Metagenome Cloning and Functional Analysis of Na+/H+ Antiporter Genes from Keke Salt Lake in China

摘要

Na+/H+ antiporters are ubiquitous membrane proteins and play a central role in cell homeostasis including pH regulation, osmoregulation, and Na+/Li+ tolerance in bacteria. The microbial communities in extremely hypersaline soil are an important resource for isolating Na+/H+ antiporter genes. A metagenomic library containing 35,700 clones was constructed by using genomic DNA obtained from the hypersaline soil samples of Keke Salt Lake in Northwest of China. Two Na+/H+ antiporters, K1-NhaD, and K2-NhaD belonging to NhaD family, were screened and cloned from this metagenome by complementing the triple mutant Escherichia coli strain KNabc (nhaA − , nhaB − , chaA −) in medium containing 0.2 M NaCl. K1-NhaD and K2-NhaD have 75.5% identity at the predicted amino acid sequence. K1-NhaD has 78% identity with Na+/H+ antiporter NhaD from Halomonas elongate at the predicted amino acid sequence. The predicted K1-NhaD is a 53.5 kDa protein (487 amino acids) with 13 transmembrane helices. K2-NhaD has 73% identity with Alkalimonas amylolytica NhaD. The predicted K2-NhaD is a 55 kDa protein (495 amino acids) with 12 transmembrane helices. Both K1-NhaD and K2-NhaD could make the triple mutant E. coli KNabc (nhaA − , nhaB − , chaA−) grow in the LBK medium containing 0.2–0.6 M Na+ or with 0.05–0.4 M Li+. Everted membrane vesicles prepared from E. coli KNabc cells carrying K1-NhaD or K2-NhaD exhibited Na+/H+ and Li+/H+ antiporter activities which were pH-dependent with the highest activity at pH 9.5. Little K+/H+ antiporter activity was also detected in vesicles form E. coli KNabc carrying K1-NhaD or K2-NhaD.