Brussowvirus SW13 requires a cell surface-associated polysaccharide to recognise its Streptococcus thermophilus host.

Four b acteriophage i nsensitive m utants (BIMs) of the dairy starter bacterium Streptococcus thermophilus UCCSt50 were isolated following a challenge with the 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 r hamnose- g lucose p olymer (Rgp) biosynthesis locus (designated here as the rgp gene cluster). 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 p oly s accharide p ellicle (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 fluorescent labelling studies. Overall, this study confirms that the rgp gene cluster of S. thermophilus encodes the biosynthetic machinery for a cell surface-associated polysaccharide which 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 which specify the surface receptor(s) is apparent. Our research identifies a single gene which 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.