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Applied and Environmental Microbiology, August 2005, p. 4176-4184, Vol. 71, No. 8
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.8.4176-4184.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Halotolerant Cyanobacterium Aphanothece halophytica Contains NapA-Type Na⁺/H⁺ Antiporters with Novel Ion Specificity That Are Involved in Salt Tolerance at Alkaline pH

Nuchanat Wutipraditkul,¹ Rungaroon Waditee,² Aran Incharoensakdi,³ Takashi Hibino,⁴ Yoshito Tanaka,⁴ Tatsunosuke Nakamura,⁵ Masamitsu Shikata,⁶ Tetsuko Takabe,¹ and Teruhiro Takabe^2,4*

Graduate School of Agricultural Science, Nagoya University, Nagoya 464-8601, Japan,¹ Research Institute of Meijo University, Nagoya 468-8502, Japan,² Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand,³ Graduate School of Environmental and Human Sciences, Meijo University, Nagoya 468-8502, Japan,⁴ Faculty of Pharmacy, Niigata University of Pharmacy and Applied Life Science, Niigata 950-2081, Japan,⁵ Genomic Research Center, Shimadzu Corporation, Kyoto 604-8511, Japan⁶

Received 6 December 2004/ Accepted 20 February 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Aphanothece halophytica is a halotolerant alkaliphilic cyanobacterium which can grow at NaCl concentrations up to 3.0 M and at pH values up to 11. The genome sequence revealed that the cyanobacterium Synechocystis sp. strain PCC 6803 contains five putative Na⁺/H⁺ antiporters, two of which are homologous to NhaP of Pseudomonas aeruginosa and three of which are homologous to NapA of Enterococcus hirae. The physiological and functional properties of NapA-type antiporters are largely unknown. One of NapA-type antiporters in Synechocystis sp. strain PCC 6803 has been proposed to be essential for the survival of this organism. In this study, we examined the isolation and characterization of the homologous gene in Aphanothece halophytica. Two genes encoding polypeptides of the same size, designated Ap-napA1-1 and Ap-napA1-2, were isolated. Ap-NapA1-1 exhibited a higher level of homology to the Synechocystis ortholog (Syn-NapA1) than Ap-NapA1-2 exhibited. Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 complemented the salt-sensitive phenotypes of an Escherichia coli mutant and exhibited strongly pH-dependent Na⁺/H⁺ and Li⁺/H⁺ exchange activities (the highest activities were at alkaline pH), although the activities of Ap-NapA1-2 were significantly lower than the activities of the other polypeptides. Only one these polypeptides, Ap-NapA1-2, complemented a K⁺ uptake-deficient E. coli mutant and exhibited K⁺ uptake activity. Mutagenesis experiments suggested the importance of Glu129, Asp225, and Asp226 in the putative transmembrane segment and Glu142 in the loop region for the activity. Overexpression of Ap-NapA1-1 in the freshwater cyanobacterium Synechococcus sp. strain PCC 7942 enhanced the salt tolerance of cells, especially at alkaline pH. These findings indicate that A. halophytica has two NapA1-type antiporters which exhibit different ion specificities and play an important role in salt tolerance at alkaline pH.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Salinity has a detrimental effect on soil microorganisms and, in general, results in decreased productivity of crop plants. Organisms that thrive in hypersaline environments possess specific mechanisms to adjust their internal osmotic status (1, 10, 21, 35). One such mechanism is the ability to accumulate low-molecular-weight organic compatible solutes, such as glycine betaine (10, 21). Another mechanism for adaptation to high salinity is exclusion of Na⁺ ions from the cells (1-4).

Aphanothece halophytica is a halotolerant cyanobacterium which can grow in a wide range of salinity conditions (0.25 to 3.0 M NaCl) and accumulate betaine concomitantly (5, 30). It also can grow at alkaline pH (pH 11.0). Na⁺/H⁺ antiporters of alkaliphilic A. halophytica may play a crucial role in Na⁺ efflux and in cytoplasmic pH homeostasis. At alkaline pH, the cells maintain a cytoplasmic pH much lower than the external pH and require unique systems to survive under these severe environmental conditions (5, 30). Indeed, previous studies have shown that ribulose-1,5-bisphosphate carboxylase/oxygenase of A. halophytica dissociates easily into large and small subunits when betaine is absent (8). A. halophytica DnaK contains a longer C-terminal segment than other DnaK/Hsp70 family members contain (12) and exhibits extremely high protein folding activity at high salinity (5). It has also been shown that an A. halophytica NhaP-type Na⁺/H⁺ antiporter has a novel ion specificity (32) and can confer tolerance to salt stress on the freshwater cyanobacterium so that it is capable of growth in seawater (30).

