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Metagenomic cloning and characterization of Na+ transporters from Huamachi Salt Lake in China
Microbiological Research 168 (2013) 119–124
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Metagenomic cloning and characterization of Na+ transporters from Huamachi Salt Lake in China Miao Gao 1 , Li Tao 1 , Sanfeng Chen ∗ State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 7 April 2012 Received in revised form 25 May 2012 Accepted 5 June 2012
Keywords: Metagenomic library Na+ transporter NhaG Ca2+ antiporter Metalophosphoesterase Multidrug resistance protein
a b s t r a c t 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. © 2012 Elsevier GmbH. All rights reserved.
1. Introduction Moderately halophilic bacteria are a class of eubacteria that grow optimally in the environments with salt concentration between 0.5 M and 2.5 M (Kushner 1985; Monteoliva-Sanchez et al. 1993). These bacteria are widely distributed in salt lakes, saline land, coastal lagoons, deserts, ocean and so on (Rodriguez-Valera 1986; Simon et al. 1994; Grant 1996; Liu et al. 2005a,b). To adapt to these environments, most of them have developed a series of sodium transport systems, which may play vital roles in the Na+ cycle that supports pH homeostasis, salt resistance, solute uptake, and motility (Padan et al. 2001, 2004; Liu et al. 2005a,b). Today, more and more halophilic species and genera are proposed in the family Halobacteriaceae, while the mechanisms of their sodium transport system or functions of related genes remain to be well understood. In general, based on source of energy, sodium transport systems can be divided into two groups: primary sodium pump and secondary transporters. Primary sodium pump directly uses
∗ Corresponding author. Tel.: +86 10 62731551; fax: +86 10 62731551. E-mail addresses: [email protected], [email protected] (S. Chen). 1 These authors contributed equally to the work. 0944-5013/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.micres.2012.06.004
chemical energy, such as adenosine triphosphate (ATP), redox energy or photon energy (light), to transport molecules across a membrane (Yamakawa et al. 1989; Häse et al. 2001). A large number of enzymes have been proposed to perform this type of transport, e.g., decarboxylases, coenzyme M methyltransferases, ATP synthases, and NADH-ubiquinone oxidoreductases (Yamakawa et al. 1989; Kaim 2001; Studer et al. 2007). Secondary Na+ /H+ antiporters are a subset of intergral membrane proteins, which play an essential role in transport molecules or ions across the membrane. In contrast to primary sodium pump, secondary antiporters use its own electrochemical gradient as a source of energy. The family of secondary Na+ /H+ antiporters as an example, contains a large number of bacterial Na+ /H+ antiporter proteins (Horn 2000; Padan 2008). In recent years, studies are mainly focus on the identification and characterization of Escherichia coli Na+ /H+ antiporter proteins, including single-subunit antiporter proteins, e.g., NhaA (Padan et al. 2009), NhaB (Herz et al. 2003), NhaD (Kurz et al. 2006), NhaG (Gouda et al. 2001), NhaP (Waditee et al. 2006). Besides, similar research has been carried out in Bacillus subtilis. Several cation/H+ antiporters, such as Mrp (Ito et al. 1999), NhaK (Fujisawa et al. 2005), Tet (L) (Cheng et al. 1996), NhaG (Gouda et al. 2001) and NhaC (Pragai et al. 2001) have been proposed to function in sodium transport system of B. subtilis.
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In this study, we isolated and characterized four putative antiporters by screening a metagenomic library which was generated with genomic DNA extracted from the hypersaline soil samples of Huamachi Salt Lake located in Northwest of China. Sequence analysis demonstrated that these four putative antiporters are new members of sodium transport system that regulates sodium homeostasis in halophilic bacteria. In addition, functional complementation assay revealed that the Na+ (Ca2+ )sensitive phenotypes of E. coli KNabc (nhaA nhaB chaA) could be restored by introducing H7-Nha into the cells, which further confirmed the Na+ /H+ transport activity of H7-Nha protein. 2. Experimental procedures 2.1. Strains and growth conditions E. coli strain KNabc (nhaA nhaB chaA) (Nozaki et al. 1996), which is unable to grow in the medium with 0.2 M NaCl, was routinely cultivated in the LBK medium containing 10 g/L tryptone, 5 g/L yeast extract, and 6.5 g/L potassium chloride at pH 7.0. E. coli strain JM109 used as the host of metagenome clone was cultivated at 37 ◦ C in Luria-Bertani medium containing 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L sodium chloride at pH 7.0. If needed, antibiotics were added at an initial concentration of 100 mg/L of ampicillin or 50 mg/L of kanamycin in the medium. 2.2. Construction of metagenomic library from Huamachi Salt Lake in China Soil samples from five spots in Huamachi Salt Lake of China were obtained for metagenomic library construction. Each sample was inoculated into a 250 mL flask with 100 mL of 8% Gibbson medium (10 g tryptone, 5 g yeast extract, 5 g casein, 2 g KCl, 3 g sodium citrate, 20 g MgSO4 ·7H2 O, 80 g NaCl in each liter) and incubated at 30 ◦ C for 2–3 days. Then 1 mL of the first cultures was transferred to the same medium for one more time. After 16 h of incubation at 30 ◦ C with shaking, all the cells were harvested by centrifugation, and DNA preparation were carried out as described by Yoon et al. (1996) with a slight modification. The DNA mixture was partially digested with Sau3A I and fragments ranging from 4 kb to 6 kb were recovered from the gel and cloned into the BamH I site of pUC118 cloning vector. The ligation product was introduced into E. coli strain JM109, and transformants were selected on LB plates with 100 mg/L ampicillin (pH 7.0). The plasmid DNA extracted from these transformants were collected and introduced into E. coli strain KNabc to construct metagenomic library. 2.3. Screening and cloning of Na+ /H+ antiporters from the metagenomic library The metagenomic library was screened via complementation experiment by checking the growth characteristics of E. coli strain KNabc (nhaA nhaB chaA) in LBK medium with 0.2 M NaCl. Clones that can grow in the medium mentioned above were selected and the DNA fragments inserted in the plasmids were sequenced further. The complete putative Na+ /H+ antiporter genes including the promoter (300 bp upstream of the ATG) and terminator region (200 bp downstream of the stop codon) were subcloned into plasmid pUC118, yielding recombinant plasmids pUCH7-nha, pUCH16-mppe, pUCH19-cap and pUCH35-mrp.
