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Keywords:

  • Halobacillus dabanensis;
  • moderately halophilic bacterium;
  • Na+/H+ antiporter;
  • nhaH

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

A gene encoding a Na+/H+ antiporter was cloned from a chromosomal DNA of Halobacillus dabanensis strain D-8T by functional complementation. Its presence enabled the antiporter-deficient Escherichia coli strain KNabc to survive in the presence of 0.2 M NaCl or 5 mM LiCl. The gene was sequenced and designated as nhaH. The deduced amino-acid sequence of NhaH consists of 403 residues with a calculated molecular mass of 43 481 Da, which was 54% identical and 76% similar to the NhaG Na+/H+ antiporter of Bacillus subtilis. The hydropathy profile was characteristic of a membrane protein with 12 putative transmembrane domains. Everted membrane vesicles prepared from E. coli cells carrying nhaH exhibited Na+/H+ as well as Li+/H+ antiporter activity, which was pH-dependent with highest activities at pH 8.5–9.0 and at pH 8.5, respectively. Moreover, nhaH confers upon E. coli KNabc cells the ability to grow under alkaline conditions.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

The Na+/H+ antiporter is ubiquitous from bacteria to mammals and plays an important role in maintaining cytoplasmic Na+ homeostasis and pH levels. In bacteria, the Na+/H+ antiporter has several roles, such as extrusion of Na+ or Li+ in exchange for H+ to keep the cytoplasm iso-osmotic with the environment and to avoid intoxication of living cells (Padan et al., 1989; Nozaki et al., 1996), establishment of an electrochemical potential of Na+ across the cytoplasmic membrane (Tsuchiya et al., 1977), and regulation and maintenance of intracellular pH homeostasis under alkaline conditions (Padan & Schuldiner, 1994). About 10 families of Na+/H+ antiporter genes have been identified in microorganisms, including nhaA in Escherichia coli (Karpel et al., 1988), Salmonella enteritidis (Pinner et al., 1992a), Vibrio alginolyticus (Nakamura et al., 1994), Vibrio cholerae (Herz et al., 2003) and Vibrio parahaemolyticus (Kuroda et al., 1994), nhaB in E. coli (Pinner et al., 1992b) and V. alginolyticus (Nakamura et al., 1996), nhaC in Bacillus pseudofirmus (formerly B. firmus) (Ito et al., 1997), nhaD in V. cholerae (Herz et al., 2003), napA in Enterococcus hirae (Waser et al., 1992), nhaP in Pseudomonas aeruginosa (Utsugi et al., 1998), Synechocystis sp. (Hamada et al., 2001) and Aphanothece halophytica (Rungaroon et al., 2001) and nhaG in Bacillus subtilis (Gouda et al., 2001). Only a single gene is involved in each of the above Na+/H+ antiporters. Another kind of Na+/H+ antiporter contains multiple subunits, such as mrp from B. subtilis (Ito et al., 1999) and mnhABCDEFG from Staphylococcus aureus (Hiramatsu et al., 1998). Furthermore, homologs of these Na+/H+ antiporters continue to emerge from genome sequence projects.

Moderately halophilic bacteria constitute a heterogeneous physiological group of microorganisms, and they can grow at a wide range of extracellular Na+ concentrations with optimal salinity between 0.5 and 2.5 M (Ventosa et al., 1998). In recent years, new species of moderately halophilic bacteria have been reported, such as Halobacillus karajensis (Amoozegar et al., 2003) and Halobacillus locisalis (Yoon et al., 2004). Halobacillus dabanensis strain D-8T (CGMCC1.3704T) is a Gram-positive spore-forming moderately halophilic bacterium isolated from Daban salt lake in Xinjiang, China, which can grow in the presence of 0.5–25% (weight in volume) NaCl in complex media under an external pH range from 5 to 11 (Liu et al., 2005). It is therefore likely that H. dabanensis strain D-8T possesses high Na+/H+ antiporter activity. In fact, almost all halophilic microorganisms have potential Na+ ion transport mechanisms to expel Na+ ions from the interior of the cells based on Na+/H+ antiporters (Oren, 1999). Physiological studies provided evidence for the existence of Na+/H+ antiporter activity in several Gram-negative moderate halophiles: for example, Salinivibrio costicola (Udagawa et al., 1986). However, no Na+/H+ antiporter has been identified at the molecular level in moderately halophilic microorganisms so far. In this study, we reported cloning and characterization of a Na+/H+ antiporter gene from H. dabanensis strain D-8T.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Bacteria and growth

