Bacteriophage-host interactions in Streptococcus thermophilus and their impact on co-evolutionary processes.

Bacteriophages (or phages) represent a persistent threat to the success and reliability of food fermentation processes. Recent reports of phages that infect Streptococcus thermophilus have highlighted the diversification of phages of this species. Phages of S. thermophilus typically exhibit a narrow range, a feature that is suggestive of diverse receptor moieties being presented on the cell surface of the host. Cell wall polysaccharides, including rhamnose-glucose polysaccharides and exopolysaccharides have been implicated as being involved in the initial interactions with several phages of this species. Following internalization of the phage genome, the host presents several defences, including CRISPR-Cas and restriction and modification systems to limit phage proliferation. This review provides a current and holistic view of the interactions of phages and their S. thermophilus host cells and how this has influenced the diversity and evolution of both entities.

Bipartite rgp Locus Diversity in Streptococcus thermophilus Corresponds to Backbone and Side Chain Differences of Its Rhamnose-Containing Cell Wall Polysaccharide.

The rhamnose-glucose polysaccharide (Rgp) of Streptococcus thermophilus represents a major cell wall component, and the gene cluster responsible for its biosynthesis (termed rgp) has recently been identified. Significant genetic diversity among these loci has previously been reported, with five distinct rgp genotypes identified (designated rgp1 through -5). In the present study, two additional genotypes were identified (designated rgp6 and rgp7) through comparative analysis of the rgp loci of 78 Streptococcus thermophilus genomes. The rgp locus of a given S. thermophilus strain encoded the biosynthetic machinery for a rhamnan-rich backbone and a variable side chain component, the latter being associated with the highly specific interactions with many bacteriophages that infect this species. The chemical structure of the Rgp from three S. thermophilus strains, representing the rgp2, -3, and -4 genotypes, was elucidated, and based on bioinformatic and biochemical analyses we propose a model for Rgp biosynthesis in dairy streptococci. Furthermore, we exploited the genetic diversity within the S. thermophilus bipartite rgp locus to develop a two-step multiplex PCR system to classify strains based on gene content associated with the biosynthesis of the variable side chain structure as well as the rhamnan backbone. IMPORTANCE Streptococcus thermophilus is present and applied in industrial and artisanal dairy fermentations for the production of various cheeses and yogurt. During these fermentations, S. thermophilus is vulnerable to phage predation, and recent studies have identified the rhamnose-glucose polymer (Rgp) as the definitive receptor for at least one problematic phage species. Detailed analysis of S. thermophilus rgp loci has revealed an unprecedented level of genetic diversity, particularly within the glycosyltransferase-encoding gene content of a given locus. Our study shows that this genetic diversity reflects the biochemical structure(s) of S. thermophilus Rgp. As such, we harnessed the genetic diversity of S. thermophilus rgp loci to develop a two-step multiplex PCR method for the classification of strain collections and, ultimately, the formation of phage-robust rational starter sets.

A structural discovery journey of streptococcal phages adhesion devices by AlphaFold2.

Successful bacteriophage infection starts with specific recognition and adhesion to the host cell surface. Adhesion devices of siphophages infecting Gram-positive bacteria are very diverse and remain, for the majority, poorly understood. These assemblies often comprise long, flexible, and multi-domain proteins, which limits their structural analyses by experimental approaches such as X-ray crystallography and electron microscopy. However, the protein structure prediction program AlphaFold2 is exquisitely adapted to unveil structural and functional details of such molecular machineries. Here, we present structure predictions of whole adhesion devices of five representative siphophages infecting Streptococcus thermophilus, one of the main lactic acid bacteria used in dairy fermentations. The predictions highlight the mosaic nature of these devices that share functional domains for which active sites and residues could be unambiguously identified. Such AlphaFold2 analyses of phage-encoded host adhesion devices should become a standard method to characterize phage-host interaction machineries and to reliably annotate phage genomes.

Needle in a Whey-Stack: PhRACS as a Discovery Tool for Unknown Phage-Host Combinations.

The field of metagenomics has rapidly expanded to become the go-to method for complex microbial community analyses. However, there is currently no straightforward route from metagenomics to traditional culture-based methods of strain isolation, particularly in (bacterio)phage biology, leading to an investigative bottleneck. Here, we describe a method that exploits specific phage receptor binding protein (RBP)-host cell surface receptor interaction enabling isolation of phage-host combinations from an environmental sample. The method was successfully applied to two complex sample types-a dairy-derived whey sample and an infant fecal sample, enabling retrieval of specific and culturable phage hosts. IMPORTANCE PhRACS aims to bridge the current divide between in silico genetic analyses (i.e., phageomic studies) and traditional culture-based methodology. Through the labeling of specific bacterial hosts with fluorescently tagged recombinant phage receptor binding proteins and the isolation of tagged cells using flow cytometry, PhRACS allows the full potential of phageomic data to be realized in the wet laboratory.

Brussowvirus SW13 Requires a Cell Surface-Associated Polysaccharide To Recognize Its Streptococcus thermophilus Host.