determined in LBK medium supplemented with different Na+ concentration (0–0.6 M). Cell density was monitored at 600 nm. The results are representative of triplicates of individual cultures. 2.5. Preparation and assays of everted membrane vesicles E. coli strain KNabc carrying the blank vector pUC118 or Na+ /H+ antiporter genes were pregrown in LBK medium at pH 7.0, and everted membrane vesicles were prepared as described by Rosen (1986). The Na+ /H+ antiport activity was determinated by the quinacrine fluorescence quenching method (Goldberg et al. 1987). pH in everted membrane vesicles (80 g of protein) was monitored with acridine orange (1 M) in a buffer containing 10 mM Tris-MES (pH 8.0), 140 mM choline chloride and 5 mM MgCl2 at pH 8.0. At the onset of the experiment, potassium l-lactate (5 mM) was added and the fluorescence quenching (Q) was recorded by Hitachi F-4500 fluorescence spectrophotometer. NaCl, KCl or LiCl (10 mM) was then added, and the new steady state of fluorescence was obtained (dequenching) and each addition was monitored. 3. Results 3.1. Construction and analysis of metagenomic library from Huamachi Salt Lake As described in Section 2, a metagenomic library was constructed by using genomic DNA obtained from the hypersaline soil samples of Huamachi Lake and with pUC118 as a vector. Based on the ability of the library-containing Na+ (Ca2+ )/H+ antiporter defective mutant E. coli KNabc (nhaA nhaB chaA) when grown on medium containing 0.2 M NaCl, a total of 41 clones conferring salt transport activity were isolated from 52,100 clones. After subcloning and sequence analysis, 4 different genes, termed H7-Nha (H7 Na+ /H+ antiporter), H16-Mppe (H16 metallophosphoesterase), H19-Cap (H19 Ca2+ antiporter) and H35-Mrp (H35 multidrug resistance protein), were obtained. Sequence comparison data indicate that the H7-nha open reading frame (ORF) is composed of 1578 bp and is predicted to encode a 525 amino acid protein (57.9 kDa), the H16-mppe ORF comprises of 838 bp and encodes a 274 aa-length protein (30.9 kDa). It is also demonstrated that the H19-Cap ORF is 1101 bp and encodes a protein of 366 amino acids with the molecular weight 39 kDa, the H35-Mrp ORF comprises of 342 bp and encodes a 113 aa-length protein. Subsequent amino acid sequence alignment indicates that H7-Nha and H19-Cap show high identity to Na+ /H+ antiporter NhaG of Bacillus subtilis (71%) and Enterobacter cancerogenus Ca2+ antiporter (99%), respectively, suggesting that H7-Nha and H19-Cap are two new members of secondary antiporter family, in which H7-Nha is grouped to NhaGtype Na+ /H+ antiporters, H19-Cap belongs to divalent cation/H+ antiporters (Fig. 1a and c). In addition, H16-Mppe was found to share 59% identity to metallophosphoesterase of Bacillus cellulosilyticus and H35-Mrp show 68% identity to multidrug resistance protein of Lysinibacillus sphaericus, implying a strong probability that these two proteins possess the primary energization capacity (Fig. 1b and d). Moreover, hydrophobicity analysis showed that all the four putative transporters except H16-Mppe contain an excess of acidic amino acids over hydrophilic residues, implying that H7Nha, H19-Cap and H35-Mrp, but not H16-Mppe are membrane proteins (Fig. 1). 3.2. Growth of E. coli KNabc carrying H7-Nha, H16-Mppe, H19-Cap or H35-Mrp in different concentrations of Na+
2.4. Growth experiments The growth of E. coli strain KNabc carrying the blank vector pUC118 or Na+ /H+ antiporter genes containing plasmid was
In order to further test roles of the four putative transporters in salt tolerance, the E. coli KNabc strains carrying H7-Nha, H16-Mppe, H19-Cap or H35-Mrp, were inoculated into liquid media containing
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Fig. 1. Sequences alignment and hydrophobicity analysis of H7-Nha (A), H16-Mppe (B), H19-Cap (C) and H35-Mrp (D). Identical and conserved regions were highlighted in dark and grey, respectively. Bs-nhe2: Na+ /H+ antiporter Nhe2 of Bacillus subtilis subsp. spizizenii ATCC 6633; Pv-nhe2: Na+ /H+ antiporter Nhe2 of Paenibacillus vortex V453; Bc-mppe: metallophosphoesterase of Bacillus cellulosilyticus DSM 2522; Bp-ppe:phosphohydrolase of Bacillus pseudofirmus OF4; Ec-cap: calcium/proton antiporter of Enterobacter cloacae ATCC 13047; Kp-cap: proton antiporter of Klebsiellapneumoniae MGH 78578; Ls-mrp: multidrug resistance protein of Lysinibacillussphaericus C3-41; Bs-mrp: multidrug resistance protein Bacillus sp. B14905.
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KNabc/pUC18 KNabc/pUCH7 Knabc/pUCH16-mppe KNabc/pUCH19-cap KNabc/pUCH35-mrp
2.5
OD600
2 1.5
H7-Nha exhibited clear fluorescence dequenching in response to Na+ at pH 9.0, but not K+ or Li+ . In contract, no fluorescence dequenching was detected in the control strain carrying empty pUC118 vector no matter which ion was added as substrate.
4. Discussion
1 0.5 0 0
0.1
0.2
0.3 0.4 NaCl (M)
0.5
0.6
Fig. 2. Growth of KNabc/H7-Nha, KNabc/H16-Mppe, KNabc/H19-Cap and KNabc/H35-Mrp in the LBK medium with different concentration of NaCl at pH 7.0. KNabc is E. coli mutant (nhaAnhaBchaA). pUCH7-nha, pUCH16-mppe, pUCH19-cap and pUCH35-mrp plasmids were generated by inserting H7-Nha, H16-Mppe, H19-Cap and H35-Mrp genes into pUC118 vector, respectively. The KNabc strain harboring empty vector pUC118 was used as the control.
