Identification, isolation and cloning for novel genes at a reasonable pace is the main driving force behind the development of unprecedented experimental approaches (Vakhlu et al., 2008). Furthermore, 99.9% of the microbial species represented in any biotope are not culturable at the moment (Streit & Schmitz, 2004; Tringe et al., 2005), which highlights the limitation of any gene discovery protocol dependent on culturing (Vakhlu et al., 2008). Thus, the diversity of enzymes with special fundamental

functions, such as Na+/H+ antiporters that usually require purification from pure culture of a specific organism before analysis, is only partially understood at present. find more Correspondingly, a large fraction of genes in the environment cannot be disclosed due to difficulties in enriching and isolating microorganisms in pure culture. Metagenomics, a culture-independent strategy, provides an access to valuable genetic resources of the microorganisms regardless of whether they can be cultured (Cowan et al., 2005; Guazzaroni et al., 2009). The various target genes have been screened by using a metagenomic library (Schmeisser

et al., 2007). In this study, we applied this methodology for the direct cloning of genes encoding Na+/H+ antiporters from the Dagong Ancient Brine Well by functional Venetoclax cost complementation of antiporter-negative mutant strain. Our results demonstrated that metagenomic DNA libraries Ponatinib are also suitable for direct cloning of functional genes encoding integral membrane proteins from a brine environment. About 10 families of Na+/H+ antiporter genes have been identified in microorganisms in the past, including a single gene of nhaA, nhaB, nhaC, nhaD, nhaG, nhaP, nhaH and chA, and multiple subunits of Mrp antiporter and MnhABCDEFG system (Hunte et al., 2005; Yang et al., 2006). In these genes, only nhaH comes from the halophilic bacteria H. dabanensis D-8T and H. aidingensis AD-6T. Although the gene m-nha cloned in the current study also comes from the halophiles,

to our knowledge it was the first Na+/H+ antiporter gene directly mined by metagenomic technology from the halophiles colonizing a high-salt environment. In single subunit Na+/H+ transporter, it is shown that the negatively charged amino acid residue Asp, localized in the membrane-spanning regions, plays an important role in the binding and transporting of cations such as H+ and Na+ in several antiporter proteins (Majernik et al., 2001). Asp-133, Asp-163 and Asp-164 were proposed to be involved in binding sodium ions in NhaA from E. coli (Inoue et al., 1995). Asp-137 of Nha from H. dabanensis D-8Tand H. aidingensis AD-6T (Yang et al., 2006; Zou et al., 2008), Asp-138 of SynnhaP from Synechocystis sp., and Asp-139 of ApnhaP from A. halophytica were also believed to be necessary for Na+/H+ antiporter activity (Hamada et al., 2001; Waditee et al.