The Hybrid Pre-CTXΦ-RS1 Prophage Genome and Its Regulatory Function in Environmental Vibrio cholerae O1 Strains

The cholera toxin genes of Vibrio cholerae are encoded by CTXΦ, a lysogenic bacteriophage. Infection with this phage plays a determinant role in toxigenicity conversion and the emergence of new clones of pathogenic V. cholerae. Multiple phage alleles, defined by sequence types of the repressor gene rstR, have been found, showing the divergence of phage genomes. Pre-CTXΦ, which is characterized by the absence of toxin genes, is predicted to be the precursor of CTXΦ. We have found a new pre-CTXΦ prophage genome (named pre-CTXZJΦ for its novel rstR allele) in nontoxigenic V. cholerae O1 isolates that were obtained during surveillance of the estuary water of the Zhujiang River. A novel hybrid genome of the helper phage RS1 was identified in an environmental strain carrying pre-CTXZJΦ in this study. The chromosomal integration and genomic arrangement of pre-CTXZJΦ and RS1 were determined. The RS2 of pre-CTXZJΦ was shown to have a function in replication, but it seemed to have lost its ability to integrate. The RstR of pre-CTXZJΦ exerted the highest repression of its own rstA promoter compared to other RstRs, suggesting rstR-specific phage superinfection immunity and potential coinfection with other pre-CTXΦ/CTXΦ alleles. The environmental strain carrying pre-CTXZJΦ could still be infected by CTXETΦ, the most common phage allele in the strains of the seventh cholera pandemic, suggesting that this nontoxigenic clone could potentially undergo toxigenicity conversion by CTXΦ infection and become a new toxigenic clone despite already containing the pre-CTXΦ prophage.