NaCl ranging from 0 M to 0.6 M, and the absorbance at 600 nm of each strain was measured after 24 h incubation at 37 ◦ C. As shown in Fig. 2, the control strain E. coli KNabc failed to grow at 0.2 M NaCl concentration, while the other strains showed varying degrees of salt-tolerant ability. The strain KNabc/H16-Mppe can tolerant and grow well in 0.2 M NaCl concentration, and the strains KNabc/H7Nha, KNabc/H19-Cap and KNabc/H35-Mrp exhibited salt-tolerant ability up to 0.6 M NaCl concentration, suggesting that the four proteins could confer varying degrees of salt-tolerant ability to the E. coli KNabc cells. Combining these results with sequence analysis data, we present an idea that the four proteins act as primary Na+ pumps or secondary Na+ /H+ antiporters in sodium transport system, when the halophilic cells are grown under salt stress condition. Since it is revealed that the strain KNabc/H7-Nha exhibited the strongest salt-tolerant ability compare to others, the property and function of protein H7-Nha was studied further. Subsequently, the secondary structure and hydrophilicity of H7Nha protein was predicted using DNAMAN software. The results show that the H7-nha gene encodes a Na+ /H+ antiporter NhaG homologue, which contains a 11 transmembrane domain (shown in Figs. 3 and 4, Table 1). 3.3. Antiport activity assays in membrane vesicles To further confirm the Na+ /H+ antiporter activity of H7-Nha, a fluorescence assay of E. coli membrane vesicles was carried out. As shown in Fig. 5 membrane vesicles from E. coli KNabc harboring
The identification of novel genes using conventional cultivation methods is severely limited, as less than 1% of environmental microorganisms can be cultured. Metagenomic library screen, a method allows to isolate certain genes directly from Metagenomic DNA, circumvents this limitation and enhances the probability of cloning particular genes of interest from extreme environments. In this study, we performed Metagenomic library screen, and identified four different types of sodium transporters that were important for salt tolerance of moderately halophilic bacteria. Our work has focused on the characterization of the protein H7-Nha, which is a secondary NhaG-type Na+ /H+ antiporter. As we know, a variety of secondary Na+ /H+ antiporters, e.g., NhaA, NhaD, Sha, Mnh, etc., have been found in various halophilic bacteria (Krulwich et al. 2009). According to the Transporter Classification Database (TCDB) (http://www.tcdb.org) operated by the Saier Lab Bioinformatics Group, monovalent cation/H+ antiporters found in bacteria are classified into different families and superfamilies. For example: bacterial NhaG, NhaP, and NhaK are grouped to the Monovalent Cation/Proton Antiporter-1 (CPA-1) Family; NhaA antiporter from E. coli belongs to CPA-2 family; Mrp, Sha, Pha, and Mnh, belong to CPA-3 family; and NhaB, NhaC and NhaD are grouped to IT superfamily (Prakash et al. 2003). Based on the TCDB classification, H7-Nha, a novel NhaG antiporter identified here, should be grouped to CPA-1 Family. Functional complementation assay reveals that three other transporters, H16-Mppe, H19-Cap, and H35-Mrp exhibit important functions in sodium exclusion. Moreover, our sequence alignment data present that H16-Mppe, H19-Cap, and H35-Mrp share high identity to metallophosphoesterase, Ca2+ antiporter, and multidrug resistance protein, respectively. Among these proteins, Ca2+ antiporter has already been verified to possess both calcium transport activity and sodium transport activity in bacteria, such as chaA gene from E. coli (Ivey et al. 1993), and similarly Dibrov (1993) demonstrated that NhaA, a Na+ /H+ antiporter from E. coli could also catalyze Ca2+ /H+ exchange. In addition, the Mrp protein was first identified in Bacillus subtilis as an important regulator that supports Na+ and alkali resistance via a Na+ /H+ antiport (Hamamoto et al. 1994; Ito et al. 2000). The complete mrp operon of B. subtilis is predicted to encode seven hydrophobic gene products (Ito et al. 1999; Kosono et al. 1999), among which the first gene, mrpA, has been proposed to be important to monovalent cation/H+ antiporter activity, but other genes of the operon are required for some combination of antiporter activity, expression, and assembly. Besides, based on the
Table 1 11 transmembrane helices of H7-Nha Na+ /H+ antiporter. No.
N terminal
Transmembrane region
C terminal
Type
Length
1 2 3 4 5 6 7 8 9 10 11
6 35 66 97 126 166 198 236 285 320 382
LHHIFQLGLILVMIAAGITAIA PIALVIVGAIIGLVNIPVLEPLK FNFVIITLFLPALLGEAALKLPF PILALAFGGTFLSFLIIGFSSLW PAAFVFAALMSATDPVSVLSIFK FNDGLAVVLFNISAFSLISYLDM WEFIKVISLGLLIGGLLGYGFSR IILFYGAFLLAESFEASGVIAVV WDVVTLLANSLVFLMVGLEITRI ILIVLIARSFAVYGSLLFLKNIP SVVLFSLVVQGLSIKPLITWLGV
27 57 88 119 148 188 220 258 307 342 404
PRIMARY PRIMARY SECONDARY PRIMARY PRIMARY SECONDARY PRIMARY PRIMARY PRIMARY PRIMARY SECONDARY
22 23 23 23 23 23 23 23 23 23 23
M. Gao et al. / Microbiological Research 168 (2013) 119–124
Fig. 3. The deduced amino acids sequence of H7-Nha.
Fig. 4. Structural model of H7-Nha Na+ /H+ antiporter. The structure models of Na+ /H+ antiporter were analyzed using SOSUI system.
