title: Metagenome cloning and functional analysis of Na⁺/H⁺ antiporter genes from Keke Salt Lake in China.
abstract: 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.
authors.affiliation: State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing, 100193, People's Republic of China.
authors.forename: Maio
authors.initials: M
authors.lastname: Gao
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: San-Feng
authors.initials: SF
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002681:China; D003001:Cloning, Molecular; D060106:Lakes / Q000032:analysis / Q000382:microbiology*; D054892:Metagenome; D008969:Molecular Sequence Data; D012965:Sodium Chloride / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D017923:Sodium-Hydrogen Exchangers; D012965:Sodium Chloride
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