Halobacillus dabanensis D-8T was grown in Gibbson medium under aerobic conditions at 35°C (Liu et al., 2005). Cells of Escherichia coli strain KNabc, lacking three major Na+/H+ antiporters (NhaA, NhaB and ChaA), were grown in a modified Luria–Bertani medium (LBK medium) consisting of 1.0% tryptone, 0.5% yeast extract and 87 mM KCl, to which NaCl or LiCl was added at indicated concentrations where necessary. Ampicillin was added to a final concentration of 50 μg mL−1 for selection and growth of transformed cells. To test the effect of pH on cell growth, the KNabc and transformant cells were grown in minimal medium containing 100 mM Tris-HCl (at indicated pHs), 20 mM (NH4)2SO4, 50 mM KCl, 1 mM K2HPO4, 0.3 mM MgSO4, 0.01 mM CaCl2 and 40 mM glycerol. Cell growth was monitored turbidimetrically at 600 nm.

Preparation of electro-competent Escherichia coli KNabc cells and electroporation

An overnight culture of E. coli KNabc cells was inoculated into 100 mL of LBK medium and grown to an optimal density of 0.4 at 600 nm. 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 electro-competent preparation. Recombinant plasmids (20–200 ng) were added to 50 μL of cell suspension and mixed thoroughly. Electroporation was carried out at a field strength of 16 kV/cm in combination with an electric resistance of 300 Ω at 25 μF in a 0.1 cm electroporation cuvette.

Cloning of the Na+/H+ antiporter gene

Chromosomal DNA was prepared from cells of H. dabanensis strain D-8T and partially digested with Sau3A1. The DNA fragments with 4–10 kb 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. Competent cells of E. coli KNabc were transformed with the ligated recombinant plasmids and were spread on agar plates containing LBK medium, 0.2 M NaCl, 1.5% agar and 100 μg mL−1 of ampicillin. The plates were incubated at 37°C for 20 h and colonies picked for further studies.

Preparation of everted membrane vesicles

Escherichia coli KNabc cells carrying the hybrid plasmid were grown in LBK medium up to the mid-exponential phase and harvested by centrifugation at 5000 g, 4°C for 10 min. Everted membrane vesicles were prepared from transformant cells of E. coli KNabc/pUC18 (as a negative control) and KNabc/pNAD04 by the French press method at 2000 psi and collected by ultracentrifugation at 100 000g for 1 h as described by Rosen (1986). The vesicles were resuspended in a buffer containing 10 mM Tris-HCl (pH 7.0), 140 mM choline chloride, 0.5 mM dithiothreitol and 250 mM sucrose, and stored at −70°C before use.

Assay of Na+/H+ antiport activity

The Na+/H+ antiporter activity of everted membrane vesicles was estimated according to the extent of the collapse of a performed proton gradient, with acridine orange as the pH indicator, as described previously (Rosen, 1986). The assay mixture contained 10 mM Tris-HCl (at the indicated pH from 6 to 9) or 10 mM Ches-KOH (pH 9.5), 140 mM choline chloride, 10 mM MgCl2, 2 μM acridine orange and 20–40 μg mL−1 protein of membrane vesicles. Potassium lactate (5 mM) was added to initiate respiration. Fluorescence was monitored with a Hitachi F-4500 fluorescence spectrophotometer (Hitachi Ltd, Tokyo, Japan) at excitation and emission wavelength of 495 and 530 nm, respectively.

