Properties and Sequence of the NhaA Na⁺/H⁺ Antiporter of Vibrio parahaemolyticus

Abstract

A gene encoding an Na⁺/H⁺ antiporter was cloned from chromosomal DNA of the slightly halophilic marine bacterium Vibrio parahaemolyticus. The host was an Escherichia coli mutant that lacked both of the two major Na⁺/H⁺ antiporters, NhaA and NhaB. Untransformed mutant cells were unable to grow in the presence of 0.6 M NaCl or 0.1 M LiCl, but Na⁺ and Li⁺ were non-toxic to cells transformed with a plasmid carrying the antiporter gene. Membrane vesicles prepared from the original E. coli mutant did not show any detectable Na⁺/H⁺ (and Li⁺/H⁺) antiport activity. However, we observed high Na⁺/H⁺ (and Li⁺/H⁺) antiport activity in membrane vesicles prepared from the transformed cells. The activity increased greatly when the pH of the assay medium was increased from 7.0 and 8.5. This property is very similar to that of the NhaA Na⁺/H⁺ antiporter of E. coli. Drastic decreases in K_m values for Li⁺ and Na⁺ were observed with membrane vesicles prepared from the transformed cells compared with those observed with V. parahaemolyticus vesicles. The amino acid sequence deduced from the nucleotide sequence of the cloned gene showed high homology (59% identity and 87% similarity) with the NhaA Na⁺/H⁺ antiporter of E. coli. Thus, we conclude that the gene we cloned and sequenced is the nhaA of V. parahaemolyticus. We also found that several regions of the NhaA protein showed sequence similarity with transport proteins from some other organisms. Such regions seem to be important for Na⁺ recognition, transport or amiloride binding.