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Fig. 5. Na+ /H+ , K+ /H+ and Li+ /H+ antiporter activities detected in membrane vesicles of E. coli KNabc/pUCH7-nha at pH 9.0. (A) Na+ /H+ antiporter activity in membrane vesicles of E. coli KNabc/pUC118 (negative control); (B) Na+ /H+ antiporter activity in membrane vesicles of E. coli KNabc/pUCH7-nha; (C) K+ /H+ antiporter activity in membrane vesicles of E. coli KNabc/pUCH7-nha; (D) Li+ /H+ antiporter activity in membrane vesicles of E. coli KNabc/pUCH7-nha.
high sequence similarity of the mrp genes to membrane-embedded subunits of energy-coupled NADH dehydrogenase complexes (Hamamoto et al. 1994; Ito et al. 1999), mrp has also been proposed to possess a capacity for primary energization (Ito et al. 2000). However, in the case of metallophosphoesterase, to date there is still no evidence for its roles in high salt resistance. Here we predict a probability for the first time that metallophosphoesterase may support Na+ resistance via Na+ or Ca2+ transport. While our studies have predicted the important functions of three putative transporters in salt resistance, much experiment need to be performed to reveal the detail mechanisms. The first task is to further confirm the sodium transport capacities of them by fluorescence assay of membrane vesicle. The second challenge is to investigate the molecular mechanisms of three transporters for salt tolerance. Acknowledgement This research is supported by National Transgenic Major Program no. 2009ZX08009-060B. References Cheng J, Guffanti AA, Wang W, Krulwich TA, Bechhofer DH. Chromosomal tetA(L) gene of Bacillus subtilis: regulation of expression and physiology of a tetA(L) deletion strain. J Bacteriol 1996;178:2853–60. Dibrov PA. Calcium transport mediated by NhaA, a Na+ /H+ antiporter from Escherichia coli. FEBS Lett 1993;336:530–4. Fujisawa M, Kusumoto A, Wada Y, Tsuchiya T, Ito M. NhaK, a novel monovalent cation/H+ antiporter of Bacillus subtilis. Arch Microbiol 2005;183:411–20. Goldberg EB, Arbel T, Chen J, Karpel R, Mackie GA, Schuldiner S, et al. Characterization of a Na+ /H+ antiporter gene of Escherichia coli. Proc Natl Acad Sci 1987;84:2615–9. Gouda T, Kuroda M, Hiramatsu T, Nozaki K, Kuroda T, Mizushima T, et al. nhaG Na+ /H+ antiporter gene of Bacillus subtilis ATCC9372, which is missing in the complete genome sequence of stain 168, and the properties of the antiporter. J Biochem 2001;130:711–7. Grant R. Prospering in dynamically-competitive environments: organizational capability as knowledge integration. Organ Sci 1996;7:375–87. Hamamoto T, Hashimoto M, Hino M, Kitada M, Seto Y, Kudo T, et al. Characterization of a gene responsible for the Na+ /H+ antiporter system of alkalophilic Bacillus species strain C-125. Mol Microbiol 1994;14:939–46. Häse CC, Fedorova ND, Galperin MY, Dibrov PA. Sodium cycle in bacterial pathogens. Evidence from cross-genome comparisons. Microbiol Mol Biol Rev 2001;65:353–70.
Herz K, Vimont S, Padan E, Berche P. Roles of NhaA, NhaB, and NhaD Na+ /H+ antiporters in survival of Vibrio cholerae in a saline environment. J Bacteriol 2003;185:1236–44. Horn J. The proton-pump inhibitors: similarities and differences. Clin Ther 2000;22:266–80. Ito M, Guffanti AA, Oudega B, Krulwich TA. mrp, a multigene, multifuctional locus in Bacillus subtilis with roles in resistance to cholate and to Na+ and in pH homeostasis. J Bacteriol 1999;181:2394–402. Ito M, Guffanti AA, Wang W, Krulwich TA. Effects of nonpolar mutations in each of the seven Bacillus subtilis mrp genes suggest complex interactions among the gene products in support of Na+ and alkali but not cholate resistance. J Bacteriol 2000;182:5663–70. Ivey DM, Guffanti AA, Zemsky J, Pinner E, Karpel R, Padan E, et al. Cloning and characterization of a putative Ca2+ /H+ antiporter gene from Escherichia coli upon functional complementation of Na+ /H+ antiporter-deficient strains by the overexpressed gene. J Biol Chem 1993;268:11296–303. Kaim G. The Na+ -translocating F1F0 ATP synthase of Propionigenium modestum: mechanochemical insights into the F0 motor that drives ATP synthesis. Biochim Biophys Acta: Bioenerg 2001;1505:94–107. Kosono S, Morotomi S, Kitada M, Kudo T. Analyses of a Bacillus subtilis homologue of the Na+ /H+ antiporter gene which is important for pH homeostasis of alkaliphilic Bacillus sp. Biochim Biophys Acta 1999;1409:171–5. Krulwich TA, Hicks DB, Ito M. Cation/proton antiporter complements of bacteria: why so large and diverse? Mol Microbiol 2009;74:257–60. Kurz M, Brunig, AN, Galinski EA. NhaD type sodium/proton-antiporter of Halomonas elongata: a salt stress response mechanism in marine habitats. Saline Systems no. 2; 2006. Kushner DJ. The halobacteriaceae. In: Woese CR, Wolfe RS, editors. The bacteria. London: Academic Press Ltd; 1985. p. 171–214. Liu J, Xue Y, Wang Q, Wei Y, Swartz TH, Hicks DB, et al. 