Physiological roles of three Na⁺/H⁺ antiporters in the halophilic bacterium Vibrio parahaemolyticus

Vibrio parahaemolyticus mutants lacking three Na⁺/H⁺ antiporters (NhaA, NhaB, NhaD) were constructed. The ΔnhaA strains showed significantly higher sensitivity to LiCl regarding their growth compared to the parental strain. The ΔnhaA and ΔnhaB strains exhibited higher sensitivities to LiCl. The mutant XACabd lacking all of the three antiporters could not grow in the presence of 500 mM LiCl at pH 7.0, or 50 mM at pH 8.5. The XACabd mutant was also sensitive to 1.0 M NaCl at pH 8.5. These results suggest that Na⁺/H⁺ antiporters, especially NhaA, are responsible for resistance to LiCl and to high concentrations of NaCl. Reduced Na ⁺/H⁺ and Li⁺/H⁺ antiport activities were observed with everted membrane vesicles of ΔnhaB strains. However, Li⁺/H⁺ antiport activities of ΔnhaB strains were two times higher than those of ΔnhaA strains when cells were cultured at pH 8.5. It seems that expression of nhaA and nhaB is dependent on medium pH to some extent. In addition, HQNO (2-heptyl-4-hydroxyquinoline N-oxide), which is a potent inhibitor of the respiratory Na⁺ pump, inhibited growth of XACabd, but not of the wild type strain. Moreover, survival rate of XACabd under hypoosmotic stress was lower than that of wild type strain. It is likely that the Na⁺/H⁺ antiporters are involved in osmoregulation under hypoosmotic stress. Based on these findings, we propose that the Na ⁺/H⁺ antiporters cooperate with the respiratory Na ⁺ pump in ionic homeostasis in V. parahaemolyticus.