Identification and Analysis of a Novel Group of Bacteriophages Infecting the Lactic Acid Bacterium Streptococcus thermophilus.
ABSTRACT: We present the complete genome sequences of four members of a novel group of phages infecting Streptococcus thermophilus, designated here as the 987 group. Members of this phage group appear to have resulted from genetic exchange events, as evidenced by their "hybrid" genomic architecture, exhibiting DNA sequence relatedness to the morphogenesis modules of certain P335 group Lactococcus lactis phages and to the replication modules of S. thermophilus phages. All four identified members of the 987 phage group were shown to elicit adsorption affinity to both their cognate S. thermophilus hosts and a particular L. lactis starter strain. The receptor binding protein of one of these phages (as a representative of this novel group) was defined using an adsorption inhibition assay. The emergence of a novel phage group infecting S. thermophilus highlights the continuous need for phage monitoring and development of new phage control measures.Phage predation of S. thermophilus is an important issue for the dairy industry, where viral contamination can lead to fermentation inefficiency or complete fermentation failure. Genome information and phage-host interaction studies of S. thermophilus phages, particularly those emerging in the marketplace, are an important part of limiting the detrimental impact of these viruses in the dairy environment.
Project description:Streptococcus thermophilus strains are among the most widely employed starter cultures in dairy fermentations, second only to those of Lactococcus lactis. The extensive application of this species provides considerable opportunity for the proliferation of its infecting (bacterio)phages. Until recently, dairy streptococcal phages were classified into two groups (cos and pac groups), while more recently, two additional groups have been identified (5093 and 987 groups). This highlights the requirement for consistent monitoring of phage populations in the industry. Here, we report a survey of 35 samples of whey derived from 27 dairy fermentation facilities in ten countries against a panel of S. thermophilus strains. This culminated in the identification of 172 plaque isolates, which were characterized by multiplex PCR, restriction fragment length polymorphism analysis, and host range profiling. Based on this characterisation, 39 distinct isolates representing all four phage groups were selected for genome sequencing. Genetic diversity was observed among the cos isolates and correlations between receptor binding protein phylogeny and host range were also clear within this phage group. The 987 phages isolated within this study shared high levels of sequence similarity, yet displayed reduced levels of similarity to those identified in previous studies, indicating that they are subject to ongoing genetic diversification.
Project description:Knowledge of phage-host interactions at a fundamental level is central to the design of rational strategies for the development of phage-resistant strains that may be applied in industrial settings. Phages infecting lactic acid bacteria, in particular Lactococcus lactis and Streptococcus thermophilus, negatively impact on dairy fermentation processes with serious economic implications. In recent years a wealth of information on structural protein assembly and topology has become available relating to phages infecting Escherichia coli, Bacillus subtilis and Lactococcus lactis, which act as models for structural analyses of dairy phages. In this review, we explore the role of model tailed phages, such as T4 and SPP1, in advancing our knowledge regarding interactions between dairy phages and their hosts. Furthermore, the potential of currently investigated dairy phages to in turn serve as model systems for this particular group of phages is discussed.
Project description:Bacteriophages are the main cause of fermentation failures in dairy plants. The majority of Streptococcus thermophilus phages can be divided into either cos- or pac-type phages and are additionally characterized by examining the V2 region of their antireceptors. We screened a large number of S. thermophilus phages from the Chr. Hansen A/S collection, using PCR specific for the cos- or pac-type phages, as well as for the V2 antireceptor region. Three phages did not produce positive results with the assays. Analysis of phage morphologies indicated that two of these phages, CHPC577 and CHPC926, had shorter tails than the traditional S. thermophilus phages. The third phage, CHPC1151, had a tail size similar to those of the cos- or pac-type phages, but it displayed a different baseplate structure. Sequencing analysis revealed the genetic similarity of CHPC577 and CHPC926 with a subgroup of Lactococcus lactis P335 phages. Phage CHPC1151 was closely related to the atypical S. thermophilus phage 5093, homologous with a nondairy streptococcal prophage. By testing adsorption of the related streptococcal and lactococcal phages to the surface of S. thermophilus and L. lactis strains, we revealed the possibility of cross-interactions. Our data indicated that the use of S. thermophilus together with L. lactis, extensively applied for dairy fermentations, triggered the recombination between phages infecting different bacterial species. A notable diversity among S. thermophilus phage populations requires that a new classification of the group be proposed.IMPORTANCEStreptococcus thermophilus is a component of thermophilic starter cultures commonly used for cheese and yogurt production. Characterizing streptococcal phages, understanding their genetic relationships, and studying their interactions with various hosts are the necessary steps for preventing and controlling phage attacks that occur during dairy fermentations.