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 2005a;187:7589–95. Liu W, Zeng J, Wang L, Dou Y, Yang S. Halobacillus dabanensis sp. nov. and Halobacillus aidingensis sp. nov., isolated from salt lakes in Xinjiang, China. Int J Syst Evol Microbiol 2005b;55:1991–6. Monteoliva-Sanchez M, Ramos-Cormenzana A, Russell NJ. The effect of salinity and compatible solutes on the biosynthesis of cyclopropane fatty acids in Pseudomonas halosaccharolytica. Microbiology 1993;139: 1877–84. Nozaki K, Inaba K, Kuroda T, Tsuda M, Tsuchiya T. Cloning and sequencing of the gene for Na+/H+ antiporter of Vibrio parahaemolyticus. Biochem Biophys Res Commun 1996;222:774–9. Padan E. The enlightening encounter between structure and function in the NhaA Na+ -H+ antiporter. Trends Biochem Sci 2008;33:435–43. Padan E, Kozachkov L, Herz K, Rimon A. NhaA crystal structure: functional–structural insights. J Exp Biol 2009;212:1593–693. Padan E, Tzubery T, Herz K, Kozachkov L, Rimon A, Galili L. NhaA of Escherichia coli, as a model of a pH-regulated Na+ /H+ antiporter. Biochem Biophys Acta: Bioenerg 2004;1658:2–13. Padan E, Venturi M, Gerchman Y, Dover N. Na+ /H+ antiporters. Biochem Biophys Acta: Bioenerg 2001;1505:144–57. Pragai Z, Eschevins C, Bron S, Harwood CR. Bacillus subtilis NhaC, an Na+ /H+ antiporter, influences expression of the phoPR operon and production of alkaline phosphatases. J Bacteriol 2001;183:2505–20. Prakash S, Cooper G, Singhi S, Saier MH. The ion transporter superfamily. Biochem Biophys Acta: Biomembr 2003;1618:79–92. Rodriguez-Valera F. The ecology and taxonomy of aerobic chemoorganotrophic halophilic eubacteria. FEMS Microbiol Lett 1986;39:17–22. Rosen BP. Recent advances in bacterial ion transport. Ann Rev Microbiol 1986;40:263–86. Simon R, Abeliovich A, Belkin S. A novel terrestrial halophilic environment: the phylloplane of Atriplex halimus, a salt-excreting plant. FEMS Microbiol Ecol 1994;14:99–109. Studer R, Dahinden P, Wang W, Auchli Y, Li X, Dimroth P. Crystal structure of the carboxyltransferase domain of the oxaloacetate decarboxylase Na+ pump from Vibrio cholerae. J Mol Biol 2007;367:547–57. Waditee R, Buaboocha T, Kato M, Hibino T, Suzuki S, Nakamura T, et al. Carboxylterminal hydrophilic tail of a NhaP type Na+ /H+ antiporter from cyanobacteria is involved in the apparent affinity for Na+ and pH sensitivity. Arch Biochem Biophys 2006;450:113–21. Yamakawa K, Oyamada H, Nakagomi O. Identification of rotaviruses by dot-blot hybridization using an alkaline phosphatase-conjugated synthetic oligonucleotide probe. Mol Cell Probe 1989;3:397–401. Yoon JH, Kim H, Kim SB, Kim HJ, Kim WY, Lee ST, et al. Identification of Saccharomonospora strains by the use of genomic DNA fragments and rRNA gene probes. Int J Syst Evol Microbiol 1996;46:502–5.
Further reading Ito M, Guffanti AA, Krulwich TA. Mrp-dependent Na+ /H+ antiporters of Bacillus exhibit characteristics that are unanticipated for completely secondary active transporters. FEBS Lett 2001;496:117–20. Kogure K, Tokuda H. Respiration-dependent primary Na+ pump in halophilic marine bacterium, Alcaligenes strain 201. FEBS Lett 1989;256:147–9.
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Metagenomic cloning and characterization of Na+ transporters from Huamachi Salt Lake in China Miao Gao 1 , Li Tao 1 , Sanfeng Chen ∗ State Key Laboratory of Agrobiotechnology, China Agricultural University, Beijing 100193, People’s Republic of China
a r t i c l e
i n f o
Article history: Received 7 April 2012 Received in revised form 25 May 2012 Accepted 5 June 2012
Keywords: Metagenomic library Na+ transporter NhaG Ca2+ antiporter Metalophosphoesterase Multidrug resistance protein
a b s t r a c t 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. © 2012 Elsevier GmbH. All rights reserved.
1. Introduction Moderately halophilic bacteria are a class of eubacteria that grow optimally in the environments with salt concentration between 0.5 M and 2.5 M (Kushner 1985; Monteoliva-Sanchez et al. 1993). These bacteria are widely distributed in salt lakes, saline land, coastal lagoons, deserts, ocean and so on (Rodriguez-Valera 1986; Simon et al. 1994; Grant 1996; Liu et al. 2005a,b). To adapt to these environments, most of them have developed a series of sodium transport systems, which may play vital roles in the Na+ cycle that supports pH homeostasis, salt resistance, solute uptake, and motility (Padan et al. 2001, 2004; Liu et al. 2005a,b). Today, more and more halophilic species and genera are proposed in the family Halobacteriaceae, while the mechanisms of their sodium transport system or functions of related genes remain to be well understood. In general, based on source of energy, sodium transport systems can be divided into two groups: primary sodium pump and secondary transporters. Primary sodium pump directly uses
∗ Corresponding author. Tel.: +86 10 62731551; fax: +86 10 62731551. E-mail addresses: [email protected], [email protected] (S. Chen). 1 These authors contributed equally to the work. 0944-5013/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.micres.2012.06.004
chemical energy, such as adenosine triphosphate (ATP), redox energy or photon energy (light), to transport molecules across a membrane (Yamakawa et al. 1989; Häse et al. 2001). A large number of enzymes have been proposed to perform this type of transport, e.g., decarboxylases, coenzyme M methyltransferases, ATP synthases, and NADH-ubiquinone oxidoreductases (Yamakawa et al. 1989; Kaim 2001; Studer et al. 2007). Secondary Na+ /H+ antiporters are a subset of intergral membrane proteins, which play an essential role in transport molecules or ions across the membrane. In contrast to primary sodium pump, secondary antiporters use its own electrochemical gradient as a source of energy. The family of secondary Na+ /H+ antiporters as an example, contains a large number of bacterial Na+ /H+ antiporter proteins (Horn 2000; Padan 2008). In recent years, studies are mainly focus on the identification and characterization of Escherichia coli Na+ /H+ antiporter proteins, including single-subunit antiporter proteins, e.g., NhaA (Padan et al. 2009), NhaB (Herz et al. 2003), NhaD (Kurz et al. 2006), NhaG (Gouda et al. 2001), NhaP (Waditee et al. 2006). Besides, similar research has been carried out in Bacillus subtilis. Several cation/H+ antiporters, such as Mrp (Ito et al. 1999), NhaK (Fujisawa et al. 2005), Tet (L) (Cheng et al. 1996), NhaG (Gouda et al. 2001) and NhaC (Pragai et al. 2001) have been proposed to function in sodium transport system of B. subtilis.
