INTRODUCTION

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High salinity is a major environmental factor that limits the growth and productivity of plants, eukaryotic microorganisms, and bacteria. Control of membrane permeability to Na⁺ ions and the counteracting K⁺ ions is the most important aspect of the acclimation of these organisms to high-salt conditions. Na⁺/H⁺ antiporters are membrane proteins that are essential for maintenance of the balance between Na⁺ and K⁺ ions in plant, fungal, and bacterial cells, in particular when the organism lacks primary Na⁺ pumps or when the Na⁺ pumps are not operative (8, 33).

Escherichia coli has at least three genes for Na⁺/H⁺ antiporters: nhaA (14, 23), nhaB (34), and chaA (19, 31). The presence of a primary Na⁺ pump has been suggested (4), but E. coli mutants deficient in all three of these genes are hypersensitive to Na⁺ and Li⁺ ions (31, 35). Saccharomyces cerevisiae has Na⁺-ATPases (17) and an Na⁺(K⁺)/H⁺ antiporter, Nha1, in the plasma membrane (5). In addition, it has been suggested that an Na⁺/H⁺ antiporter in yeast, designated Nhx1, functions to remove Na⁺ ions from the cytosol by sequestering these ions in a prevacuolar compartment (28, 29). In contrast, it is well established that high-affinity K⁺ channels that restrict the influx of Na⁺ ions determine the capacity of plant cells to tolerate high-salt stress (38). Moreover, Apse et al. (1) demonstrated that a vacuolar Na⁺/H⁺ antiporter in Arabidopsis thaliana, AtNHX1, which is homologous to Nhx1 of S. cerevisiae, also participates in the acclimation of A. thaliana to high-salt conditions. Shi et al. (40) proposed recently that SOS1 of A. thaliana, a homolog of Na⁺/H⁺ antiporters in plasma membranes, might play a role in Na⁺/K⁺ homeostasis.

We chose cyanobacteria as a model system for studies of the molecular mechanisms of the responses of plants to high-salt stress for the following reasons. (i) Cyanobacteria perform oxygenic photosynthesis using photosystems similar to those in plant chloroplasts. (ii) The structure and the lipid compositions of cyanobacterial membranes resemble those of chloroplasts of higher plants and algae (49). (iii) Cyanobacterial cells exhibit more obvious responses to salt stress than do plant cells, and they can be exposed directly to changes in external salt conditions, demonstrating a pronounced ability to acclimate to new conditions. (iv) Some strains of unicellular cyanobacteria, such as Synechocystis sp. strain PCC 6803 (hereafter "Synechocystis") and Synechococcus sp. strain PCC 7942, are naturally transformable and can easily be modified by transformation and gene targeting (15). (v) The entire nucleotide sequence of the Synechocystis genome has been determined (22). Moreover, cyanobacteria themselves are unusual in that they contain thylakoid membranes in addition to the outer and cytoplasmic membranes. The thylakoid membranes provide sites for photosynthesis and a variety of metabolic pathways. The unusual structural and functional features of cyanobacterial cells led us to postulate that the systems that regulate ion fluxes across membranes in cyanobacterial cells might differ from those in other types of cells.

Cyanobacterial cells actively extrude Na⁺ ions via the actions of Na⁺/H⁺ antiporters. They maintain low intracellular concentrations of Na⁺ ions and relatively high intracellular concentrations of K⁺ ions (36). Therefore, they must have transport systems that discriminate between K⁺ and Na⁺ ions. When cyanobacterial cells are grown under high-salt conditions, the pH gradient-dependent (pH-dependent) transport of Na⁺ ions across the cytoplasmic membrane is enhanced (7, 30). Respiratory activity and the activity of cytochrome c oxidase are also enhanced under high-salt conditions (13, 20, 26). These observations provide circumstantial evidence for the electron transport-driven extrusion of Na⁺ ions by an Na⁺/H⁺ antiporter in cyanobacterial cells. There have been extensive studies of the molecular aspects of salt-inducible proteins (2, 6) and of salt-regulated genes (3, 48). However, Na⁺/H⁺ antiporters and other transporters involved in the efflux of Na⁺ ions have not yet been identified in cyanobacteria.

In the present study, we attempted to identify the Na⁺/H⁺ antiporters in Synechocystis. This cyanobacterium has five putative genes for homologs of Na⁺/H⁺ antiporters (22). We used a mutant of E. coli that was deficient in Na⁺/H⁺ antiporters to characterize these cyanobacterial genes by functional complementation. We demonstrate here that Synechocystis has at least two genes that encode low-affinity and high-affinity Na⁺/H⁺ antiporters, respectively.