The genome sequence of Synechocystis sp. strain PCC 6803 revealed the presence of five putative Na⁺/H⁺ antiporter genes (9). Of the five proteins encoded by these genes, two (Syn-NhaP1 and Syn-NhaP2) are homologous to NhaP of Pseudomonas aeruginosa and three (Syn-NapA1, Syn-NapA2, and Syn-NapA3) are homologous to NapA of Enterococcus hirae (3, 4, 7, 33). Originally, NapA was designated an Na⁺/H⁺ antiporter different from Escherichia coli NhaA (34). NhaP antiporters exhibit some homology to eukaryotic antiporters, such as SOS1 and NHX1 from plants and NHE1 from animals (29, 32).

NapA is a member of the monovalent cation-proton antiporter 2 (CPA-2) family (22). In Arabidopsis plants, 35 putative CPA-2 antiporter genes have been assigned based on the genome sequence (13). A CPA-2 antiporter has not been reported for mammalian cells. In prokaryotic cells, the members of this family include a putative iron transport protein, MagA, from Magnetospirillum sp. strain AMB-1 (15), KefB and KefC from E. coli, which are K⁺ efflux system activated by glutathione (14), and Na⁺/H⁺ antiporter protein NapA from E. hirae (34). Putative antiporters important in germination of Bacillus megaterium (GrmA) (26) and Bacillus cereus (GerN) (23, 27) are members of the CPA-2 family and most closely resemble NapA.

The physiological and functional properties of NapA-type antiporters are largely unknown. One of the napA-type antiporter genes in Synechocystis sp. strain PCC 6803 (sll0689, nhaS3), here designated Syn-napA1, has been proposed to be essential for the survival of this organism since site-directed null mutants could not be isolated (3, 7, 33). However, no information is currently available on NapA-type antiporters in other cyanobacteria. Because of these findings, we were interested in isolating a homologous gene from A. halophytica to characterize its functional properties. Here, we show that A. halophytica contains at least two genes (Ap-napA1-1 and Ap-napA1-2) homologous to Syn-napA1. Although Ap-NapA1-1 and Ap-NapA1-2 had Na⁺/H⁺ and Li⁺/H⁺ exchange activities, Ap-NapA1-2 exhibited K⁺ uptake activity. In contrast to NhaP1, the exchange activities of these NapA1 antiporters were strongly pH dependent, and the highest activity was observed at alkaline pH. An important role for NapA1 antiporters in salt tolerance at an alkaline pH was demonstrated.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Strains and culture conditions.
A. halophytica cells were grown photoautotropically in BG11 liquid medium plus 18 mM NaNO₃ and Turk Island salt solution at 28°C as previously described (5). Synechocystis sp. strain PCC 6803 and Synechococcus sp. strain PCC 7942 cells were grown at 30°C under continuous fluorescent white light (40 microeinsteins m^–2 s^–1) in BG11 liquid medium supplemented with 10 mM HEPES-KOH and bubbled with 3% CO₂. E. coli DH5 was grown at 37°C in LB medium. E. coli TO114 cells, in which Na⁺/H⁺ antiporter genes (i.e., nhaA, nhaB, and chaA) were deleted, were grown at 37°C in LBK medium (18). E. coli LB650 was grown at 37°C in minimal medium as previously described (16). Ampicillin, erythromycin, kanamycin, and chloramphenicol were added to final concentrations of 50, 150, 30, 30 and µg ml^–1, respectively, whereas isopropyl-ß-D-thiogalactopyranoside (IPTG) was not added. The growth medium pH was adjusted with KOH or HCl. Cell growth of E. coli and cell growth of cyanobacteria were monitored by measuring the light scattering at 620 and 730 nm, respectively. For the growth experiments with E. coli, E. coli TO114 or LB 650 cells in the late logarithmic phase were transferred into fresh medium (LB medium for TO114 cells or minimum medium for LB 650 cells) at an initial optical density at 620 nm (OD₆₂₀) of 0.02. The medium was supplemented with KCl, NaCl, or LiCl as indicated below. Growth of the cells was measured by determining the OD₆₂₀ after 9 h unless indicated otherwise. Growth curves were constructed by using the averages of at least three independent measurements.

Isolation of napA1 genes.
The napA1 genes from A. halophytica, Ap-napA1-1 and Ap-napA1-2, were amplified by PCR using primers ApNapA1-1-F and ApNapA1-1-R and primers ApNapA1-2-F and ApNapA1-2-R, respectively. The sequences of all the primers are shown in Table 1. The Syn-napA1 gene from Synechocystis sp. strain PCC 6803 was amplified by with primers SynNapA1-F and SynNapA1-R. The amplified fragments were ligated into the EcoRV restriction site of pBSK+ (Stratagene, La Jolla, CA) and sequenced. Next, the inserts were transferred into the NcoI/SalI sites of pTrcHis2C (Invitrogen, Carlsbad, CA). The resulting plasmids, pApNapA1-1, pApNapA1-2, and pSynNapA1, encode Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1, respectively, fused in frame to six histidines at the C terminus. These plasmids were transferred first to E. coli DH5 and then to TO114 cells in which the nhaA, nhaB, and chaA genes were deleted (4, 18, 32).

