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 ) has recently been identified. Significant genetic diversity among these loci has previously been reported, with five distinct genotypes identified (designated through - ). In the present study, two additional genotypes were identified (designated and ) through comparative analysis of the loci of 78 Streptococcus thermophilus genomes. The 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 , , 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 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. 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 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 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.