DNA manipulation and sequence analysis

Preparation of plasmid DNA, extraction of total DNA, restriction enzyme digestion and ligation were carried out as described by Sambrook et al. (1989). DNA sequencing was performed by China Bioasia Bio-Technology Sequencing Co. Ltd (Beijing, China). Databank searches were performed through the National Center for Biotechnology Information (NCBI) using the website http:/www.ncbi.nlm.nih.gov/blast. Alignment and hydropathy analysis were performed with the dnaman sequence analysis software.

Protein content determination

Protein content in everted membrane vesicles was determined by the method of Lowry et al. (1951) with bovine serum albumin as a standard.

Nucleotide sequence accession number

The nucleotide sequence reported in this paper has been submitted to GenBank with accession number DQ167194.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

Cloning of the Na+/H+ antiporter gene

Mutant cells of Escherichia coli KNabc lacking all three major Na+/H+ antiporters facilitated cloning of the Na+/H+ antiporter gene(s) from Halobacillus dabanensis D-8T. By functional complementation tests, 20 candidate recombinant plasmids that enabled the KNabc cells to grow in the presence of 0.2 M NaCl were obtained and were designated as pNAD01 to pNAD20. These 20 positive clones transformed with recombinant plasmids showed almost similar growth rates at 0.2 M NaCl, and relatively low growth rates at 5 mM LiCl (data not shown). The effects of NaCl concentration on the growth of transformant cells and of E. coli KNabc/pUC18, containing only empty vector (pUC18), were determined. All 20 transformant clones grew well in liquid medium containing 0.2 M NaCl, while cells of E. coli KNabc/pUC18 did not. These transformants could survive even in the presence of 0.25 M NaCl (Fig. 1). Twenty hybrid plasmids were sequenced, which revealed that they carried a common DNA fragment of a putative Na+/H+ antiporter gene.

image

Figure 1.  Growth of Escherichia coli strains in the presence of NaCl. Cells of E. coli KNabc/pUC18 (○) and KNabc/pNAD04 (•) were grown in Luria–Bertani medium in the presence of 0–0.25 M NaCl. Each data point represents the average of three independent determinations.

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Many species of Gram-positive and Gram-negative bacteria contain multiple Na+/H+ antiporter genes, such as nhaA, nhaB and chaA in E. coli (Karpel et al., 1988; Pinner et al., 1992a, b; Ivey et al., 1993), and the five Na+/H+ antiporter genes, nhaC (previously yheL), mleN (previously yqkL), nhaG, tetA(L) and mrp in Bacillus subtilis (Cheng et al., 1994; Ito et al., 1999; Gouda et al., 2001). As H. dabanensis strain D-8T can grow in the presence of NaCl concentrations up to 25% and grow at a pH up to 11, it is most likely that more than one Na+/H+ antiporter gene exists in the strain. However, only one Na+/H+ antiporter gene was obtained from H. dabanensis strain D-8T by several batches of screening. Perhaps other Na+/H+ antiporter gene(s) were not expressed, or were expressed at an insufficient level in E. coli KNabc cells, or the expressed antiporter(s) exhibited too low or even no activity. Plasmid pNAD04, carrying a moderate size of DNA insert, was selected for further analysis.

