Abstract

Metagenomic DNA libraries prepared from the Dagong Ancient Brine Well were screened for the NhaD type Na⁺/H⁺ antiporter gene by functional complementation with the antiporter-deficient Escherichia coli KNabc strain. One NhaD type clone with Na⁺ and alkaline pH resistance phenotype was obtained and its inserted Na⁺/H⁺ antiporter gene was designated as m-nhaD. The deduced amino-acid sequence of M-NhaD protein consists of 330 residues with a calculated molecular weight of 34999 Da and a predicted isoelectric point of 7.02, which is homologous to NhaD from Klebsiella pneumoniae 342(93%) and Yersinia frederiksenii ATCC 33641 (87%) but with only 77 and 76% sequence coverage, respectively. M-NhaD had a hydropathy profile with 9 putative transmembrane domains and a long carboxyl terminal hydrophilic tail of 31 amino acid residues different from the other NhaD. The m-nhaD gene could successfully express in the E. coli KNabc and its production rendered the E. coli KNabc with both the resistance up to 1.0 M Na⁺and the ability to grow under pH 8.5 - 10 alkaline condition. The difference in structure and function suggested that the m-nhaD was a novel NhaD type Na⁺/H⁺ antiporter gene and it was an attractive candidate gene of salt tolerance genetic breeding.

Key words: Metagenomic library, NhaD antiporter gene, halophiles, Dagong Ancient Brine Well.