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TABLE 1. Primers used for isolation and expression of Na+/H+ antiporter genes

Construction of Ap-NapA1-1 mutants.
The amino acid Glu129 in Ap-NapA1-1 was changed to Asp, Gln, and His by PCR mutagenesis as previously described (31). Briefly, the 5'- and 3'-terminal parts of Ap-napA1-1 were amplified with primers ApNapA1-1-F and ApE129DQ-R and primers Ap129DQ-F and ApNapA1-1-R using pApNapA1-1 as the template. After the primers were removed, two PCR-amplified fragments were mixed, heated, annealed, and used as the templates for amplification with primers ApNapA1-1-F and ApNapA1-1-R. The PCR product was ligated into the EcoRV site of pBSK+ and sequenced. The E129D and E129Q mutants were transferred to pTrcHis2C and used for transformation of TO114. E142D, E142Q, D225E, D225N, D226E, and D226N mutants were constructed essentially by the same method.

Antiporter activity.
Everted membrane vesicles were prepared from cells grown in LBK medium at pH 7.0 as previously described (17, 32). Briefly, E. coli cells were harvested by centrifugation at 3,000 x g for 10 min at 4°C and then washed with TCDS suspension buffer (10 mM Tris-HCl, pH 7.5, 0.14 M choline chloride, 0.5 mM dithiothreitol, 0.25 M sucrose). The pellet was suspended in 10 ml TCDS buffer and applied to a French pressure cell (4,000 lb/in²). The solution was then centrifuged at 12,000 x g for 10 min at 4°C. The supernatant was finally centrifuged at 110,000 x g for 60 min at 4°C, and the pellet was suspended in 500 µl TCDS buffer. The antiporter activity was assayed by monitoring the changes in pH (pH) (transmembrane [TMx pH gradient) after addition of salt to the 2-ml reaction mixture containing 10 mM Tris-HCl, 5 mM MgCl₂, 0.14 M choline chloride, 1 µM acridine orange, and everted membrane vesicles (50 µg of protein) (4, 17, 32). The pH was monitored by using acridine orange fluorescence with excitation at 492 nm and emission at 525 nm. Before addition of salt, Tris-DL-lactate (5 mM) was added to initiate fluorescence quenching due to respiration. Lactate energized the vesicles, causing accumulation of H⁺ intravesicularly and subsequent accumulation of the dye, resulting in fluorescence quenching. Salt (5 mM) was then added to dequench the fluorescence due to the excretion of H⁺ by antiporters. Finally, 25 mM NH₄Cl was added to dissipate the pH.

K⁺-depleted cells.
Cells harvested from 8 ml of culture were suspended at a concentration of 0.5 mg of cell protein per ml of buffer containing 25 mM HEPES-NaOH, pH 7.5 and 1 mM EDTA-NaOH, pH 7.5. This suspension was gently shaken for 10 min at 37°C. Subsequently, the cells were centrifuged and washed three times with the same buffer containing 50 mM NaCl (suspension buffer). The cells were then suspended at concentration of 5 to 10 mg of cell protein per ml of suspension buffer and shaken at 37°C until the start of the experiment.

Detection of K⁺ uptake with an ion analyzer.
K⁺-depleted cells were suspended at a concentration of 5 to 10 mg of cell protein per ml of suspension buffer. After addition of 10 mM glucose, the suspension was shaken for 10 min at 37°C, and KCl was then added at the concentrations indicated below. At different times, a 1-ml sample was withdrawn from the suspension and then immediately subjected to centrifugation. The cell pellet was suspended in 1 ml of distilled water and boiled for 5 min. After removal of cell debris by centrifugation, the K⁺ content in the supernatant was determined with a Shimadzu PIA-1000 personal ion analyzer.

Overexpression of Ap-NapA1-1 in a freshwater cyanobacterium.
An expression plasmid for Ap-NapA1-1 which contained its own promoter was constructed as previously described (30). The 400-bp promoter region of Ap-napA1-1 was amplified from the genomic DNA of A. halophytica using primers ApNapA1Pro-F and ApNapA1Pro-R and ligated into the NcoI site of pApNapA1-1. After the orientation of the promoter was checked by sequencing, the full length of Ap-napA1-1 (containing the promoter and the His tag) was amplified with primers ApNapA1Pro-F and HisBamHI, blunt ended, and ligated into the BamHI-digested site of E. coli-Synechococcus shuttle vector pUC303-Bm (30). The resulting plasmid was designated pUC303-ApNapA1-1 and was used to transform Synechococcus sp. strain PCC 7942 cells (30). For the salt stress experiments, Synechococcus cells were subcultured in BG11 medium as described above together with 10 µg ml^–1 streptomycin. Cells in the late logarithmic phase were transferred into fresh medium containing various concentrations of NaCl (0 to 0.5 M).

