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The genes encoding cholera toxin (ctxA andctxB ) are encoded in the genome of CTXφ, a filamentous phage that infectsVibrio cholerae . To study the evolutionary history of CTXφ, we examined genome diversity in CTXφs derived from a variety of epidemic and nonepidemicVibrio sp. natural isolates. Among these were threeV. cholerae strains that contained CTX prophage sequences but not thectxA andctxB 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 missingctxAB . Sequence analysis of these three plasmids revealed that they lacked the upstream control region normally found 5′ ofctxA , as well as thectxAB promoter region and coding sequences. These findings are consistent with the hypothesis that a CTXφ precursor that lackedctxAB simultaneously acquired the toxin genes and their regulatory sequences. To assess the evolutionary relationships among additional CTXφs, two CTXφ-encoded genes,orfU andzot , were sequenced from 13V. cholerae and 4V. mimicus isolates. Comparative nucleotide sequence analyses revealed that the CTXφs derived from classical and El TorV. cholerae isolates comprise two distinct lineages within otherwise nearly identical chromosomal backgrounds (based onmdh sequences). These findings suggest that nontoxigenic precursors of the twoV. cholerae O1 biotypes independently acquired distinct CTXφs.

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