Evolutionary Relationships of Pathogenic Clones of Vibrio cholerae by Sequence Analysis of Four Housekeeping Genes

ABSTRACT Studies of the Vibrio cholerae population, using molecular typing techniques, have shown the existence of several pathogenic clones, mainly sixth-pandemic, seventh-pandemic, and U.S. Gulf Coast clones. However, the relationship of the pathogenic clones to environmental V. cholerae isolates remains unclear. A previous study to determine the phylogeny of V. cholerae by sequencing the asd (aspartate semialdehyde dehydrogenase) gene of V. cholerae showed that the sixth-pandemic, seventh-pandemic, and U.S. Gulf Coast clones had very differentasd sequences which fell into separate lineages in theV. cholerae population. As gene trees drawn from a single gene may not reflect the true topology of the population, we sequenced the mdh (malate dehydrogenase) and hlyA(hemolysin A) genes from representatives of environmental and clinical isolates of V. cholerae and found that the mdhand hlyA sequences from the three pathogenic clones were identical, except for the previously reported 11-bp deletion inhlyA in the sixth-pandemic clone. Identical sequences were obtained, despite average nucleotide differences in the mdhand hlyA genes of 1.52 and 3.25%, respectively, among all the isolates, suggesting that the three pathogenic clones are closely related. To extend these observations, segments of the recAand dnaE genes were sequenced from a selection of the pathogenic isolates, where the sequences were either identical or substantially different between the clones. The results show that the three pathogenic clones are very closely related and that there has been a high level of recombination in their evolution.

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