Other methods.
The nucleotide sequences were determined using an ABI310 genetic analyzer (Applied Biosystems, Foster City, CA). Cellular ions were determined with a Shimadzu PIA-1000 personal ion analyzer. The protein content was determined by Lowry's method as described previously (5, 32). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analysis were carried out as described previously (4, 32). An antibody raised against His₆ (six-His tag) was obtained from R&D Systems (Minneapolis, MN). The hydropathy profile of proteins was predicted using the computer-assisted procedure performed as described by Kyte and Doolittle (6, 11). The possible TM structure of Ap-NapA1 and Syn-NapA1 was predicted with the computer program TopPredII (6, 11).

Nucleotide sequence accession numbers.
Nucleotide sequence data for Ap-napA1-1 and Ap-napA1-2 have been deposited in the DDBJ databases under accession numbers AB193603 and AB193604, respectively.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cloning of NapA1-type antiporter genes from A. halophytica.
Although only one napA1-type antiporter gene, Syn-napA1, has been reported for Synechocystis sp. strain PCC 6803, two open reading frames homologous to Syn-napA1 were found in the shotgun clones of A. halophytica. The two genes were isolated by PCR amplification and were sequenced as described in Materials and Methods. The predicted gene products (Ap-NapA1-1 and Ap-NapA1-2) each consist of 467 amino acids, and they have molecular masses of 48,113 and 48,566, respectively (Fig. 1A). The ClustalW analysis (Fig. 1B) showed that Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 could be classified as different Na⁺/H⁺ antiporter proteins than Syn-NapA2, Syn-NapA3, Syn-NhaP1, and Syn-NhaP2. Among the NapA1-type antiporters, Ap-NapA1-1 exhibited high levels of homology to Syn-NapA1 from Synechocystis sp. strain PCC 6803 (67%) and to Ap-NapA1-2 (64%). By contrast, Ap-NapA1-1 exhibited low levels of homology to GrmA from B. megaterium (37%) (26), GerN from B. cereus (37%) (27), and NapA from E. hirae (34%) (34). Analysis of the hydropathy plot and TM prediction program suggested that there are 11 putative TM spanning segments in these antiporters (Fig. 1A). All six antiporters shown in Fig. 1A contain two consecutive Asp residues in a putative TM6 segment (Asp225 and Asp226 in the case of Ap-NapA1-1). Moreover, the trimer Gly-Leu-Glu in the loop region connecting TM segments (i.e., TM3 and TM4) (Gly140-Leu141-Glu142 in the case of Ap-NapA1-1) is also conserved (Fig. 1A).

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FIG. 1. Comparison of the deduced amino acid sequences of cation-proton antiporters. (A) Alignment of the deduced amino acid sequences of six cation-proton antiporters. The sequences were aligned with the program ClustalW. The amino acid residues conserved in all sequences are indicated by asterisks. Predicted membrane-spanning regions are indicated above the alignment. Site-directed mutated amino acid residues in Ap-NapA1-1 are enclosed in boxes. (B) Phylogenetic analysis of cation-proton antiporters. Multiple-sequence alignment and generation of the phylogenetic tree were performed with the ClustalW and TreeView software, respectively. The accession numbers for various antiporters are as follows: AB193603 for Ap-NapA1-1, AB193604 for Ap-NapA1-2, D64001 for Synechocystis sp. strain PCC 6803 Syn-NapA1 (slr0689), AF246294 for B. cereus GerN, U17283 for B. megaterium putative spore germination apparatus protein (GrmA), and M81961 for E. hirae NapA.

Expression of Ap-NapA1-1 and Ap-NapA1-2 in E. coli and complementation of the Na⁺- and Li⁺-sensitive phenotypes.
To examine the functional properties of Ap-NapA1-1 and Ap-NapA1-2, the genes encoding these proteins were expressed in salt-sensitive E. coli mutant TO114 cells in which the nhaA, nhaB, and chaA genes were disrupted. As shown in Fig. 2A, Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 could be expressed in TO114 cells. The level of expression of Ap-NapA1-1 was considerably lower than the levels of expression of Ap-NapA1-2 and Syn-NapA1. Figures 2B and C show the growth of E. coli TO114 cells in LB medium containing 30 mM KCl and various concentrations of NaCl or LiCl. TO114 cells transformed with the control plasmid could not grow in medium containing NaCl at a concentration higher than 0.2 M, whereas the cells transformed with Ap-napA1-1, Ap-napA1-2, and Syn-napA1 could grow (Fig. 2B). Similar results were obtained for growth in medium containing LiCl at a concentration higher than 4 mM (Fig. 2C). These results clearly indicate that Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 could complement the NaCl- and LiCl-sensitive phenotypes of E. coli TO114 cells.

