In M-Nha, most Asp residues (14/19) were predicted to be in the hydrophobic region, while the alignment of M-Nha with Na+/H+ antiporters of another six microorganisms indicated that three aspartates, including Asp-138, Asp-167 and Asp-224, were conserved in M-Nha (Fig. 3). The protein encoded by m-nha gene showed a high similarity of 92%, 86% and 62% to NhaH from H. dabanensis D-8T, H. aidingensis AD-6T and B.

subtilis, respectively. Interestingly, M-Nha has a long carboxyl terminal hydrophilic tail (140 amino acid residues), similar to Nhap and NhaG type Na+/H+ antiporters, whereas NhaH does not. It was reported that both the ion specificity and activity of an Na+/H+ antiporter were partially determined by the structural properties of the C-terminal hydrophilic tail (Hamada et al., 2001; Waditee et al., 2001). NhaG from B. subtilis possesses a hydrophilic segment with >100 amino acid selleck residues at the carboxyl terminal region (Gouda et al., 2001), and such a long hydrophilic domain is not present in any other microbial Na+/H+ antiporter except SynNhaP (NhaS1) in Synechocystis sp. (Hamada et al., 2001) and ApnhaP in A. halophytica (Waditee et al., 2001). The activities of NhaG decreased

when 26 residues in the C-terminal of the protein were lost (Gouda et al., 2001), and 56 residues in the C-terminal region of SynNhaP were necessary for antiporter activity (Hamada et al., 2001). Hydropathy analysis BYL719 usually showed that the Na+/H+ antiporter had 10–12 hydrophobic and also probably membrane-spanning regions (Majernik et al., 2001; Yang et al., 2006). Our results also revealed that m-nha gene product fits well into this model. The NhaH PIK3C2G and NhaG had 12 TMS, but M-Nha had only 10 TMS, although they all had high similarity of amino acid sequence. Consequently, the mechanism of ion transport by M-Nha from the Dagong Ancient Brine Well should be different from that of NhaH, NhaG and SynNhaP. With the differences of amino acid sequence and the putative secondary structure of the protein encoded

by m-nha from those Na+/H+ antiporter genes reported previously, it can be proposed that m-nha is a novel Na+/H+ antiporter gene. This study was significant in not only helping us understand the necessity of the existence of Na+/H+ antiporter in the Dagong Ancient Brine Well to maintain the intracellular environment homeostasis for halophiles, but also enriches our knowledge about the different mechanisms of Na+/H+ antiporter in halophiles in such an extreme environment. We thank Dr Terry A. Krulwich (Department of Biochemistry, Mount Sinai School of Medicine of the City University, New York) and Prof. Susheng Yang (Department of Microbiology, College of Biological Sciences, China Agricultural University, Beijing, China) for donating the strain E. coli KNabc.