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In this study, we isolated and characterized four putative antiporters by screening a metagenomic library which was generated with genomic DNA extracted from the hypersaline soil samples of Huamachi Salt Lake located in Northwest of China. Sequence analysis demonstrated that these four putative antiporters are new members of sodium transport system that regulates sodium homeostasis in halophilic bacteria. In addition, functional complementation assay revealed that the Na+ (Ca2+ )sensitive phenotypes of E. coli KNabc (nhaA nhaB chaA) could be restored by introducing H7-Nha into the cells, which further confirmed the Na+ /H+ transport activity of H7-Nha protein. 2. Experimental procedures 2.1. Strains and growth conditions E. coli strain KNabc (nhaA nhaB chaA) (Nozaki et al. 1996), which is unable to grow in the medium with 0.2 M NaCl, was routinely cultivated in the LBK medium containing 10 g/L tryptone, 5 g/L yeast extract, and 6.5 g/L potassium chloride at pH 7.0. E. coli strain JM109 used as the host of metagenome clone was cultivated at 37 ◦ C in Luria-Bertani medium containing 10 g/L tryptone, 5 g/L yeast extract, and 10 g/L sodium chloride at pH 7.0. If needed, antibiotics were added at an initial concentration of 100 mg/L of ampicillin or 50 mg/L of kanamycin in the medium. 2.2. Construction of metagenomic library from Huamachi Salt Lake in China Soil samples from five spots in Huamachi Salt Lake of China were obtained for metagenomic library construction. Each sample was inoculated into a 250 mL flask with 100 mL of 8% Gibbson medium (10 g tryptone, 5 g yeast extract, 5 g casein, 2 g KCl, 3 g sodium citrate, 20 g MgSO4 ·7H2 O, 80 g NaCl in each liter) and incubated at 30 ◦ C for 2–3 days. Then 1 mL of the first cultures was transferred to the same medium for one more time. After 16 h of incubation at 30 ◦ C with shaking, all the cells were harvested by centrifugation, and DNA preparation were carried out as described by Yoon et al. (1996) with a slight modification. The DNA mixture was partially digested with Sau3A I and fragments ranging from 4 kb to 6 kb were recovered from the gel and cloned into the BamH I site of pUC118 cloning vector. The ligation product was introduced into E. coli strain JM109, and transformants were selected on LB plates with 100 mg/L ampicillin (pH 7.0). The plasmid DNA extracted from these transformants were collected and introduced into E. coli strain KNabc to construct metagenomic library. 2.3. Screening and cloning of Na+ /H+ antiporters from the metagenomic library The metagenomic library was screened via complementation experiment by checking the growth characteristics of E. coli strain KNabc (nhaA nhaB chaA) in LBK medium with 0.2 M NaCl. Clones that can grow in the medium mentioned above were selected and the DNA fragments inserted in the plasmids were sequenced further. The complete putative Na+ /H+ antiporter genes including the promoter (300 bp upstream of the ATG) and terminator region (200 bp downstream of the stop codon) were subcloned into plasmid pUC118, yielding recombinant plasmids pUCH7-nha, pUCH16-mppe, pUCH19-cap and pUCH35-mrp.
determined in LBK medium supplemented with different Na+ concentration (0–0.6 M). Cell density was monitored at 600 nm. The results are representative of triplicates of individual cultures. 2.5. Preparation and assays of everted membrane vesicles E. coli strain KNabc carrying the blank vector pUC118 or Na+ /H+ antiporter genes were pregrown in LBK medium at pH 7.0, and everted membrane vesicles were prepared as described by Rosen (1986). The Na+ /H+ antiport activity was determinated by the quinacrine fluorescence quenching method (Goldberg et al. 1987). pH in everted membrane vesicles (80 g of protein) was monitored with acridine orange (1 M) in a buffer containing 10 mM Tris-MES (pH 8.0), 140 mM choline chloride and 5 mM MgCl2 at pH 8.0. At the onset of the experiment, potassium l-lactate (5 mM) was added and the fluorescence quenching (Q) was recorded by Hitachi F-4500 fluorescence spectrophotometer. NaCl, KCl or LiCl (10 mM) was then added, and the new steady state of fluorescence was obtained (dequenching) and each addition was monitored. 3. Results 3.1. Construction and analysis of metagenomic library from Huamachi Salt Lake As described in Section 2, a metagenomic library was constructed by using genomic DNA obtained from the hypersaline soil samples of Huamachi Lake and with pUC118 as a vector. Based on the ability of the library-containing Na+ (Ca2+ )/H+ antiporter defective mutant E. coli KNabc (nhaA nhaB chaA) when grown on medium containing 0.2 M NaCl, a total of 41 clones conferring salt transport activity were isolated from 52,100 clones. After subcloning and sequence analysis, 4 different genes, termed H7-Nha (H7 Na+ /H+ antiporter), H16-Mppe (H16 metallophosphoesterase), H19-Cap (H19 Ca2+ antiporter) and H35-Mrp (H35 multidrug resistance protein), were obtained. Sequence comparison data indicate that the H7-nha open reading frame (ORF) is composed of 1578 bp and is predicted to encode a 525 amino acid protein (57.9 kDa), the H16-mppe ORF comprises of 838 bp and encodes a 274 aa-length protein (30.9 kDa). It is also demonstrated that the H19-Cap ORF is 1101 bp and encodes a protein of 366 amino acids with the molecular weight 39 kDa, the H35-Mrp ORF comprises of 342 bp and encodes a 113 aa-length protein. Subsequent amino acid sequence alignment indicates that H7-Nha and H19-Cap show high identity to Na+ /H+ antiporter NhaG of Bacillus subtilis (71%) and Enterobacter cancerogenus Ca2+ antiporter (99%), respectively, suggesting that H7-Nha and H19-Cap are two new members of secondary antiporter family, in which H7-Nha is grouped to NhaGtype Na+ /H+ antiporters, H19-Cap belongs to divalent cation/H+ antiporters (Fig. 1a and c). In addition, H16-Mppe was found to share 59% identity to metallophosphoesterase of Bacillus cellulosilyticus and H35-Mrp show 68% identity to multidrug resistance protein of Lysinibacillus sphaericus, implying a strong probability that these two proteins possess the primary energization capacity (Fig. 1b and d). Moreover, hydrophobicity analysis showed that all the four putative transporters except H16-Mppe contain an excess of acidic amino acids over hydrophilic residues, implying that H7Nha, H19-Cap and H35-Mrp, but not H16-Mppe are membrane proteins (Fig. 1). 3.2. Growth of E. coli KNabc carrying H7-Nha, H16-Mppe, H19-Cap or H35-Mrp in different concentrations of Na+
