Abstract
In this study, metagenomic DNA was screened for the Na+/H+ antiporter gene from the halophilic bacteria in Daban Salt Lake by selection in Escherichia coli KNabc lacking three major Na+/H+ antiporters. One gene designated as Hb_nhaD encoding a novel NhaD-type Na+/H+ antiporter was finally cloned. The presence of Hb_NhaD conferred tolerance of E. coli KNabc to up to 0.5 M NaCl and 0.2 M LiCl, and an alkaline pH. Hb_NhaD has the highest identity (70.6 %) with a putative NhaD-type Na+/H+ antiporter from an uncharacterized Clostridiaceae species, and also has lower identity with known NhaD-type Na+/H+ antiporters from Halomonas elongata (20.8 %), Alkalimonas amylolytica (19.0 %), Vibrio parahaemolyticus (18.9 %) and Vibrio cholerae (18.7 %). pH-dependent Na+(Li+)/H+ antiport activity was detected from everted membrane vesicles prepared from E. coli KNabc carrying Hb_nhaD. Hb_NhaD exhibited very high Na+(Li+)/H+ antiport activity over a wide pH range from 6.5 to 9.0 with the highest activity at pH 7.0 which is significantly different from those of the above known NhaD-type Na+/H+ antiporters. Also, the apparent K m values of Hb_NhaD for Na+ and Li+ at pH 7.0 were determined to be 1.31 and 2.16, respectively. Based on the above results, we proposed that Hb_NhaD should be categorized as a novel NhaD-type Na+/H+ antiporter.
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References
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410
Cheng J, Guffanti AA, Krulwich TA (1994) The chromosomal tetracycline resistance locus of Bacillus subtilis encodes a Na+/H+ antiporter that is physiologically important at elevated pH. J Biol Chem 269:27365–27371
Claros MG, von Heijne G (1994) TopPred II: an improved software for membrane protein structure predictions. Comput Appl Biosci 10:685–686
Edgar R, Bibi E (1997) MdfA, an Escherichia coli multidrug resistance protein with an extraordinarily broad spectrum of drug recognition. J Bacteriol 179:2274–2280
Gao M, Wang L, Chen S (2012) Metagenome Cloning and functional analysis of Na+/H+ antiporter genes from Keke Salt Lake in China. Curr Microbiol 64:179–184
Gerchman Y, Olami Y, Rimon A, Taglicht D, Schuldiner S, Padan E (1993) Histidine-226 is part of the pH sensor of NhaA, a Na+/H+ antiporter in Escherichia coli. Proc Natl Acad Sci USA 90:1212–1216
Gouda T, Kuroda M, Hiramatsu T, Nozaki K, Kuroda T, Mizushima T, Tsuchiya T (2001) nhaG Na+/H+ antiporter gene of Bacillus subtilis ATCC, which is missing in the complete genome sequence of strain, and properties of the antiporter. J Biochem 130:711–717
Herz K, Vimont S, Padan E, Berche P (2003) Roles of NhaA, NhaB, and NhaD Na+/H+ antiporters in survival of Vibrio cholerae in a saline environment. J Bacteriol 185:1236–1244
Hiramatsu T, Kodama K, Kuroda T, Mizushima T, Tsuchiya T (1998) A putative multisubunit Na+/H+ antiporter from Staphylococcus aureus. J Bacteriol 180:6642–6648
Ito M, Guffanti AA, Aemsky J, Mackivey D, Krulwich TA (1997) Role of the nhaC-encoded Na+/H+ antiporter of alkaliphilic Bacillus firmus OF4. J Bacteriol 63:3851–3857
Ito M, Guffanti AA, Oudega B, Krulwich TA (1999) mrp, a multigene, multifunctional locus in Bacillus subtilis with roles in resistance to cholate and to Na+ and in pH homeostasis. J Bacteriol 181:2394–2402
Ito M, Guffanti AA, Krulwich TA (2001) Mrp-dependent Na+/H+ antiporters of Bacillus exhibit characteristics that are unanticipated for completely secondary active transporters. FEBS Lett 496:117–120
Jiang J, Wei W, Du B, Li X, Wang L, Yang S (2004) Salt-tolerance genes involved in cation efflux and osmoregulation of Sinorhizobium fredii RT19 detected by isolation and characterization of Tn5 mutants. FEMS Microbiol Lett 239:139–146
Jiang J, Wang L, Zhang H, Wu H, Huang H, Yang L (2013) Putative paired small multidrug resistance family proteins PsmrAB, the homolog of YvdSR, actually function as a novel two-component Na+/H+ antiporter. FEMS Microbiol Lett 338:31–38
Karpel R, Olami Y, Taglicht D, Schuldiner S, Padan E (1988) Sequencing of the gene ant which affects the Na+/H+ antiporter activity in Escherichia coli. J Biol Chem 263:10408–10414
Kurz M, Brünig ANS, Galinski EA (2006) NhaD type sodium/proton-antiporter of Halomonas elongata: a salt stress response mechanism in marine habitats? Saline Syst 2:10
Lewinson O, Padan E, Bibi E (2004) Alkalitolerance: a biological function for a multidrug transporter in pH homeostasis. Proc Natl Acad Sci USA 101:14073–14078
Liu J, Xue Y, Wang Q et al (2005a) The activity profile of the NhaD-Type Na+(Li+)/H+ antiporter from the soda lake Haloalkaliphile Alkalimonas amylolytica is adaptive for the extreme environment. J Bacteriol 187:7589–7595
Liu W, Zeng J, Wang L, Dou Y, Yang S (2005b) Halobacillus dabanensis sp. nov. and Halobacillus aidingensis sp. nov., isolated from salt lakes in Xinjiang, China. Int J Syst Evol Microbiol 55:1991–1996