Four bacteriophage-insensitive mutants (BIMs) of the dairy starter bacterium Streptococcus thermophilus UCCSt50 were isolated following challenge with Brussowvirus SW13. The BIMs displayed an altered sedimentation phenotype. Whole-genome sequencing and comparative genomic analysis of the BIMs uncovered mutations within a family 2 glycosyltransferase-encoding gene (orf06955UCCSt50) located within the variable region of the cell wall-associated rhamnose-glucose polymer (Rgp) biosynthesis locus (designated the rgp gene cluster here). Complementation of a representative BIM, S. thermophilus B1, with native orf06955UCCSt50 restored phage sensitivity comparable to that of the parent strain. Detailed bioinformatic analysis of the gene product of orf06955UCCSt50 identified it as a functional homolog of the Lactococcus lactis polysaccharide pellicle (PSP) initiator WpsA. Biochemical analysis of cell wall fractions of strains UCCSt50 and B1 determined that mutations within orf06955UCCSt50 result in the loss of the side chain decoration from the Rgp backbone structure. Furthermore, it was demonstrated that the intact Rgp structure incorporating the side chain structure is essential for phage binding through fluorescence labeling studies. Overall, this study confirms that the rgp gene cluster of S. thermophilus encodes the biosynthetic machinery for a cell surface-associated polysaccharide that is essential for binding and subsequent infection by Brussowviruses, thus enhancing our understanding of S. thermophilus phage-host dynamics. IMPORTANCE Streptococcus thermophilus is an important starter culture bacterium in global dairy fermentation processes, where it is used for the production of various cheeses and yogurt. Bacteriophage predation of the species can result in substandard product quality and, in rare cases, complete fermentation collapse. To mitigate these risks, it is necessary to understand the phage-host interaction process, which commences with the recognition of, and adsorption to, specific host-encoded cell surface receptors by bacteriophage(s). As new groups of S. thermophilus phages are being discovered, the importance of underpinning the genomic elements that specify the surface receptor(s) is apparent. Our research identifies a single gene that is critical for the biosynthesis of a saccharidic moiety required for phage adsorption to its S. thermophilus host. The acquired knowledge provides novel insights into phage-host interactions for this economically important starter species.

Cell wall polysaccharides of Gram positive ovococcoid bacteria and their role as bacteriophage receptors.

Gram-positive bacterial cell walls are characterised by the presence of a thick peptidoglycan layer which provides protection from extracellular stresses, maintains cell integrity and determines cell morphology, while it also serves as a foundation to anchor a number of crucial polymeric structures. For ovococcal species, including streptococci, enterococci and lactococci, such structures are represented by rhamnose-containing cell wall polysaccharides, which at least in some instances appear to serve as a functional replacement for wall teichoic acids. The biochemical composition of several streptococcal, lactococcal and enterococcal rhamnose-containing cell wall polysaccharides have been elucidated, while associated functional genomic analyses have facilitated the proposition of models for individual biosynthetic pathways. Here, we review the genomic loci which encode the enzymatic machinery to produce rhamnose-containing, cell wall-associated polysaccharide (Rha cwps) structures of the afore-mentioned ovococcal bacteria with particular emphasis on gene content, biochemical structure and common biosynthetic steps. Furthermore, we discuss the role played by these saccharidic polymers as receptors for bacteriophages and the important role phages play in driving Rha cwps diversification and evolution.

Revisiting the host adhesion determinants of Streptococcus thermophilus siphophages.

Available 3D structures of bacteriophage modules combined with predictive bioinformatic algorithms enabled the identification of adhesion modules in 57 siphophages infecting Streptococcus thermophilus (St). We identified several carbohydrate-binding modules (CBMs) in so-called evolved distal tail (Dit) and tail-associated lysozyme (Tal) proteins of St phage baseplates. We examined the open reading frame (ORF) downstream of the Tal-encoding ORF and uncovered the presence of a putative p2-like receptor-binding protein (RBP). A 21 Å resolution electron microscopy structure of the baseplate of cos-phage STP1 revealed the presence of six elongated electron densities, surrounding the core of the baseplate, that harbour the p2-like RBPs at their tip. To verify the functionality of these modules, we expressed GFP- or mCherry-coupled Tal and putative RBP CBMs and observed by fluorescence microscopy that both modules bind to their corresponding St host, the putative RBP CBM with higher affinity than the Tal-associated one. The large number of CBM functional domains in St phages suggests that they play a contributory role in the infection process, a feature that we previously described in lactococcal phages and beyond, possibly representing a universal feature of the siphophage host-recognition apparatus.

A cell wall-associated polysaccharide is required for bacteriophage adsorption to the Streptococcus thermophilus cell surface.

Streptococcus thermophilus strain ST64987 was exposed to a member of a recently discovered group of S. thermophilus phages (the 987 phage group), generating phage-insensitive mutants, which were then characterized phenotypically and genomically. Decreased phage adsorption was observed in selected bacteriophage-insensitive mutants, and was partnered with a sedimenting phenotype and increased cell chain length or aggregation. Whole genome sequencing of several bacteriophage-insensitive mutants identified mutations located in a gene cluster presumed to be responsible for cell wall polysaccharide production in this strain. Analysis of cell surface-associated glycans by methylation and NMR spectroscopy revealed a complex branched rhamno-polysaccharide in both ST64987 and phage-insensitive mutant BIM3. In addition, a second cell wall-associated polysaccharide of ST64987, composed of hexasaccharide branched repeating units containing galactose and glucose, was absent in the cell wall of mutant BIM3. Genetic complementation of three phage-resistant mutants was shown to restore the carbohydrate and phage resistance profiles of the wild-type strain, establishing the role of this gene cluster in cell wall polysaccharide production and phage adsorption and, thus, infection.

Biodiversity of Streptococcus thermophilus Phages in Global Dairy Fermentations.

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.

A Decade of Streptococcus thermophilus Phage Evolution in an Irish Dairy Plant.

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.