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FIG. 2. Effects of NaCl and LiCl on the growth of E. coli cells expressing Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1. (A) Immunoblot analyses of pApNapA1-1-, pApNapA1-2-, and pSynNapA1-expressing cells. Lane 1, pApNapA1-1-expressing cells; lane 2, pApNapA1-2-expressing cells; lane 3, pSynNapA1-expressing cells; lane 4, pTrcHis2C control cells. In each lane, 50 µg membrane proteins was loaded. Antiporter proteins were detected using an antibody raised against the His tag. (B) LB medium containing 30 mM KCl and different concentrations of NaCl. (C) LB medium containing 30 mM KCl and different concentrations of LiCl. The control TO114 cells and transformant TO114 cells expressing Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 in the exponential phase were transferred to growth medium containing the different salts and pH. Each value is the average of three independent measurements of OD620 at 9 h.

Antiporter activities of Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 at various pHs.
To examine whether Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 exhibit exchange activities, everted membrane vesicles of TO114 cells were prepared (17, 32). Cells transformed with the control plasmid exhibited essentially no Na⁺/H⁺ and Li⁺/H⁺ antiporter activities at all pHs examined (pH 6.0 to 9.0) (Fig. 3A and B). However, Na⁺/H⁺ exchange activities were observed in the cells expressing Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 at pH 7.0 to 9.0 (Fig. 3A). Ap-NapA1-2 exhibited exchange activities for both Na⁺/H⁺ and Li⁺/H⁺ antiporters that were lower than those of Ap-NapA1-1 and Syn-NapA1 (Fig. 3A and B). We could not detect Mg²⁺/H⁺ exchange activity at any pH examined, although low Ca²⁺/H⁺ and K⁺/H⁺ exchange activities were observed at pH 7.0 to 8.0 and at pH 7.0 to 9.0, respectively, as shown for Ap-NapA1-1 (Fig. 3C) and Ap-NapA1-2 (Fig. 3D).

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FIG. 3. Cation-proton exchange activities measured by the acridine orange fluorescence quenching method. The control TO114 cells and transformed TO114 cells expressing Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 were grown in LBK medium, from which everted membrane vesicles were prepared. The antiporter activity was measured as described in Materials and Methods. (A and B) Na+/H+ and Li+/H+ antiporter activities of Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1, respectively. (C and D) Na+/H+, K+/H+, Mg2+/H+ and Ca2+/H+ antiporter activities of Ap-NapA1-1 and Ap-NapA1-2, respectively. The final concentration of salts was 5 mM. Each value is the average of three independent measurements.

Interestingly, Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 exhibited pH-dependent activities. The exchange activities of these antiporters increased with increasing pH (Fig. 3). This is more clearly shown in Fig. 4. The NhaP-type antiporter from A. halophytica, Ap-NhaP1, exhibited high Na⁺/H⁺ exchange activities at pH 6.0 to 9.0 (Fig. 4A). By contrast, Ap-NapA1-1 had no Na⁺/H⁺ exchange activity at pH 6.0, and its activity increased with increasing pH; the optimum pH was around 8.5 (Fig. 4A). Ap-NapA1-1 exhibited a similar pH dependence for Li⁺/H⁺ exchange, whereas Ap-NhaP1 showed very little or no activity at all pHs examined (Fig. 4B).

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FIG. 4. Comparison of Na+/H+ (A) and Li+/H+ (B) exchange activities of Ap-NapA1-1 and Ap-NhaP1. The experimental conditions were the same as those described in the legend to Fig. 3. Each value is the average of three independent measurements.

The kinetic parameters were also examined. At pH 8.5, the K_m values for Na⁺ were 0.8, 1.8, and 0.5 mM for Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1, respectively, and those for Li⁺ were 0.05, 0.3, and 0.02 mM, respectively. The K_m values for Na⁺ and Li⁺ at pH 7.0 were similar to those at pH 8.5 (data not shown). Although it is well documented that amiloride inhibits the Na⁺/H⁺ exchange activities of antiporters from animals (19) and plants (2), Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 were not sensitive to 0.1 mM amiloride for both the Na⁺/H⁺ and Li⁺/H⁺ exchange reactions (data not shown).

Potassium uptake activity and complementation of potassium uptake-deficient E. coli mutant by Ap-NapA1-2.
Recently, it has been shown that the spore germination protein GerN from Bacillus is an Na⁺/H⁺-K⁺ antiporter and can complement K⁺ uptake-deficient E. coli (23). We therefore tested whether Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 could transport K⁺ by performing complementation experiments with K⁺ uptake-deficient E. coli LB650 cells (16). It was found that the growth of control and transformed cells expressing Ap-NapA1-1 and Syn-NapA1 was essentially the same as that of the cells expressing pTrcHis2C (Fig. 5A). By contrast, the growth of Ap-NapA1-2-expressing cells was more rapid than the growth of the cells expressing Ap-NapA1-1 and Syn-NapA1 when the growth medium contained 10 and 15 mM KCl (Fig. 5A). The positive control cells with the pKT66 plasmid carrying the K⁺ transport gene grew even in the defined medium containing 3 mM KCl. These results indicate that Ap-NapA1-2 could partially complement the K⁺ uptake-deficient mutant.

