Roles of NhaA, NhaB, and NhaD Na+/H+ antiporters in survival of Vibrio cholerae in a saline environment.
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.
Herz
Katia
K
Department of Microbial and Molecular Ecology, Institute of Life Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel.
Vimont
Sophie
S
Padan
Etana
E
Berche
Patrick
P
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003470:Culture Media; D029968:Escherichia coli Proteins; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D017422:Sequence Analysis, DNA; D012965:Sodium Chloride / Q000494:pharmacology*; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014734:Vibrio cholerae / Q000254:growth & development* / Q000502:physiology
D001426:Bacterial Proteins; D003470:Culture Media; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; C102839:NhaA protein, E coli; C491047:NhaB protein, E coli; C111423:NhaD protein, Vibrio; D017923:Sodium-Hydrogen Exchangers; D012965:Sodium Chloride