Metagenomic cloning and characterization of Na⁺ transporters from Huamachi Salt Lake in China.

Moderately halophilic bacteria are a kind of extreme environment microorganism that can tolerate moderate salt concentrations ranging from 0.5M to 2.5M. Here, via a metagenomic library screen, we identified four putative Na(+) transporters, designated H7-Nha, H16-Mppe, H19-Cap and H35-Mrp, from moderately halophilic community in the hypersaline soil of Huamachi Salt Lake, China. Functional complementation observed in a Na(+)(Ca(2+))/H(+) antiporter-defective Escherichia coli mutant (KNabc) suggests that the four putative Na(+) transporters could confer cells a capacity of Na(+) resistance probably by enhancing Na(+) or Ca(2+) efflux, but not Li(+) or K(+) exchange. Blastp analysis of the deduced amino-acid sequences indicates that H7-Nha has 71% identity to the NhaG Na(+)/H(+) antiporter of Bacillus subtilis, while H19-Cap shows 99% identity to Enterobacter cloacae Ca(2+) antiporter. Interestingly, H16-Mppe shares 59% identity to the metallophosphoesterase of Bacillus cellulosilyticus and H35-Mrp shows 68% identity to multidrug resistance protein of Lysinibacillus sphaericus. This is the first report that predicts a potential role of metallophosphoesterase in Na(+) resistance in halophilic bacteria. Furthermore, everted membrane vesicles prepared from E. coli cells harboring H7-Nha exhibit Na(+)/H(+) antiporter activity, but not Li(+) (K(+))/H(+) antiporter activity, confirming that H7-Nha supports Na(+) resistance mainly via Na(+)/H(+) antiport. Our report also demonstrates that metagenomic library screen is a convenient and effective way to explore more novel types of Na(+) transporters.