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FIG. 5. KCl dependence on growth and K + uptake in E. coli cells expressing Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1. (A) Growth of K+ uptake-deficient control LB650 cells and cells expressing pKT66 (positive control), Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 in minimum medium containing different concentrations of KCl. (B) K + uptake of cells expressing pKT66. Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 were detected with K+-depleted cells as described in Material and Methods. KCl (2 mM) was added at zero time. K + contents of the cells were measured as described in Materials and Methods. Each value is the average of three measurements.

We further determined the initial rate of K⁺ uptake, and the results are shown in Fig. 5B. Cells expressing Ap-NapA1-2 could take up K⁺, although the uptake rate was about one-half that of the positive control cells. In contrast, the K⁺ uptake rates of the cells expressing Ap-NapA1-1and Syn-NapA1 were almost the same as those of the cells bearing pTrcHis2C. These results indicated that Ap-NapA1-2, but not Ap-NapA1-1 and Syn-NapA1, could mediate K⁺ uptake.

Potassium significantly affected Na⁺/H⁺ and Li⁺/H⁺ exchange activities of Ap-NapA1-2.
To test whether the antiporters could use K⁺ instead of H⁺ as a coupling ion, we performed a fluorescence assay using everted membrane vesicles. The everted membrane vesicles were prepared with TCDS buffer without addition of extra K⁺. Under these conditions, the intracellular K⁺ concentration of the everted membranes was 1 mM (25). Next, we tested the effects of K⁺ in the assay buffer on fluorescence dequenching. If K⁺ and H⁺ can compete for binding inside everted membrane vesicles, then the efflux of K⁺ would be affected by K⁺ in the assay medium, which in turn would affect the efflux of H⁺. As shown in Fig. 6A, in the everted membrane vesicles expressing Ap-NapA1-2, the fluorescence dequenching upon addition of NaCl was low when choline chloride (140 mM) was included in the assay medium. By contrast, the fluorescence dequenching was significantly higher when choline chloride was replaced with KCl (140 mM) (Fig. 6B). However, in the case of Ap-NapA1-1, the fluorescence dequenching was relatively high in both choline chloride and KCl medium (Fig. 6C and D). Similar results were obtained for Li⁺-induced dequenching (Fig. 6E to H). These data suggest that Ap-NapA1-2, but not Ap-NapA1-1, mediates the exchange activity between Na⁺ and H⁺, as well as between Na⁺ and K⁺, and that Na⁺ could be replaced by Li⁺.

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FIG. 6. Effects of K+ on Na+/H+ and Li+/H+ exchange activities. Everted membrane vesicles were prepared using TCDS buffer. For panels A, C, E, and G, the assay medium contained 140 mM choline chloride. For panels B, D, F, and H, KCl (140 mM) replaced choline chloride in the assay medium. (A, B, E, and F) Ap-NapA1-2-expressing cells; (C, D, G, and H) Ap-NapA1-1-expressing cells. (A to D) Na+/H+ exchange activity; (E to H) Li+/H+ exchange activity.

Acidic amino acid residues Asp225, Asp226, and Glu129 in the TM segment are essential for the activity.
Alignment of the deduced amino acid sequences (Fig. 1A) and a topological model of Ap-NapA1-1 (Fig. 7) suggested that Asp225 and Asp226 are the only conserved charged amino acid residues in TM segments in the six antiporters. The function of acidic amino acid residues in TM domains has not been reported previously for any NapA-type antiporter. Therefore, we examined the effects of mutations replacing Asp225 and Asp226 with Asn and Glu. The D225N and D225E mutants did not exhibit the Na⁺/H⁺ and Li⁺/H⁺ antiporter activities at all pHs tested, and these two mutants could not complement the Na⁺- and Li⁺-sensitive phenotypes of TO114 (data not shown). Essentially the same results were obtained for D226E and D226N mutants (data not shown). These results indicate that not only the negative charges but also the side chains of Asp225 and Asp226 are crucial for the Na⁺/H⁺ and Li⁺/H⁺ exchange activities.

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FIG. 7. Schematic secondary structure model of Ap-NapA1-1. Acidic and basic amino acid residues in the loop regions are indicated by circled minus and plus signs, respectively. The conserved amino acid residues Gly140-Glu142 and Asp225-Asp226 are shown. E129 and K383 are charged amino acid residues not conserved among NapA antiporters.

Figure 1A shows that Glu129 in Ap-NapA1-1 is a highly conserved charged amino acid residue in TM segments. In six antiporters, Glu is replaced with Asn only in GerN. We tested the role of Glu129 in Ap-NapA1-1. Mutation of Glu129 to Gln or Asp abolished the Na⁺/H⁺ and Li⁺/H⁺ exchange activities (data not shown), indicating that Glu129 in the TM3 segment of Ap-NapA1-1 is also essential for the exchange activity.