Sequence and characteristics of the Na+/H+ antiporter gene

As shown in Fig. 2, the length of the DNA insert of pNAD04 was 5362 bp, and contained five intact open reading frames (ORF1–5). One of the ORFs, ORF2, was predicted to encode a Na+/H+ antiporter by NCBI BLAST, and we designed this ORF nhaH (Na+/H+ antiporter of H. dabanensis). The deduced amino-acid sequence of nhaH showed that NhaH consisted of 403 amino-acid residue with a predicted molecular mass of 43 481 Da and a pI of 5.63. The most abundant amino-acid residues of this protein was Leu (66/403), followed by Val (40/403), Glu (38/403) and Ile (37/403). The least abundant residue was Cys (1/403). Among the 403 residues, 275 were hydrophobic, indicating that NhaH is of low polarity. This is consistent with the idea that the Na+/H+ antiporter is an integral membrane protein. Among the hydrophobic regions, 24 residues were charged, 18 of these were acidic and six were basic, resulting in an excess of 12 negatively charged residues in the putative 12 transmembrane regions. It has been shown that the negatively charged amino-acid residue Asp localized in the membrane-spanning regions plays an important role in the translocation of Na+ in several Na+/H+ antiporter proteins. Asp-138 of SynnhaP from Synechocystis sp. and Asp-139 of ApnhaP from Aphanothece halophytica were identified as necessary for Na+/H+ antiporter activity (Hamada et al., 2001; Rungaroon et al., 2001), and replacement of these residues inactivated the Na+/H+ antiporters. In NhaH, most Asp residues (11/14) are predicted to be in the hydrophobic regions, and alignment of NhaH with Na+/H+ antiporters of five microorganisms indicated that three aspartates, Asp-137, Asp-166 and Asp-224, were conserved in NhaH (Fig. 3).

image

Figure 2.  Genetic organization of the DNA insert of pNAD04. Possible promoter sequences (−35 region and −10 region), a putative Shine–Dalgarno sequence (SD), the putative initiation code ATG and termination code TGA of orf2 (nhaH, accession number DQ167194) are underlined. A possible terminator following the orf2 is indicated by inverted arrows, and * represents a stop codon.

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image

Figure 3.  Alignment of the predicted amino-acid sequence of the Na+/H+ antiporter NhaH from Halobacillus dabanensis D-8 with NhaG from Bacillus subtilis and NhaP from Aphanothece halophytica, Pseudomonas aeruginosa, Synechocystis sp. and Vibrio parahaemolyticus. The amino-acid residues conserved in all sequences are highlighted in black, and conservative substitutions are shown in gray. Three conserved Asp residues are indicated by the downward arrows. The predicted 12 transmembrane domains are marked with bold lines above the alignment.

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A homology search revealed that NhaH had the highest homology (54% identity and 76% similarity) with the NhaG Na+/H+ antiporter from B. subtilis, and a little lower similarity (34–36% identity) to the NhaP or putative Na+/H+ antiporters of Vibrio vulnificus, Pseudomonas aeruginosa, Synechocystis sp. PCC 6803, Anabaena variabilis, Archaeoglobus fulgidus DSM 4304 and A. halophytica (Fig. 3). NhaH also showed significant similarity to Na+/H+ antiporters from plants such as Arabidopsis thaliana (NHX7 or SOS1, 27% identity) and Oryza sativa (SOS1 or NHA1, 26% identity). In addition, ORF1 was predicted to encode a protein of 295 amino acids showing the highest homology (74% identity) to the ferrichrome ABC transporter (ABC-binding protein) of B. subtilis: ORF3 (729 bp), ORF4 (741 bp) and ORF5 (678 bp) were predicted to encode proteins showing high homology to a transcriptional regulator (46% identity) of Chromobacterium violaceum; NADPH-flavin oxidoreductase (54% identity) of Oceanobacillus iheyensis HTE831; and a protein with unknown function (21% identity) of O. iheyensis HTE831.

It was reported that the ion specificity and activity of a Na+/H+ antiporter are partly determined by the structural properties of the C-terminal hydrophilic tail (Hamada et al., 2001; Rungaroon et al., 2001). NhaG from B. subtilis possesses a hydrophilic segment with more than 100 amino-acid 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 for SynNhaP (NhaS1) of Synechocystis sp. (Hamada et al., 2001) and ApNhaP in A. halophytica (Rungaroon et al., 2001). The activities of NhaG decreased when the C-terminal 26 residues of the protein were missing (Gouda et al., 2001), and the 56 residues of the C-terminal region of SynNhaP were necessary for antiporter activity (Hamada et al., 2001). The hydropathy profile indicated that only nine hydrophilic amino-acid residues (395PLIKKLGMI403) are present at the C-terminal region of NhaH. Hence, the mechanism of ion transport for NhaH may be different from that of NhaG and SynNhaP.