	MATERIALS AND METHODS

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Nomenclature of genes. We refer to the putative genes for Na⁺/H⁺ antiporters in Synechocystis as nhaS1 (slr1727 in the designation system proposed by Kaneko et al. [22]), nhaS2 (sll0273), nhaS3 (sll0689), nhaS4 (slr1595), and nhaS5 (slr0415).

Bacterial strains and growth conditions. E. coli TO114 (W3110 nhaA::Km^r nhaB::Em^r chaA::Cm^r) (31) was generously provided by H. Kobayashi (Chiba University, Chiba, Japan). It was used as the host for complementation tests with cyanobacterial genes. Cells were grown in modified Luria-Bertani medium (39) that consisted of 1.0% tryptone (Difco, Detroit, Mich.), 0.5% yeast extract (Difco), and 100 mM KCl (LBK medium; pH 6.8). For selection and growth of transformed cells, ampicillin was added to 50 µg ml¹.

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Construction of plasmids for expression of nhaS genes in E. coli Plasmid pGM42 (14) was kindly provided by E. Padan (Hebrew University of Jerusalem, Jerusalem, Israel). This plasmid is a derivative of pBR322 and includes a 4.2-kbp segment of the chromosomal DNA of E. coli that contains the nhaA gene under control of the Na⁺-inducible promoter nhaAp plus the nhaR gene for the positive trans-acting regulator of the nhaA gene (11). Plasmids for expression of each of the five nhaS genes in E. coli were constructed from pGM42 as shown in Fig. 1A. (i) The SphI site in pGM42 was deleted to yield plasmid pGM42A by digestion with SphI and NruI, blunting with a DNA blunting kit (Takara Shuzo Co. Ltd., Tokyo, Japan), and self-ligation. (ii) A 6.7-kbp fragment that contained the nhaAp promoter, the nhaR gene, and the pBR322 backbone was amplified by PCR with pGM42A as template, the forward primer P1 (CGTCCATCAGTTTGAgAGctCGGTTTACCG, corresponding to nucleotides +1153 to +1182, counted from the site of initiation of translation of the nhaA gene, which was designated +1), and the reverse primer P2 (GATGCAGATGTTgCAtgcTTTATTTCTCTTTCAGG, complementary to nucleotides +13 to 19). (Italicized portions represent restriction sites for SphI [GCATGC] and SacI [GAGCTC]; nucleotides that differ from the ones in the template are lowercased.) The nhaA gene was also amplified with pGM42A as template, the forward primer A1 (CCTGAAAGAGAAATAAAgcaTGcAACATCTGCATC, corresponding to nucleotides 19 to +13, counted from the site of initiation of translation of the nhaA gene, which was designated +1), and the reverse primer A2 (CGGTAAACCGagCTcTCAAACTGATGGACG, complementary to nucleotides +1182 to +1153). (iii) The 6.7-kbp fragment and the nhaA gene were digested with SphI and SacI and ligated to yield plasmid pGM42B. (iv) A 2.2-kbp StuI-StuI fragment of pGM42A was replaced by the corresponding part of pGM42B. The resultant plasmid, designated pRnhaA, was identical to pGM42A except that it contained an SphI site at the site of initiation of translation of the nhaA gene (nucleotides 2 to +4) and a SacI site just downstream of the nhaA gene (nucleotides +1168 to +1173). (v) The various nhaS genes were amplified with the chromosomal DNA isolated from Synechocystis as template and the following synthetic oligonucleotides as primers: forward primer CAgCaTGcATACAGCGGTCAACGA (corresponding to nucleotides 4 to +20, counted from the site of initiation of translation of the nhaS1 gene, which was designated +1) and reverse primer aagagctcCTAGGATGGTTCGGCCACAT (complementary to nucleotides +1584 to +1565) for the nhaS1 gene, forward primer CTgCATGcCTTAAGCTCCCTGTGC (corresponding to nucleotides 4 to +19, counted from the site of initiation of translation of the nhaS2 gene, which was designated +1) and reverse primer TTgAGcTCGTCAGTCATCCTGCAGG (complementary to nucleotides +1632 to +1608) for the nhaS2 gene, forward primer ttgcATGcTTATGAACCCATTGCTCCCTC (corresponding to nucleotides +1 to +25, counted from the site of initiation of translation of the nhaS3 gene, which was designated +1) and reverse primer ttgagctcCTAATCTGGGGTGGGAACTG (complementary to nucleotides +1386 to +1367) for the nhaS3 gene, forward primer AAgcATGcACACCAATACTTTACTGCTAATT (corresponding to nucleotides 4 to +27, counted from the site of initiation of translation of the nhaS4 gene, which was designated +1) and reverse primer ttgaGcTcTTAATGGGCTGGGGCAGGAT (complementary to nucleotides +1237 to +1214) for the nhaS4 gene, and forward primer ttgcATGcATGGCCTATTCGCACCAATTC (corresponding to nucleotides +1 to +25, counted from the site of initiation of translation of the nhaS5 gene, which was designated +1) and reverse primer aagagctcCTAGGCGTAGGGATCGCCA (complementary to nucleotides +2097 to +2079) for the nhaS5 gene. (vi) The nhaA gene in pRnhaA was removed by digestion with SphI and SacI, and an amplified nhaS gene was inserted. The resultant plasmids were designated pRnhaS1, pRnhaS2, pRnhaS3, pRnhaS4, and pRnhaS5. (vii) To generate another set of plasmids that did not contain the nhaR gene, the plasmids pRnhaS1, pRnhaS2, pRnhaS3, pRnhaS4, pRnhaS5, and pRnhaA were further digested with Ecl136II (an isozyme of SacI) and PshA1 and self-ligated. The resultant plasmids were designated pnhaS1, pnhaS2, pnhaS4, pnhaS5, and pnhaA. We failed to generate pnhaS3. All the amplified fragments and the ligated junctions were verified by determination of nucleotide sequences.

