Expression of the Na+/H+ antiporter gene (g1-nhaC) of alkaliphilic bacillus sp. G1 in escherichia coli

Abstract

A Na+/H+ antiporter gene was isolated from alkaliphilic Bacillus sp. G1. The full-length sequence of the Na+/H+ antiporter gene was obtained using a genome walking method, and designated as g1-nhaC. An ORF preceded by a promoter-like sequence and a Shine—Dalgarno sequence, and followed by a terminator-like sequence was identified. The deduced amino acid sequence consists of 535 amino acids, and a calculated molecular mass of 57 776 Da. g1-nhaC was subsequently cloned into pET22b(+) and expressed in Escherichia coli BL21 (DE3). Recombinant E. coli harboring the g1-nhaC gene was able to grow in modified L medium at various concentrations of NaCl (0.2-2.0 M) at different pH values. The recombinant bacteria grew well in the medium with concentrations of NaCl as high as 1.75 M at pH 8.0-9.0. Minimal growth was observed at 2.0 M NaCI, pH 8.0-9.0. At pH 10, the recombinant bacteria grew well in a medium with a low concentration of NaCl (0.2 M). These results suggested that the g1-NhaC antiporter from Bacillus sp. G1 plays a role in Na+ extrusion at lower pH values and in pH homeostasis at pH 10 under Na+ -limiting conditions.