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Journal of Bacteriology, October 2000, p. 5530-5538, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

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

E. Fidelma Boyd,dagger Andrew J. Heilpern, and Matthew K. Waldor*

Howard Hughes Medical Institute and Division of Geographic Medicine and Infectious Diseases, Tufts-New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111

Received 10 March 2000/Accepted 10 July 2000

The genes encoding cholera toxin (ctxA and ctxB) are encoded in the genome of CTXphi , a filamentous phage that infects Vibrio cholerae. To study the evolutionary history of CTXphi , we examined genome diversity in CTXphi s 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 CTXphi 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 CTXphi precursor that lacked ctxAB simultaneously acquired the toxin genes and their regulatory sequences. To assess the evolutionary relationships among additional CTXphi s, two CTXphi -encoded genes, orfU and zot, were sequenced from 13 V. cholerae and 4 V. mimicus isolates. Comparative nucleotide sequence analyses revealed that the CTXphi s 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 CTXphi s.

* Corresponding author. Mailing address: Division of Geographic Medicine and Infectious Diseases, New England Medical Center #041, 750 Washington St., Boston, MA 02111. Phone: (617) 636-7618. Fax: (617) 636-5292. E-mail: mwaldor{at}lifespan.org.

dagger Present address: Department of Microbiology, University College Cork, Cork, Ireland.

Journal of Bacteriology, October 2000, p. 5530-5538, Vol. 182, No. 19
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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