View larger version (50K): [in this window] [in a new window]   FIG. 1.   Plasmids used for expression of nhaS genes in E. coli TO114. (A) Construction of vector plasmids. Solid lines represent the pBR322 backbone and the flanking regions of the nhaA and nhaR genes of E. coli. P, Na+-inducible promoter of the nhaA gene (nhaAp). Restriction sites: Nr, NruI; Sp, SphI; St, StuI; Sc, SacI; Ph, PshAI; Ec, Ecl136II. For details, see Materials and Methods. (B) Plasmid characteristics. Plasmids pnhaS1, pnhaS2, pRnhaS3, and pnhaS4 were successfully introduced into TO114 cells (indicated by the absence of shading).

Isolation of RNA. E. coli cells were grown in LBK medium to the early exponential phase of growth (OD₆₀₀, 0.4). Aliquots of the culture were withdrawn, mixed immediately with an equal volume of ice-cold ethanol that contained 5% (wt/vol) phenol, and centrifuged at 3,000 × g for 10 min. Each pellet was washed with 50 mM Tris-HCl (pH 8.0) and 100 mM EDTA and then resuspended in 600 µl of 50 mM Tris-HCl (pH 8.0)-5 mM EDTA-0.25% sodium dodecyl sulfate (SDS). The suspension was mixed with 600 µl of acid phenol (a mixture of 50% phenol, 48% chloroform, and 2% isoamyl alcohol [vol/vol], buffered with an equal volume of 50 mM sodium acetate, pH 5.2), and the mixture was incubated at 65°C for 5 min to disrupt the cells. Total nucleic acids were extracted three times with acid phenol and precipitated in ethanol. Total RNA was separated from DNA by precipitation twice in LiCl and stored at 80°C.

DNA probes. The DNA fragments used for the preparation of probes for Northern blotting analysis were generated by excision from the nhaS genes that had been amplified by PCR as described above: nhaS1 (with HincII, nucleotides +13 to +648), nhaS2 (with SphI and NcoI, nucleotides +1 to +545), nhaS3 (with SphI and EcoRI, nucleotides +1 to +652), and nhaS4 (with SphI and BstEII, nucleotides +1 to +576). The resultant DNA fragments were labeled with [-³²P]dCTP using a BcaBEST labeling kit (Takara Shuzo).

Northern blotting. Fifteen micrograms of total RNA was fractionated by electrophoresis on a 1.2% agarose gel that contained 6.3% formaldehyde in 3-(N-morpholino)propanesulfonic acid buffer, pH 7.0 (39), and bands of RNA were transferred to a nylon membrane (NEN Life Science Products, Boston, Mass.). The membrane was baked at 80°C for 2 h and then incubated for 2 h at 65°C in a solution of 0.5 M sodium phosphate buffer (pH 7.2), 5% SDS, 5× Denhardt's reagent (39), and 100 µg of denatured salmon sperm DNA ml¹. Then the DNA probe was added (2 × 10⁵ cpm ml¹), and hybridization was allowed to proceed for 16 h at 65°C. After a 1-h wash at 55°C in a solution of 0.05 M sodium phosphate buffer (pH 7.2) and 0.5% SDS, the membrane was exposed to an X-ray film (Eastman Kodak Company, Rochester, N.Y.).