Project description:Despite the persistent and costly problem caused by (bacterio)phage predation of Streptococcus thermophilus in dairy plants, DNA sequence information relating to these phages remains limited. Genome sequencing is necessary to better understand the diversity and proliferative strategies of virulent phages. In this report, whole genome sequences of 40 distinct bacteriophages infecting S. thermophilus were analyzed for general characteristics, genomic structure and novel features. The bacteriophage genomes display a high degree of conservation within defined groupings, particularly across the structural modules. Supporting this observation, four novel members of a recently discovered third group of S. thermophilus phages (termed the 5093 group) were found to be conserved relative to both phage 5093 and to each other. Replication modules of S. thermophilus phages generally fall within two main groups, while such phage genomes typically encode one putative transcriptional regulator. Such features are indicative of widespread functional synteny across genetically distinct phage groups. Phage genomes also display nucleotide divergence between groups, and between individual phages of the same group (within replication modules and at the 3' end of the lysis module)-through various insertions and/or deletions. A previously described multiplex PCR phage detection system was updated to reflect current knowledge on S. thermophilus phages. Furthermore, the structural protein complement as well as the antireceptor (responsible for the initial attachment of the phage to the host cell) of a representative of the 5093 group was defined. Our data more than triples the currently available genomic information on S. thermophilus phages, being of significant value to the dairy industry, where genetic knowledge of lytic phages is crucial for phage detection and monitoring purposes. In particular, the updated PCR detection methodology for S. thermophilus phages is highly useful in monitoring particular phage group(s) present in a given whey sample. Studies of this nature therefore not only provide information on the prevalence and associated threat of known S. thermophilus phages, but may also uncover newly emerging and genomically distinct phages infecting this dairy starter bacterium.
Project description:Comparative genomics has proven useful in exploring the biodiversity of phages and understanding phage-host interactions. This knowledge is particularly useful for phages infecting Streptococcus thermophilus, as they constitute a constant threat during dairy fermentations. Here, we explore the genetic diversity of S. thermophilus phages to identify genetic determinants with a signature for host specificity, which could be linked to the bacterial receptor genotype. A comparative genomic analysis was performed on 142 S. thermophilus phage genomes, 55 of which were sequenced in this study. Effectively, 94 phages were assigned to the group cos (DT1), 36 to the group pac (O1205), six to the group 5093, and six to the group 987. The core genome-based phylogeny of phages from the two dominating groups and their receptor binding protein (RBP) phylogeny corresponded to the phage host-range. A role of RBP in host recognition was confirmed by constructing a fluorescent derivative of the RBP of phage CHPC951, followed by studying the binding of the protein to the host strain. Furthermore, the RBP phylogeny of the cos group was found to correlate with the host genotype of the exocellular polysaccharide-encoding operon. These findings provide novel insights towards developing strategies to combat phage infections in dairies.
Project description:Lactococcus Ceduovirus (formerly c2virus) bacteriophages are among the three most prevalent phage types reported in dairy environments. Phages from this group conduct a strictly lytic lifestyle and cause substantial losses during milk fermentation processes, by infecting lactococcal host starter strains. Despite their deleterious activity, there are limited research data concerning Ceduovirus phages. To advance our knowledge on this specific phage group, we sequenced and performed a comparative analysis of 10 new Lactococcus lactis Ceduovirus phages isolated from distinct dairy environments. Host range studies allowed us to distinguish the differential patterns of infection of L. lactis cells for each phage, and revealed a broad host spectrum for most of them. We showed that 40% of the studied Ceduovirus phages can infect both cremoris and lactis strains. A preference to lyse strains with the C-type cell wall polysaccharide genotype was observed. Phage whole-genome sequencing revealed an average nucleotide identity above 80%, with distinct regions of divergence mapped to several locations. The comparative approach for analyzing genomic data and the phage lytic spectrum suggested that the amino acid sequence of the orf8-encoded putative tape measure protein correlates with host range. Phylogenetic studies revealed separation of the sequenced phages into two subgroups. Finally, we identified three types of phage origin of replication regions, and showed they are able to support plasmid replication without additional phage proteins.
Project description:Receptors on the cell surfaces of bacterial hosts are essential during the infection cycle of bacteriophages. To date, the phage receptors of the industrial relevant dairy starter bacterium Streptococcus thermophilus remain elusive. Thus, we set out to identify cell surface structures that are involved in host recognition by dairy streptococcal phages. Five industrial S. thermophilus strains sensitive to different phages (pac type, cos type, and the new type 987), were selected to generate spontaneous bacteriophage-insensitive mutants (BIMs). Of these, approximately 50% were deselected as clustered regularly interspaced short palindromic repeat (CRISPR) mutants, while the other pool was further characterized to identify receptor mutants. On the basis of genome sequencing data, phage resistance in putative receptor mutants was attributed to nucleotide changes in genes encoding glycan biosynthetic pathways. Superresolution structured illumination microscopy was used to visualize the interactions between S. thermophilus and its phages. The phages were either regularly distributed along the cells or located at division sites of the cells. The cell wall structures mediating the latter type of phage adherence were further analyzed via phenotypic and biochemical assays. Altogether, our data suggested that phage adsorption to S. thermophilus is mediated by glycans associated with the bacterial cell surface. Specifically, the pac-type phage CHPC951 adsorbed to polysaccharides anchored to peptidoglycan, while the 987-type phage CHPC926 recognized exocellular polysaccharides associated with the cell surface.IMPORTANCE Streptococcus thermophilus is widely used in starter cultures for cheese and yoghurt production. During dairy fermentations, infections of bacteria with bacteriophages result in acidification failures and a lower quality of the final products. An understanding of the molecular factors involved in phage-host interactions, in particular, the phage receptors in dairy bacteria, is a crucial step for developing better strategies to prevent phage infections in dairy plants.