Na+/H+ antiporter activity in everted membrane vesicles

Na+/H+ antiporter activity with everted membrane vesicles prepared from cells of E. coli strains KNabc/pUC18 and KNabc/pNAD04 was determined by measuring the dequenching of acridine orange fluorescence upon addition of NaCl and LiCl. As shown in Fig. 4, both Na+/H+ and Li+/H+ antiporter activities were detected in membrane vesicles from KNabc/pNAD04, while no Na+/H+ or Li+/H+ antiporter activities were detected in those from KNabc/pUC18.

image

Figure 4.  Na+/H+ antiporter and Li+/H+ antiporter activities in membrane vesicles. The activities measurements for Na+/H+ antiporter (a) and Li+/H+ antiporter (b) were performed at pH8.5 in everted membrane vesicles prepared from cells of Escherichia coli KNabc/pUC18 or KNabc/pNAD04 by the French press method. At the time points indicated by downward arrows, potassium lactate (final concentration 5 mM) was added to the assay mixture to initiate fluorescence quenching. At the time point indicated by upward arrows, NaCl (final concentration 5 mM) or LiCl (final concentration 5 mM) was added to the assay mixture. Fluorescence quenching is shown in arbitrary units.

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The effect of pH on the activity of Na+/H+ as well as Li+/H+ antiporter was also measured. NhaH exhibited Na+/H+ antiporter activity at a wide range of pH between 6.5 and 9.5, whereas no Li+/H+ antiporter activity was measured below pH 7.5 (Fig. 5). Optimal pH for the Na+/H+ and Li+/H+ antiport activity was 8.5–9.0 and 8.5, respectively, which was different from that of NhaG in B. subtilis (Gouda et al., 2001). Furthermore, Na+/H+ antiporter activity was higher than Li+/H+ antiporter activity, and the Km values for Na+ and Li+ were 0.83 and 2.14 mM, respectively. This suggested that Na+ was a better substrate than Li+ for the antiporter.

image

Figure 5.  pH profile of the Na+/H+ antiporter activity of NhaH. The antiporter activities were measured by the fluorescence quenching method. Na+/H+ antiporter activity (▴) and Li+/H+ antiporter activity (▪) were measured at the indicated pH. The wavelength of excitation light was 495 nm and fluorescence was monitored at 530 nm. Each value represents the average of three independent determinations.

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Resistance of KNabc/pNAD04 to pH

The wide range of pH of NhaH antiport activity of H. dabanensis D-8T suggests that cells of KNabc/pNAD04 may be able to grow in a wide pH range. To test the effect of pH on cell growth, E. coli KNabc/pUC18 and KNabc/pNAD04 were grown in a minimal medium in the presence of 0.2 M NaCl at different pH values from 7 to 9. As shown in Fig. 6, the growth of E. coli KNabc/pUC18 was greatly reduced under alkaline conditions, especially at pH above 8.0, compared with that below neutral pH, whereas the Na+/H+ antiporter gene nhaH of H. dabanensis D-8T confers upon E. coli KNabc cells the ability to grow under alkaline conditions.

image

Figure 6.  Effects of pH on cell growth of Escherichia coli KNabc/pUC18 (○) and KNabc/pNAD04 (•). Cells were grown in a minimal medium supplemented with 40 mM glycerol at the indicated pH. Each data point represents the average of three independent determinations.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References

We are grateful to Dr Terry A. Krulwich (Department of Biochemistry, Mount Sinai School of Medicine of the City University, New York) for the kind gift of Escherichia coli strain KNabc. This work was supported by the Chinese National Program for High Technology Research and Development (2003AA241150).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
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