome analysis of three mutants of Lactococcus cremoris 3107, which are resistant to the P335 group phage TP901‐1 due to mutations that affect TP901‐1 DNA release. Through genetic complementation and phage infection assays, a predicted lactococcal three‐component glycosylation system (TGS) was shown to be required for TP901‐1 infection. Major cell wall saccharidic components were analysed, but no differences were found. However, heterologous gene expression experiments indicate that this TGS is involved in the glucosylation of a cell envelope‐associated component that triggers TP901‐1 DNA release. To date, a saccharide modification has not been implicated in the DNA delivery process of a Gram‐positive infecting phage