RS1 Satellite Phage Promotes Diversity of Toxigenic Vibrio cholerae by Driving CTX Prophage Loss and Elimination of Lysogenic Immunity

In El Tor biotype strains of toxigenic Vibrio cholerae, the CTX phi prophage often resides adjacent to a chromosomally integrated satellite phage genome, RS1, which produces RS1 phi particles by using CTX prophage-encoded morphogenesis proteins. RS1 encodes RstC, an antirepressor against the CTX phi repressor RstR, which cooperates with the host-encoded LexA protein to maintain CTX phi lysogeny. We found that superinfection of toxigenic El Tor strains with RS1 phi, followed by inoculation of the transductants into the adult rabbit intestine, caused elimination of the resident CTX prophage-producing nontoxigenic derivatives at a high frequency. Further studies using recA deletion mutants and a cloned rstC gene showed that the excision event was recA dependent and that introduction of additional copies of the cloned rstC gene instead of infection with RS1 phi was sufficient to enhance CTX phi elimination. Our data suggest that once it is excised from the chromosome, the elimination of CTX prophage from host cells is driven by the inability to reestablish CTX phi lysogeny while RstC is overexpressed. However, with eventual loss of the additional copies of rstC, the nontoxigenic derivatives can act as precursors of new toxigenic strains by acquiring the CTX prophage either through reinfection with CTX phi or by chitin-induced transformation. These results provide new insights into the role of RS1 phi in V. cholerae evolution and the emergence of highly pathogenic clones, such as the variant strains associated with recent devastating epidemics of cholera in Asia, sub-Saharan Africa, and Haiti.