, 2008);

however, no Na+/H+ antiporters have been identified at the molecular level in the extremophiles colonizing the Dagong Ancient Brine Well. In recent years, metagenomic libraries have been widely used for mining novel genes HKI-272 mw or products of pharmacological importance directly from some environments without necessarily cultivating microorganisms first (Cardenas & Tiedje, 2008; Vakhlu et al., 2008). Many novel genes have also been identified with this approach (Cowan et al., 2005; Schmeisser et al., 2007). In this study, we constructed a metagenomic library by directly extracting DNA from the brine in the Dagong Ancient Brine Well. Screening of Na+/H+ antiporters was performed by function complementation of the antiporter-deficient Escherichia coli strain KNabc that lacks three major genes, nhaA, nhaB and chaA, coding Na+/H+ antiporters (Nozaki et al., 1996). After the identification of the Na+/H+ antiporter genes, the structure and function of the protein it encoded were analyzed. This is the first report of the identification of a novel Na+/H+ antiporter gene from a metagenome from a special man-made ancient hypersaline environment. The halophile genomic DNAs were prepared from the brine in the Dagong Ancient Brine Well using methods originally described by Moon with modifications (Moon et al., 2004). Briefly, 100-mL samples were GSK2126458 mouse centrifuged at 14 000 g and 4 °C for

10 min, and the slurry was resuspended with 5 mL phosphate-buffered saline (pH 7.5) centrifuged at 5 g for 2 min at room temperature. The dispersion was again centrifuged at 14 000 g and 4 °C for 2 min. The bacterial cell pellets obtained were directly used for extracting environmental DNA using the Ultra-Clean Soil DNA Kit (Mo Bio Laboratories, Solana Beach, CA). Total DNA was subsequently subjected to electrophoresis in 0.8% agarose gels and stored

at −20 °C. An overnight culture of E. coli KNabc was inoculated into 100 mL of a modified Luria–Bertani medium (LBK medium) consisting of 1.0% tryptone, 0.5% yeast extract Florfenicol and 87 mM KCl, and then grown at 37 °C under aerobic conditions to an OD600 nm of 0.4. Cells were harvested by centrifugation at 4000 g for 10 min at 4 °C and washed three times in 10 mL of ice-cold sterile 10% glycerol solution before electrocompetent preparation (Yang et al., 2006). The halophile genomic DNAs were partially digested with Sau3AI to produce 1.5–6 kbp fragments. These DNA fragments were separated by agarose electrophoresis and ligated into pUC18, which had been digested with BamHI and dephosphorylated with bacterial alkaline phosphatase, using T4 DNA ligase (Mayumi et al., 2008). The ligated recombinant plasmids (20–200 ng) were added to 50 μL of competent cells of E. coli KNabc suspension and mixed thoroughly. Electroporation was carried out at field strength of 16 kV cm−1 in combination with an electric resistance of 300 Ω at 25 mF in a 0.1-cm electroporation cuvette.