2.4. Growth experiments The growth of E. coli strain KNabc carrying the blank vector pUC118 or Na+ /H+ antiporter genes containing plasmid was
In order to further test roles of the four putative transporters in salt tolerance, the E. coli KNabc strains carrying H7-Nha, H16-Mppe, H19-Cap or H35-Mrp, were inoculated into liquid media containing
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Fig. 1. Sequences alignment and hydrophobicity analysis of H7-Nha (A), H16-Mppe (B), H19-Cap (C) and H35-Mrp (D). Identical and conserved regions were highlighted in dark and grey, respectively. Bs-nhe2: Na+ /H+ antiporter Nhe2 of Bacillus subtilis subsp. spizizenii ATCC 6633; Pv-nhe2: Na+ /H+ antiporter Nhe2 of Paenibacillus vortex V453; Bc-mppe: metallophosphoesterase of Bacillus cellulosilyticus DSM 2522; Bp-ppe:phosphohydrolase of Bacillus pseudofirmus OF4; Ec-cap: calcium/proton antiporter of Enterobacter cloacae ATCC 13047; Kp-cap: proton antiporter of Klebsiellapneumoniae MGH 78578; Ls-mrp: multidrug resistance protein of Lysinibacillussphaericus C3-41; Bs-mrp: multidrug resistance protein Bacillus sp. B14905.
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KNabc/pUC18 KNabc/pUCH7 Knabc/pUCH16-mppe KNabc/pUCH19-cap KNabc/pUCH35-mrp
2.5
OD600
2 1.5
H7-Nha exhibited clear fluorescence dequenching in response to Na+ at pH 9.0, but not K+ or Li+ . In contract, no fluorescence dequenching was detected in the control strain carrying empty pUC118 vector no matter which ion was added as substrate.
4. Discussion
1 0.5 0 0
0.1
0.2
0.3 0.4 NaCl (M)
0.5
0.6
Fig. 2. Growth of KNabc/H7-Nha, KNabc/H16-Mppe, KNabc/H19-Cap and KNabc/H35-Mrp in the LBK medium with different concentration of NaCl at pH 7.0. KNabc is E. coli mutant (nhaAnhaBchaA). pUCH7-nha, pUCH16-mppe, pUCH19-cap and pUCH35-mrp plasmids were generated by inserting H7-Nha, H16-Mppe, H19-Cap and H35-Mrp genes into pUC118 vector, respectively. The KNabc strain harboring empty vector pUC118 was used as the control.
NaCl ranging from 0 M to 0.6 M, and the absorbance at 600 nm of each strain was measured after 24 h incubation at 37 ◦ C. As shown in Fig. 2, the control strain E. coli KNabc failed to grow at 0.2 M NaCl concentration, while the other strains showed varying degrees of salt-tolerant ability. The strain KNabc/H16-Mppe can tolerant and grow well in 0.2 M NaCl concentration, and the strains KNabc/H7Nha, KNabc/H19-Cap and KNabc/H35-Mrp exhibited salt-tolerant ability up to 0.6 M NaCl concentration, suggesting that the four proteins could confer varying degrees of salt-tolerant ability to the E. coli KNabc cells. Combining these results with sequence analysis data, we present an idea that the four proteins act as primary Na+ pumps or secondary Na+ /H+ antiporters in sodium transport system, when the halophilic cells are grown under salt stress condition. Since it is revealed that the strain KNabc/H7-Nha exhibited the strongest salt-tolerant ability compare to others, the property and function of protein H7-Nha was studied further. Subsequently, the secondary structure and hydrophilicity of H7Nha protein was predicted using DNAMAN software. The results show that the H7-nha gene encodes a Na+ /H+ antiporter NhaG homologue, which contains a 11 transmembrane domain (shown in Figs. 3 and 4, Table 1). 3.3. Antiport activity assays in membrane vesicles To further confirm the Na+ /H+ antiporter activity of H7-Nha, a fluorescence assay of E. coli membrane vesicles was carried out. As shown in Fig. 5 membrane vesicles from E. coli KNabc harboring
The identification of novel genes using conventional cultivation methods is severely limited, as less than 1% of environmental microorganisms can be cultured. Metagenomic library screen, a method allows to isolate certain genes directly from Metagenomic DNA, circumvents this limitation and enhances the probability of cloning particular genes of interest from extreme environments. In this study, we performed Metagenomic library screen, and identified four different types of sodium transporters that were important for salt tolerance of moderately halophilic bacteria. Our work has focused on the characterization of the protein H7-Nha, which is a secondary NhaG-type Na+ /H+ antiporter. As we know, a variety of secondary Na+ /H+ antiporters, e.g., NhaA, NhaD, Sha, Mnh, etc., have been found in various halophilic bacteria (Krulwich et al. 2009). According to the Transporter Classification Database (TCDB) (http://www.tcdb.org) operated by the Saier Lab Bioinformatics Group, monovalent cation/H+ antiporters found in bacteria are classified into different families and superfamilies. For example: bacterial NhaG, NhaP, and NhaK are grouped to the Monovalent Cation/Proton Antiporter-1 (CPA-1) Family; NhaA antiporter from E. coli belongs to CPA-2 family; Mrp, Sha, Pha, and Mnh, belong to CPA-3 family; and NhaB, NhaC and NhaD are grouped to IT superfamily (Prakash et al. 2003). Based on the TCDB classification, H7-Nha, a novel NhaG antiporter identified here, should be grouped to CPA-1 Family. Functional complementation assay reveals that three other transporters, H16-Mppe, H19-Cap, and H35-Mrp exhibit important functions in sodium exclusion. Moreover, our sequence alignment data present that H16-Mppe, H19-Cap, and H35-Mrp share high identity to metallophosphoesterase, Ca2+ antiporter, and multidrug resistance protein, respectively. Among these proteins, Ca2+ antiporter has already been verified to possess both calcium transport activity and sodium transport activity in bacteria, such as chaA gene from E. coli (Ivey et al. 1993), and similarly Dibrov (1993) demonstrated that NhaA, a Na+ /H+ antiporter from E. coli could also catalyze Ca2+ /H+ exchange. In addition, the Mrp protein was first identified in Bacillus subtilis as an important regulator that supports Na+ and alkali resistance via a Na+ /H+ antiport (Hamamoto et al. 1994; Ito et al. 2000). The complete mrp operon of B. subtilis is predicted to encode seven hydrophobic gene products (Ito et al. 1999; Kosono et al. 1999), among which the first gene, mrpA, has been proposed to be important to monovalent cation/H+ antiporter activity, but other genes of the operon are required for some combination of antiporter activity, expression, and assembly. Besides, based on the
Table 1 11 transmembrane helices of H7-Nha Na+ /H+ antiporter. No.