Project description:Phages of Streptococcus thermophilus present a major threat to the production of many fermented dairy products. To date, only a few studies have assessed the biodiversity of S. thermophilus phages in dairy fermentations. In order to develop strategies to limit phage predation in this important industrial environment, it is imperative that such studies are undertaken and that phage-host interactions of this species are better defined. The present study investigated the biodiversity and evolution of phages within an Irish dairy fermentation facility over an 11-year period. This resulted in the isolation of 17 genetically distinct phages, all of which belong to the so-called cos group. The evolution of phages within the factory appears to be influenced by phages from other dairy plants introduced into the factory for whey protein powder production. Modular exchange, primarily within the regions encoding lysogeny and replication functions, was the major observation among the phages isolated between 2006 and 2016. Furthermore, the genotype of the first isolate in 2006 was observed continuously across the following decade, highlighting the ability of these phages to prevail in the factory setting for extended periods of time. The proteins responsible for host recognition were analyzed, and carbohydrate-binding domains (CBDs) were identified in the distal tail (Dit), the baseplate proteins, and the Tail-associated lysin (Tal) variable regions (VR1 and VR2) of many isolates. This supports the notion that S. thermophilus phages recognize a carbohydrate receptor on the cell surface of their host.IMPORTANCE Dairy fermentations are consistently threatened by the presence of bacterial viruses (bacteriophages or phages), which may lead to a reduction in acidification rates or even complete loss of the fermentate. These phages may persist in factories for long periods of time. The objective of the current study was to monitor the progression of phages infecting the dairy bacterium Streptococcus thermophilus over a period of 11 years in an Irish dairy plant so as to understand how these phages evolve. A focused analysis of the genomic region that encodes host recognition functions highlighted that the associated proteins harbor a variety of carbohydrate-binding domains, which corroborates the notion that phages of S. thermophilus recognize carbohydrate receptors at the initial stages of the phage cycle.
Project description:Lactococcal dairy starter strains are under constant threat from phages in dairy fermentation facilities, especially by members of the so-called 936, P335, and c2 species. Among these three phage groups, members of the P335 species are the most genetically diverse. Here, we present the complete genome sequences of two P335-type phages, Q33 and BM13, isolated in North America and representing a novel lineage within this phage group. The Q33 and BM13 genomes exhibit homology, not only to P335-type, but also to elements of the 936-type phage sequences. The two phage genomes also have close relatedness to phages infecting Enterococcus and Clostridium, a heretofore unknown feature among lactococcal P335 phages. The Q33 and BM13 genomes are organized in functionally related clusters with genes encoding functions such as DNA replication and packaging, morphogenesis, and host cell lysis. Electron micrographic analysis of the two phages highlights the presence of a baseplate more reminiscent of the baseplate of 936 phages than that of the majority of members of the P335 group, with the exception of r1t and LC3.
Project description:Virulent lactococcal phages are still a major risk for milk fermentation processes as they may lead to slowdowns and low-quality fermented dairy products, particularly cheeses. Some of the phage control strategies used by the industry rely on heat treatments. Recently, a few Lactococcus lactis phages were found to be highly thermo-resistant. To identify the genetic determinant(s) responsible for the thermal resistance of lactococcal phages, we used the virulent phage CB14 (of the Lactococcus lactis 936 [now Sk1virus] phage group) to select for phage mutants with increased heat stability. By treating phage CB14 to successive low and high temperatures, we were able to select two CB14 derivatives with increased heat stability. Sequencing of their genome revealed the same nucleotide sequences as the wild-type phage CB14, except for a same-sized deletion (120 bp) in the gene coding for the tape measure protein (TMP) of each phage mutant, but at a different position. The TMP protein sequences of these mutant phages were compared with their homologues in other wild-type L. lactis phages with a wide diversity in heat stability. Comparative analysis showed that the same nucleotide deletion appears to have also occurred in the gene coding for the TMP of highly thermo-resistant lactococcal phages P1532 and P680. We propose that the TMP is, in part, responsible for the heat stability of the highly predominant lactococcal phages of the Sk1virus group.IMPORTANCE Virulent lactococcal phages still represent a major risk for milk fermentation as they may lead to slowdowns and low-quality fermented dairy products. Heat treatment is one of the most commonly used methods to control these virulent phages in cheese by-products. Recently, a few Lactococcus lactis phages, members of the Sk1virus group, have emerged with high thermal stability. To our knowledge, the genetic determinant(s) responsible for this thermal resistance in lactococcal phages is unknown. A better understanding of the thermal stability of these emerging virulent lactococcal phages is needed to improve industrial control strategies. In this work, we report the identification of a phage structural protein that is involved in the heat stability of a virulent Sk1virus phage. Identifying such a genetic determinant for heat stability is a first step in understanding the emergence of this group of thermostable phages.