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 0.5 M to 2.5 M. 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+(Ca2+)/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 Ca2+ 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 Ca2+ 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.