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

文献类型： 外文期刊

第一作者： Gao, Maio

作者： Gao, Maio;Wang, Lei;Chen, San-feng;Gao, Maio

作者机构：

期刊名称：CURRENT MICROBIOLOGY （ 影响因子：2.188； 五年影响因子：2.197 ）

ISSN： 0343-8651

年卷期： 2012 年 64 卷 2 期

页码：

收录情况： SCI

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.

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