Roles of NhaA, NhaB, and NhaD Na+/H+ antiporters in survival of Vibrio cholerae in a saline environment

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.

FIG. 1.

(A) Genetic organization of the nhaB locus of V. cholerae O1. (B) Clustal W alignment of NhaB of E. coli, V. alginolyticus (V algino), V. parahaemolyticus (V parahae), and V. cholerae N18. Asterisks indicate amino acid identity, and dots indicate amino acid similarity. Helical structures spanning the membrane are indicated with open boxes and are numbered. They were deduced by comparison with the NhaB two-dimensional model of V. alginolyticus (9). The putative amiloride-binding site is indicated with a black circle.

FIG. 2.

Na⁺/H⁺ antiporter activity of Vc-NhaA. (A) EP432/pBR322-Vc-NhaA or EP432/pGM36 was grown in LBK (pH 7.5), and everted membrane vesicles were isolated. ΔpH was monitored in everted membrane vesicles (50 μg of protein) with acridine orange (0.5 μM) at pH 8.5 in a buffer containing 140 mM KCl, 5 mM MgCl₂, 50 mM BTP. At the onset of the experiment, Tris-d-lactate (2 mM) or ATP (2 mM) was added (arrow pointing down) and the fluorescence quenching (Q) was recorded. NaCl (10 mM, arrows pointing up) was then added, and the new steady state of fluorescence was obtained (dequenching) after each addition was monitored. (B) The pH-dependent Na⁺/H⁺ antiport activity of the Vc-NhaA antiporter (closed squares) compared to that of the Ec-NhaA antiporter (opened squares). Membrane vesicles were prepared and assayed as described in the legend to panel A, but the reaction mixtures were titrated to the identical pH with KOH. (C) The pH-dependent Li⁺/H⁺ antiport activity of the Vc-NhaA antiporter (closed squares) compared to that of the Ec-NhaA antiporter (open squares).

FIG. 3.

The role of V. cholerae antiporters in the stationary phase. N18 (open bars) or NABD mutant (closed bars) was grown for 16 h on LBK-BTP medium and was exposed for 3 h to the indicated pHs and various concentrations of NaCl or LiCl (0.2 to 0.8 M). Bacteria were then plated on LBK agar to determine CFU counts.