Site-directed mutagenesis of Glu142 in the loop region of Ap-NapA1-1.
Figures 1 and 7 indicate that the tripeptide Gly-Leu-Glu in the loop region connecting the TM3 and TM4 segments is conserved among the six antiporters. We therefore investigated the effects of mutation of Glu142 to Gln and Asp on the exchange activities. The E142Q mutant lost the Na⁺/H⁺ and Li⁺/H⁺ exchange activities at all pHs tested and could not complement the Na⁺- and Li⁺-sensitive phenotypes of TO114 cells (data not shown). The E142D mutant exhibited drastically reduced Na⁺/H⁺ and Li⁺/H exchange activities but could complement the Na⁺- and Li⁺-sensitive phenotypes with slower growth than the growth with wild-type Ap-NapA1-1 (data not shown). These results suggest that the negative charge on Glu142 and also partially its size have an important role in the salt tolerance.

Overexpression of Ap-NapA1-1 conferred salt tolerance on the freshwater cyanobacterium Synechococcus sp. strain PCC 7942.
We have shown previously that overexpression of Ap-NhaP1 can confer salt tolerance on freshwater Synechococcus sp. strain PCC 7942, making it able to grow even in seawater (30). To examine the potential of Ap-NapA1-1 for abiotic stress tolerance, we overexpressed Ap-NapA1-1 and Ap-NhaP1 in freshwater Synechococcus sp. strain PCC 7942. As shown in Fig. 8A, both the wild-type and transformed cells could grow at almost the same rate in BG11 medium. However, in BG11 medium containing 0.4 M NaCl, the wild-type cells could not grow, whereas the cells expressing Ap-NapA1-1 and Ap-NhaP1 could grow under the same conditions (Fig. 8B). When the growth medium contained 0.5 M NaCl, only the cells expressing Ap-NhaP could grow (Fig. 8C). These results indicate that overexpression of Ap-NapA1-1 could confer salt tolerance on a freshwater cyanobacterium. However, the potential of Ap-NapA1-1 for salt tolerance was lower than that of Ap-NhaP1.

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FIG. 8. Salt tolerance of Synechococcus sp. strain PCC 7942 cells expressing Ap-NapA1-1 and Ap-NhaP1. Freshwater Synechococcus sp. strain PCC 7942 cells transformed with the vector only (control), with Ap-NapA1-1, and with Ap-NhaP1 were grown in BG11 medium (A) or in BG11 medium containing 0.4 M NaCl (B) or 0.5 M NaCl (C) at pH 7.0. Each value is the average of three independent measurements.

Overexpression of Ap-NapA1-1 conferred salt tolerance on the freshwater cyanobacterium Synechococcus sp. strain PCC 7942 at alkaline pH.
Since the exchange activities of Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 were high at alkaline pH, we examined the salt tolerance of Synechococcus sp. strain PCC 7942 cells at an alkaline pH. As shown in Fig. 9A, both the wild-type and transformed cells could grow at almost the same rate in BG11 medium at pH 7.0 and 9.0. When 0.3 M NaCl was present in BG 11 medium, faster growth was observed for the cells expressing Ap-NapA1-1 at pH 9.0 but not at pH 7.0 (Fig. 9A and B). These results suggest that Ap-NapA1-1 has an important role in salt tolerance at alkaline pH.

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FIG. 9. Salt tolerance of Synechococcus sp. strain PCC 7942 cells expressing Ap-NapA1-1. Freshwater Synechococcus sp. strain PCC 7942 cells transformed with the vector only (control) and with Ap-NapA1-1 were grown in BG11 medium (open symbols) or in BG11 medium containing 0.3 M NaCl (solid symbols) at pH 7.0 (A) and pH 9.0 (B). Each value is the average of three independent measurements.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this paper, we show that the halotolerant cyanobacterium A. halophytica has two antiporters with the same polypeptide size, Ap-NapA1-1 and Ap-NapA1-2. Based on the finding that the antiporter-deficient E. coli TO114 mutant cells became Na⁺ and Li⁺ tolerant after transformation with Ap-napA1-1 and Ap-napA1-2 (Fig. 2) and the finding that membrane vesicles of these transformants exhibited the Na⁺/H⁺ and Li⁺/H⁺ exchange activities (Fig. 3), these genes could be identified as the Na⁺/H⁺ and Li⁺/H⁺ antiporter genes. However, the growth complementation of the K⁺ uptake-deficient mutant (Fig. 5A), the K⁺ uptake activity (Fig. 5B), and the effects of KCl on the exchange activity (Fig. 6) revealed that Ap-NapA1-2 has unique properties; namely, K⁺ can partially replace H⁺. To our knowledge, the replacement of H⁺ with K⁺ has not been reported previously for any transporter from photosynthetic organisms, such as plants and cyanobacteria. The more rapid growth of E. coli cells expressing Ap-NapA1-2 than of the other expressing cells in the presence of 0.6 M NaCl (Fig. 2B) and 0.1 M LiCl (Fig. 2C) might have been due to the uptake of K⁺ in the former cells (Fig. 5).

