The streptococcal phase-variable type I restriction modification system SsuCC20p dictates the methylome of Streptococcus suis impacting the transcriptome and virulence in a zebrafish larvae infection model

Phase-variable type I restriction modification(RM) systems are epigenetic regulatory systems that have been identifiedin numerous human bacterial pathogens. We previously showed that an emerging zoonotic lineage of Streptococcus suis acquired a phase-variable type I RM system, named SsuCC20p. The SsuCC20p locus was identifiedin the genome of multiple streptococcal species, indicating that it is not restricted to S. suis and can be acquired through horizontal gene transfer. We demonstrate that SsuCC20p phase variability relies on a recombinase present within the locus. SsuCC20p is the single RM system responsible for the genome methylation profilesthat were detected in the representative zoonotic S. suis isolate 861160 in vitro. In addition, we show that, contrary to previous observations, hsdS genes located downstream of hsdM and the recombinase gene contribute to the SsuCC20p genome methylation profile.SsuCC20p locked mutants expressing a single hsdS each show a unique genome methylation profileand, when grown in human serum, have distinct transcriptomes. In a zebrafishlarvae infection model, we observed significantdifferencesin virulence between the locked mutants and a corresponding shift in hsdS allele distribution in the wild type. These data indicate that the streptococcal phase-variable type I RM system SsuCC20p can impact bacterial fitnessvia epigenetic regulation of gene expression, which impacts the virulence of S. suis in the zebrafishlarvae infection model. IMPORTANCE Phase variation allows a single strain to produce phenotypic diverse subpopulations. Phase-variable restriction modification(RM) systems are systems that allow for such phase variation via epigenetic regulation of gene expression levels. The phase-variable RM system SsuCC20p was found in multiple streptococcal species and was acquired by an emerging zoonotic lineage of Streptococcus suis. We show that the phase variability of SsuCC20p is dependent on a recombinase encoded within the SsuCC20p locus. We characterized the genome methylation profilesof the differentphases of SsuCC20p and demonstrated the consequential impact on the transcriptome and virulence in a zebrafishinfection model. Acquiring mobile genetic elements containing epigenetic regulatory systems, like phase-variable RM systems, enables bacterial pathogens to produce diverse phenotypic subpopulations that are better adapted to specific(host) environments encountered during infection.