Expression of the Na+/H+ antiporter gene (g1-nhaC) of alkaliphilic Bacillus sp. G1 in Escherichia coli.
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 NaCl, 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.
Liew
Chong Wai
CW
Department of Bioprocess Engineering, Faculty of Chemical and Natural Resources Engineering, Universiti Teknologi Malaysia, Skudai, Johor, Malaysia.
Illias
Rosli Md
RM
Mahadi
Nor Muhammad
NM
Najimudin
Nazalan
N
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003470:Culture Media / Q000737:chemistry; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000502:physiology*; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013728:Terminator Regions, Genetic
D001426:Bacterial Proteins; D003470:Culture Media; D004269:DNA, Bacterial; D017923:Sodium-Hydrogen Exchangers; D012965:Sodium Chloride