One of the striking properties of Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 antiporters is their pH dependence and roles in salt tolerance at alkaline pH (Fig. 3, 4, and 9). Figures 3 and 4 show that the Na⁺/H⁺ and Li⁺/H⁺ exchange activities of these NapA1-type antiporters significantly increased with increasing pH. This is in sharp contrast to the previous results for Ap-NhaP1 and Syn-NhaP1, which exhibit high exchange activities over a wide pH range (pH 5.0 to 9.0) (5, 7). The pH dependence of Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1 was different from that of E. hirae NapA (34) and that of B. cereus GerN (27), but it was similar to that of the E. coli NhaA antiporter (25). The exchange activity of NapA decreased with increasing pH (34), whereas that of GerN was constant. In NhaA from E. coli (20) and Helicobacter pylori (28), several amino acid residues involved in pH sensing have been identified. Therefore, it would be interesting to identify the amino acid residues involved in pH sensing in Ap-NapA1-1, Ap-NapA1-2, and Syn-NapA1.

Two consecutive Asp residues in TM6 (Asp225 and Asp226 in the case of Ap-NapA1-1) are conserved in all six NapA-type antiporters shown in Fig. 1. The role of these residues has not been examined previously. Site-directed mutagenesis of Asp225 and Asp226 indicated that not only the negative charge on the TM segment but also the configuration of the side chain is important for the exchange activity; i.e., replacement of Asp with Glu resulted in a loss of exchange activity. A change of one of the two Asp residues decreased the exchange activity (see above), indicating that two consecutive aspartates (Asp225 and Asp226) in TM6 are required for the exchange activity. The importance of Asp in the TM has been documented for several Na⁺/H⁺ antiporters; the important Asp residues include Asp163 and Asp164 in E. coli NhaA (25), Asp155 and Asp156 in Vibrio alginolyticus NhaA (17), and Asp138 in Syn-NhaP1 (4). Several Na⁺/H⁺ antiporters do not contain two consecutive negatively charged amino acids in the TM segment. Nonetheless, all six NapA1-type antiporters shown in Fig. 1A have consecutive Asp residues in the TM domain. The molecular mechanisms of consecutive Asp residues for Na⁺/H⁺ exchange activity are an interesting issue.

The tripeptide Gly-Leu-Glu in the loop region connecting TM segments is conserved in the six antiporters shown in Fig. 1. Previously, the role of this peptide has not been examined in any NapA-type antiporter. We therefore examined the role of Glu142 in Ap-NapA1-1 in exchange activity and found that Glu is essential and cannot be replaced by Gln (see above). However, Asp could partially replace Glu. These results suggest that the negative charge on the third amino acid residue in the tripeptide is crucial for exchange activity.

Due to the failure to obtain a complete deletion of the Syn-napA1 gene, Syn-napA1 has been proposed to be essential for the survival of Synechocystis sp. strain PCC 6803 (3, 7, 33). This situation contrasts with the results for Arabidopsis, in which an NhaP homologous gene, SOS1, is crucial and contributes to salt tolerance, although Arabidopsis plants have many CPA-2-type antiporters which are homologous to NapA (24). Moreover, it should be noted that the NhaP-type antiporters Ap-NhaP1 and Syn-NhaP1 have high Na⁺/H⁺ exchange activities over a wide range of pHs (pH 6 and 9) and complement an Na⁺-sensitive E. coli mutant (4, 32), and overexpression of Ap-NhaP1 in a freshwater cyanobacterium conferred salt tolerance on Synechococcus sp. strain PCC 7942 cells capable of growth in seawater (30). The data in Fig. 8 indicate that the salt tolerance of Synechococcus sp. strain PCC 7942 cells conferred by Ap-NapA1-1 is lower than that conferred by Ap-NhaP1. These data strongly suggest that Syn-NhaP1 or Ap-NhaP1 could replace Syn-NapA1. Therefore, it is still not clear why the Syn-napA1 gene could not be deleted. Further studies are required to understand the physiological role of Ap-NapA1-1 and Ap-NapA1-2.

 
This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan and the High-Tech Research Center of Meijo University. N.W. and A.I. were supported by the Thai government through the Thailand-Japan Technology Transfer Project.

We thank Eiko Tsunekawa for her expert technical assistance.

 
* Corresponding author. Mailing address: Research Institute of Meijo University, Tenpaku-ku, Nagoya 468-8502, Japan. Phone: 81-52-838-2277. Fax: 81-52-832-1545. E-mail: takabe{at}ccmfs.meijo-u.ac.jp.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, August 2005, p. 4176-4184, Vol. 71, No. 8
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Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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