Roles of NhaA, NhaB, and NhaD Na + /H + Antiporters in Survival of Vibrio cholerae in a Saline Environment

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Abstract
Vibrio cholerae , the causative agent of cholera, is a normal inhabitant of aquatic environments, where it survives in a wide range of conditions of pH and salinity. In this work, we investigated the role of three Na + /H + antiporters on the survival of V. cholerae in a saline environment. We have previously cloned the Vc- nhaA gene encoding the V. cholerae homolog of Escherichia coli . Here we identified two additional antiporter genes, designated Vc- nhaB and Vc -nhaD , encoding two putative proteins of 530 and 477 residues, respectively, highly homologous to the respective antiporters of Vibrio species and E. coli. We showed that both Vc-NhaA and Vc-NhaB confer Na + resistance and that Vc-NhaA displays an antiport activity in E. coli , which is similar in magnitude, kinetic parameters, and pH regulation to that of E. coli NhaA. To determine the roles of the Na + /H + antiporters in V. cholerae , we constructed nhaA , nhaB , and nhaD mutants (single, double, and triple mutants). In contrast to E. coli , the inactivation of the three putative antiporter genes (Vc- nhaABD) in V. cholerae did not alter the bacterial exponential growth in the presence of high Na + concentrations and had only a slight effect in the stationary phase. In contrast, a pronounced and similar Li + -sensitive phenotype was found with all mutants lacking Vc- nhaA during the exponential phase of growth and also with the triple mutant in the stationary phase of growth. By using 2- n -nonyl-4-hydroxyquinoline N -oxide, a specific inhibitor of the electron-transport-linked Na + pump NADH-quinone oxidoreductase (NQR), we determined that in the absence of NQR activity, the Vc-NhaA Na + /H + antiporter activity becomes essential for the resistance of V. cholerae to Na + at alkaline pH. Since the ion pump NQR is Na + specific, we suggest that its activity masks the Na + /H + but not the Li + /H + antiporter activities. Our results indicate that the Na + resistance of the human pathogen V. cholerae requires a complex molecular system involving multiple antiporters and the NQR pump.