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 different asd sequences which fell into separate lineages in the V. 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 mdh and hlyA sequences from the three pathogenic clones were identical, except for the previously reported 11-bp deletion in hlyA in the sixth-pandemic clone. Identical sequences were obtained, despite average nucleotide differences in the mdh and 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 recA and 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.

FIG. 1

Polymorphic sites within the mdh (A) and hlyA (B) genes of V. cholerae isolates. (A) Polymorphic sites within unique mdh sequences. M793 represents the sixth-pandemic, seventh-pandemic, and U.S. Gulf Coast clones, M535 also represents M553, M557, also represents M558, and M559 also represents M560. (B) Polymorphic sites within the 1,038-bp segment of the hlyA gene. M793 represents the seventh-pandemic and U.S. Gulf Coast clones, M642 represents the sixth-pandemic clone, and M555 also represents M559 and M560. Numbering of the polymorphic sites (vertical format) are from the first position of the sequence segment. The position within the codon for each polymorphic site is shown below the sequences. Asterisks indicate polymorphic sites which are informative.

FIG. 2

Phylogenetic tree for the mdh gene (A) and for the hlyA gene with (B) and without (C) the 60-bp region of recombination. The mdh tree was rooted with the partial mdh sequence of strain M547. The hlyA trees were rooted with the vmhA gene sequence of V. mimicus. Bootstrap values are percentages of 1,000 computer-generated trees and are shown at the nodes. Values of less than 50 are not shown.

FIG. 3

Polymorphic sites within the 1,041-bp fragment of recA (A) and the 1,067-bp fragment of dnaE (B) of selected V. cholerae isolates. (A) M793 represents the seventh-pandemic and U.S. Gulf Coast clones, and M642 represents the sixth-pandemic clone. (B) M793 represents the sixth- and seventh-pandemic clones, and M794 represents the U.S. Gulf Coast clone. See the legend to Fig. 1 for further details.

FIG. 4

Representation of the genetic identities of four housekeeping genes between the pathogenic clones of V. cholerae. Genes within the triangle are identical to the adjacent pathogenic clones. Genes outside the triangle are different between adjacent pathogenic clones; percent nucleotide differences are in parentheses.