N terminal
Transmembrane region
C terminal
Type
Length
1 2 3 4 5 6 7 8 9 10 11
6 35 66 97 126 166 198 236 285 320 382
LHHIFQLGLILVMIAAGITAIA PIALVIVGAIIGLVNIPVLEPLK FNFVIITLFLPALLGEAALKLPF PILALAFGGTFLSFLIIGFSSLW PAAFVFAALMSATDPVSVLSIFK FNDGLAVVLFNISAFSLISYLDM WEFIKVISLGLLIGGLLGYGFSR IILFYGAFLLAESFEASGVIAVV WDVVTLLANSLVFLMVGLEITRI ILIVLIARSFAVYGSLLFLKNIP SVVLFSLVVQGLSIKPLITWLGV
27 57 88 119 148 188 220 258 307 342 404
PRIMARY PRIMARY SECONDARY PRIMARY PRIMARY SECONDARY PRIMARY PRIMARY PRIMARY PRIMARY SECONDARY
22 23 23 23 23 23 23 23 23 23 23
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Fig. 3. The deduced amino acids sequence of H7-Nha.
Fig. 4. Structural model of H7-Nha Na+ /H+ antiporter. The structure models of Na+ /H+ antiporter were analyzed using SOSUI system.
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Fig. 5. Na+ /H+ , K+ /H+ and Li+ /H+ antiporter activities detected in membrane vesicles of E. coli KNabc/pUCH7-nha at pH 9.0. (A) Na+ /H+ antiporter activity in membrane vesicles of E. coli KNabc/pUC118 (negative control); (B) Na+ /H+ antiporter activity in membrane vesicles of E. coli KNabc/pUCH7-nha; (C) K+ /H+ antiporter activity in membrane vesicles of E. coli KNabc/pUCH7-nha; (D) Li+ /H+ antiporter activity in membrane vesicles of E. coli KNabc/pUCH7-nha.
high sequence similarity of the mrp genes to membrane-embedded subunits of energy-coupled NADH dehydrogenase complexes (Hamamoto et al. 1994; Ito et al. 1999), mrp has also been proposed to possess a capacity for primary energization (Ito et al. 2000). However, in the case of metallophosphoesterase, to date there is still no evidence for its roles in high salt resistance. Here we predict a probability for the first time that metallophosphoesterase may support Na+ resistance via Na+ or Ca2+ transport. While our studies have predicted the important functions of three putative transporters in salt resistance, much experiment need to be performed to reveal the detail mechanisms. The first task is to further confirm the sodium transport capacities of them by fluorescence assay of membrane vesicle. The second challenge is to investigate the molecular mechanisms of three transporters for salt tolerance. Acknowledgement This research is supported by National Transgenic Major Program no. 2009ZX08009-060B. References Cheng J, Guffanti AA, Wang W, Krulwich TA, Bechhofer DH. Chromosomal tetA(L) gene of Bacillus subtilis: regulation of expression and physiology of a tetA(L) deletion strain. J Bacteriol 1996;178:2853–60. Dibrov PA. Calcium transport mediated by NhaA, a Na+ /H+ antiporter from Escherichia coli. FEBS Lett 1993;336:530–4. Fujisawa M, Kusumoto A, Wada Y, Tsuchiya T, Ito M. NhaK, a novel monovalent cation/H+ antiporter of Bacillus subtilis. Arch Microbiol 2005;183:411–20. Goldberg EB, Arbel T, Chen J, Karpel R, Mackie GA, Schuldiner S, et al. Characterization of a Na+ /H+ antiporter gene of Escherichia coli. Proc Natl Acad Sci 1987;84:2615–9. Gouda T, Kuroda M, Hiramatsu T, Nozaki K, Kuroda T, Mizushima T, et al. nhaG Na+ /H+ antiporter gene of Bacillus subtilis ATCC9372, which is missing in the complete genome sequence of stain 168, and the properties of the antiporter. J Biochem 2001;130:711–7. Grant R. Prospering in dynamically-competitive environments: organizational capability as knowledge integration. Organ Sci 1996;7:375–87. Hamamoto T, Hashimoto M, Hino M, Kitada M, Seto Y, Kudo T, et al. Characterization of a gene responsible for the Na+ /H+ antiporter system of alkalophilic Bacillus species strain C-125. Mol Microbiol 1994;14:939–46. Häse CC, Fedorova ND, Galperin MY, Dibrov PA. Sodium cycle in bacterial pathogens. Evidence from cross-genome comparisons. Microbiol Mol Biol Rev 2001;65:353–70.
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Further reading Ito M, Guffanti AA, Krulwich TA. Mrp-dependent Na+ /H+ antiporters of Bacillus exhibit characteristics that are unanticipated for completely secondary active transporters. FEBS Lett 2001;496:117–20. Kogure K, Tokuda H. Respiration-dependent primary Na+ pump in halophilic marine bacterium, Alcaligenes strain 201. FEBS Lett 1989;256:147–9.