Measurement of Na⁺/H⁺ antiport activities of IMVs. Cells were grown in LBK medium to the middle of the exponential phase of growth (OD₆₀₀, 1.5). Inverted membrane vesicles (IMVs) were prepared with a French pressure cell (SLM Instruments, Inc., Urbana, Ill.) as described previously (37). The Na⁺/H⁺ antiport activities of IMVs were estimated from the extent of the collapse of a preformed proton gradient, with acridine orange as the pH indicator, essentially as described previously (14). The assay solution consisted of 140 mM choline chloride, 5 mM MgCl₂, 1 µM acridine orange, and 10 mM Tris titrated with 2-(N-morpholino)ethanesulfonic acid (MES; pH 8.5). In some cases choline chloride was replaced by 140 mM KCl. An aliquot corresponding to 20 µg of vesicle protein was added to 2 ml of the assay solution that was being stirred in a cuvette. Fluorescence from acridine orange was monitored in a fluorometer (model RF-5000; Shimadzu, Kyoto, Japan). The wavelength of excitation light was 495 nm, and fluorescence was monitored at 530 nm. Addition of Tris-D-lactate to a final concentration of 2 mM energized the IMVs and resulted in quenching of the fluorescence. Subsequent addition of NaCl or LiCl resulted in restoration of fluorescence. The initial rate of this restoration, as measured during the 2-s interval that followed the addition of NaCl or LiCl at various concentrations was taken as the Na⁺/H⁺ antiport activity, which was expressed in arbitrary units (fluorescence units s¹ mg of protein¹). IMVs from pBR322⁺ cells (negative control) had low Na⁺/H⁺ antiport activity, which was taken as the background activity. For calculations of kinetic parameters, the Na⁺/H⁺ antiport activity of IMVs prepared from pBR322⁺ cells was subtracted from the activity of IMVs prepared from nhaA⁺, nhaS1⁺, and nhaS3⁺ cells.

Evaluation of the sensitivity of cell growth to salt stress. Transformed cells that had been grown in LBK medium were spread on plates prepared with 1.0% tryptone, 0.5% yeast extract, and 1.5% agar (Difco; LBn solid medium) that had been supplemented with various concentrations of NaCl or LiCl, in addition to KCl, for evaluation of the sensitivity of cell growth to high concentrations of Na⁺ and Li⁺ ions. For evaluation of the sensitivity of cell growth to depletion of K⁺ ions, we used plates of LBn solid medium that had been supplemented with various concentrations of KCl. LBn solid medium by itself contained 20 mM Na⁺ ions and 5 mM K⁺ ions. Formation of colonies was examined after incubation for 24 h at 37°C.

Targeted mutagenesis of the nhaS genes in Synechocystis. Plasmid pAM1573, which contained a chloramphenicol resistance (Cm^r) gene cartridge, and plasmid pAM1303, which contained a spectinomycin resistance (Sp^r) gene cartridge, were kindly provided by S. S. Golden (Texas A&M University, College Station, Tex.). The nhaS genes that had been amplified by PCR, as described above, were subcloned into the TA cloning site of plasmid pT7Blue (Novagen, Madison, Wis.). For construction of a plasmid with a disrupted nhaS1 gene, the region between the BbsI and StuI sites of the nhaS1 gene in pT7Blue was removed and the ends of the cleaved plasmid were blunted with the DNA blunting kit. The cleaved and blunted plasmid was ligated with a kanamycin resistance (Km^r) gene cartridge, which had been excised by SmaI from plasmid pUC-KIXX (Pharmacia, Uppsala, Sweden). The resultant plasmid was designated pnhaS1::Km^r.

Concentrations of proteins in IMVs. The concentrations of proteins in IMVs were determined as described elsewhere (9).

	RESULTS

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Expression of nhaS genes in E. coli TO114. We transformed E. coli TO114 cells with plasmids that contained individual nhaS genes (Fig. 1B). The resultant transformed cells were grown on solid LBK medium supplemented with 50 µg of ampicillin ml¹. We obtained colonies only when cells had been transformed with pnhaS1, pnhaS2, pRnhaS3, or pnhaS4. Transformation with plasmids pRnhaS1, pRnhaS2, pRnhaS4, pRnhaS5, and pnhaS5 failed to yield colonies under our selection conditions. Thus, transformation of cells with the nhaS5 gene was unsuccessful. We also obtained TO114 cells that harbored pBR322, pRnhaA, or pnhaA.

