Molecular Analyses of a Putative CTXφ Precursor and Evidence for Independent Acquisition of Distinct CTXφs by Toxigenic Vibrio cholerae

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Abstract
The genes encoding cholera toxin ( ctxA and ctxB ) are encoded in the genome of CTXφ, a filamentous phage that infects Vibrio cholerae . To study the evolutionary history of CTXφ, we examined genome diversity in CTXφs derived from a variety of epidemic and nonepidemic Vibrio sp. natural isolates. Among these were three V. cholerae strains that contained CTX prophage sequences but not the ctxA and ctxB genes. These prophages each gave rise to a plasmid form whose genomic organization was very similar to that of the CTXφ replicative form, with the exception of missing ctxAB . Sequence analysis of these three plasmids revealed that they lacked the upstream control region normally found 5′ of ctxA , as well as the ctxAB promoter region and coding sequences. These findings are consistent with the hypothesis that a CTXφ precursor that lacked ctxAB simultaneously acquired the toxin genes and their regulatory sequences. To assess the evolutionary relationships among additional CTXφs, two CTXφ-encoded genes, orfU and zot , were sequenced from 13 V. cholerae and 4 V. mimicus isolates. Comparative nucleotide sequence analyses revealed that the CTXφs derived from classical and El Tor V. cholerae isolates comprise two distinct lineages within otherwise nearly identical chromosomal backgrounds (based on mdh sequences). These findings suggest that nontoxigenic precursors of the two V. cholerae O1 biotypes independently acquired distinct CTXφs.