Characterization of VPI pathogenicity island and CTX phi prophage in environmental strains of Vibrio cholerae

Environmental isolates of Vibrio cholerae of eight randomly amplified polym orphic DNA (RAPD) fingerprint types from Calcutta, India, that were unusual in containing toxin-coregulated pilus or cholera toxin genes but not O1 or O139 antigens of epidemic strains were studied by PCR and sequencing to ga in insights into V. cholerae evolution. We found that each isolate containe d a variant form of the VPI pathogenicity island. Distinguishing features i ncluded (i) four new alleles of tcpF (which encodes secreted virulence prot ein; its exact function is unknown), 20 to 70% divergent (at the protein le vel) from each other and canonical tcpF; (ii) a new allele of toxT (virulen ce regulatory gene), 36% divergent (at the protein level) in its 5' half an d nearly identical in its 3' half to canonical toxT, (iii) a new tcpA (pili n) gene; and (iv) four variant forms of a regulatory sequence upstream of W T. Also found were transpositions of an IS903-related element and function- un known genes to sites in VPI. Cholera toxin (ctx) genes were found in iso lates of two RAPD types, in each case embedded in CTX phi -like prophages. Fragments that are inferred to contain only putative repressor, replication , and integration genes were present in two other RAPD types. New possible prophage repressor and replication genes were also identified. Our results show marked genetic diversity in the virulence-associated gene clusters fou nd in some nonepidemic V. cholerae strains, suggest that some of these gene s contribute to fitness in nature, and emphasize the potential importance o f interstrain gene exchange in the evolution of this species.