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Nakamura T, Enomoto H, Unemoto T (1996) Cloning and sequencing of the nhaB gene encoding an Na+/H+ antiporter from Vibrio alginolyticus. Biochim Biophys Acta 1275:157–160
Nozaki K, Kuroda T, Mizushima T, Tsuchiya T (1998) A new Na+/H+ antiporter, NhaD, of Vibrio parahaemolyticus. Biochim Biophys Acta 1369:213–220
Oren A (1999) Bioenergetic aspects of halophilism. Microbiol Mol Biol Rev 63:334–348
Padan E, Schuldiner S (1994) Molecular physiology of Na+/H+ antiporters, key transporters in circulation of Na+ and H+ in cells. Biochim Biophys Acta 1185:129–151
Padan E, Venturi M, Gerchman Y, Dover N (2001) Na+/H+ antiporters. Biochim Biophys Acta 1505:144–157
Padan E, Bibi E, Ito M, Krulwich TA (2005) Alkaline pH homeostasis in bacteria: new insights. Biochim Biophys Acta 177:67–88
Pinner E, Padan E, Schuldiner S (1992) Cloning, sequencing and expression of the nhaB gene, encoding a Na+/H+ antiporter in Escherichia coli. J Biol Chem 267:11064–11068
Quinn MJ, Resch CT, Sun J, Lind EJ, Dibrov P, Häse CC (2012) NhaP1 is a K+(Na+)/H+ antiporter required for growth and internal pH homeostasis of Vibrio cholerae at low extracellular pH. Microbiology 158:1094–1105
Rosen BP (1986) Ion extrusion system in Escherichia coli. Methods Enzymol 125:328–336
Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425
Sambrook J, Fritsch EF, Maniatis T (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor
Utsugi J, Inaba K, Kuroda T, Tsuda M, Tsuchiya T (1998) Cloning and sequencing of a novel Na+/H+ antiporter gene from Pseudomonas aeruginosa. Biochim Biophys Acta 1398:330–334
Ventosa A, Nieto JJ, Oren A (1998) Biology of moderately halophilic aerobic bacteria. Microbiol Mol Biol Rev 62:504–544
Waser M, Hess BD, Davies K, Solioz M (1992) Cloning and disruption of a putative Na+/H+ antiporter gene of Enterococcus hirae. J Biol Chem 267:5396–5400
Wu H, Wang Z, Yang L (2010) The selective enrichment and diversity analysis of halophilic bacteria in sedimental sample from Daban Salt Lake in Xinjiang. Microbiol China 37:956–961
Yang L, Jiang J, Wei W, Zhang B, Wang L, Yang S (2006a) The pha2 gene cluster involved in Na+ resistance and adaption to alkaline pH in Sinorhizobium fredii RT19 encodes a monovalent cation/proton antiporter. FEMS Microbiol Lett 262:172–177
Yang L, Jiang J, Zhang B, Zhao B, Wang L, Yang S (2006b) A primary sodium pump gene of the moderate halophile Halobacillus dabanensis exhibits secondary antiporter properties. Biochem Biophys Res Commun 346:612–617
Yang L, Jiang J, Zhao B et al (2006c) A Na+/H+ antiporter gene of the moderately halophilic bacterium Halobacillus dabanensis D-8T: cloning and molecular characterization. FEMS Microbiol Lett 255:89–95
Acknowledgments
We are so grateful to Dr Terry A. Krulwich (Department of Pharmacology and Systems Therapeutics, Mount Sinai School of Medicine of the City University, New York) for the kind gift of E. coli strain KNabc. We are also so thankful to Editor Dr. Sonja-Verena Albers and two anonymous reviewers for such valuable suggestions about the improvement of our manuscript. This work was supported by National Natural Science Foundation of China (Grant No. 30960009, 31000055 and 31370087), Fund for Fostering Talents in Basic Science of the National Natural Science Foundation of China (Grant No. J1210069), Natural Science Foundation of Heilongjiang Province of China (Grant No. LC201025), Key Project of Returned Overseas Chinese Scholars of Heilongjiang Province of China (Grant No. 1251HZ001), China Postdoctoral Science Foundation (Grant No. 20100471244 and 201104408), Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (Grant No. 2012940), Heilongjiang Postdoctoral Funds (Grant No. LBH-TZ1107 and LBH-Z10239), Science and Technology Innovation Fund of Harbin City of China (Grant No. 2012RFLXN004), Doctor Start-up Fund of Northeast Agricultural University (Grant No. 2009RC23), Key Laboratory Open Fund of Soybean Biology of Ministry of Education (Grant No. SB11A05) and Key Laboratory Open Fund of Agricultural Biological Functional Genes of Heilongjiang Province (Grant No. NSGJ2012-09).
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Communicated by S. Albers.
H. Zhang and Z. Wang contributed equally to this work.
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Zhang, H., Wang, Z., Wang, L. et al. Cloning and identification of a novel NhaD-type Na+/H+ antiporter from metagenomic DNA of the halophilic bacteria in soil samples around Daban Salt Lake. Extremophiles 18, 89–98 (2014). https://doi.org/10.1007/s00792-013-0600-2
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DOI: https://doi.org/10.1007/s00792-013-0600-2