Rho factor mediates flagellum and toxin phase variation and impacts virulence in Clostridioides difficile.

Author summary Phenotypic heterogeneity maintained by phase variation allows bacterial subpopulations to overcome potentially detrimental stresses in the environment, contributing to bacterial survival. Phase variation of flagella and toxins inC.difficilesuggests that maintaining heterogeneity of their production may be important for survival and virulence. In this study, we identified Rho as atrans-acting factor that mediates the differential gene expression that imparts heterogeneity in flagellum and toxin production. These results reveal a new role for Rho-mediated transcription termination in regulation of gene expression. The intestinal pathogenClostridioides difficileexhibits heterogeneity in motility and toxin production. This phenotypic heterogeneity is achieved through phase variation by site-specific recombination via the DNA recombinase RecV, which reversibly inverts the "flagellar switch" upstream of theflgBoperon. ArecVmutation prevents flagellar switch inversion and results in phenotypically locked strains. The orientation of the flagellar switch influences expression of theflgBoperon post-transcription initiation, but the specific molecular mechanism is unknown. Here, we report the isolation and characterization of spontaneous suppressor mutants in the non-motile, non-toxigenicrecV flgOFF background that regained motility and toxin production. The restored phenotypes corresponded with increased expression of flagellum and toxin genes. The motile suppressor mutants contained single-nucleotide polymorphisms (SNPs) inrho, which encodes the bacterial transcription terminator Rho factor. Analyses using transcriptional reporters indicate that Rho contributes to heterogeneity in flagellar gene expression by preferentially terminating transcription offlgOFF mRNA within the 5' leader sequence. Additionally, Rho is important for initial colonization of the intestine in a mouse model of infection, which may in part be due to the sporulation and growth defects observed in therhomutants. Together these data implicate Rho factor as a regulator of gene expression affecting phase variation of important virulence factors ofC.difficile.