A novel pre-CTX prophage in the Vibrio cholerae serogroup O139 strain

In Vibrio cholerae, the lysogenic bacteriophage CTXΦ carries the cholera toxin genes ctxAB, which can be transferred from toxigenic strains to nontoxigenic strains through infection and lysogenic conversion of CTXΦ. This phage also has the precursor genome which does not harbor ctxAB, named pre-CTXΦ. Based on the sequences of the transcriptional regulator-encoding gene rstR alleles in CTXΦ/pre-CTXΦ, multiple types of these prophages have been classified and identified in toxigenic and nontoxigenic V. cholerae strains. In this study, by combining the short-read and long-read sequencing approaches of next generation sequencing, we obtained the complete genome sequence of the studied V. cholerae toxigenic serogroup O139 strain and identified the CTXΦ and a pre-CTXΦ genome type encoding a novel rstR allele, pre-CTX^ZHJΦ. This pre-CTX prophage integrates into the small chromosome of the V. cholerae host strain and coexists with a typical CTX^ETΦ prophage present in the large chromosome, which is commonly present in the seventh pandemic serogroup O1 and toxigenic serogroup O139 strains. RstR^ZHJ could bind to the ig-2 region in the RstAB promotor in the pre-CTX^ZHJΦ genome, and could repress the expression of its own rstAB genes but could not repress rstAB expression in CTX^ETΦ and CTX^classΦ, suggesting that the V. cholerae strains carrying the pre-CTX^ZHJΦ prophage cannot prevent the infection of these epidemic CTXΦs, hence have the potentiality to become toxigenic by acquiring and lysogenic conversion of CTXΦs. Our study identified a novel pre-CTXΦ type, and presents the new evidence for the complexity and diversity of the CTXΦ/pre-CTXΦ family in V. cholerae.