View larger version (100K): [in this window] [in a new window]   FIG. 2.   Northern blotting analysis of the expression of nhaS genes in transformed E. coli TO114 cells. Total RNA was extracted from cells that had been grown in LBK medium. Results are shown for nhaS1 transcripts in nhaS1+ cells (lane 1), nhaS2 transcripts in nhaS2+ cells (lane 2), nhaS3 transcripts in nhaS3+ cells (lane 3), and nhaS4 transcripts in nhaS4+ cells (lane 4). The positions of the expected transcripts are indicated (arrow). The lower panels show bands that correspond to 16S and 23S rRNAs on each gel, as revealed after staining with ethidium bromide prior to blotting. Three independent experiments yielded essentially the same results.

Na⁺/H⁺ antiport activities of IMVs. We measured the Na⁺/H⁺ antiport activity of IMVs prepared from transformed cells as the Na⁺-mediated and Li⁺-mediated net efflux of protons, which we monitored by observing changes in the fluorescence of acridine orange. Since IMVs from pnhaA/TO114 and pRnhaA/TO114 cells had almost the same Na⁺/H⁺ antiport activity (data not shown), we used pRnhaA/TO114 cells in further experiments as the positive control, referring to them as nhaA⁺ cells. We used pBR322/TO114 (pBR322⁺) cells as the negative control.

View larger version (12K): [in this window] [in a new window]   FIG. 3.   Activities of the Na+/H+ antiport system in IMVs prepared from transformed cells. IMVs were prepared from cells that had been grown in LBK medium. Activity was assayed in a solution that consisted of 5 mM MgCl2, 1 µM acridine orange, and 10 mM Tris titrated with MES (pH 8.5) and supplemented with 140 mM choline chloride (A) or 140 mM KCl (B). Arrowheads, time at which 5 mM NaCl was added to the assay solution.

View larger version (10K): [in this window] [in a new window]   FIG. 4.   Activities of the Li+/H+ antiport system in IMVs prepared from transformed cells. Experiments were carried out as described in the legend to Fig. 3 except that 5 mM LiCl was added to the assay solution instead of 5 mM NaCl.

Sensitivity of cell growth to high concentrations of Na⁺ and Li⁺ ions. Table 2 shows the maximum concentrations of Na⁺ and Li⁺ ions that allowed growth of transformed cells on solid LBn medium prepared with 5, 25, 105, or 305 mM K⁺ ions. In the presence of 105 mM K⁺ ions, growth of pBR322⁺ cells was inhibited at 120 mM Na⁺ ions and at 3 mM Li⁺ ions, and it was completely arrested at 170 mM Na⁺ ions and at 5 mM Li⁺ ions. In contrast, nhaS1⁺ and nhaS3⁺ cells were able to grow at 570 mM Na⁺ ions and 10 mM Li⁺ ions and at 420 mM Na⁺ ions and 70 mM Li⁺ ions, respectively, in the presence of 105 mM K⁺ ions. These results were consistent with the restored Na⁺/H⁺ antiport activity in the membranes isolated from the respective cell lines. nhaS1⁺ and nhaS3⁺ cells retained their high tolerance to Na⁺ and Li⁺ ions when the concentration of K⁺ ions was decreased to 5 mM.

Sensitivity of cell growth to depletion of K⁺ ions. pBR322⁺ cells did not grow in the presence of 5 mM K⁺ ions, the background level, even in the absence of additional Na⁺ and Li⁺ ions (Table 2), an observation that was consistent with previous reports on a nhaAnhaB strain of E. coli (16, 47) and was probably due to the inability of these cells to maintain intracellular concentrations of Na⁺ ions at an appropriate level when the ratio of K⁺ ions to Na⁺ ions in the medium was low (16). To elucidate the effect of transformation on the sensitivity to depletion of K⁺ ions, we examined the growth of transformed cells at various concentrations of K⁺ ions. pBR322⁺ cells required at least 20 mM K⁺ ions; nhaS1⁺ and nhaS3⁺ cells grew at 5 mM K⁺ ions, the background level, as did nhaA⁺ cells. nhaS4⁺ cells also exhibited a lower requirement for K⁺ ions (6 mM) than that of pBR322⁺ cells. In contrast, the requirement of nhaS2⁺ cells for K⁺ ions did not differ significantly from that of pBR322⁺ cells.

Disruption of nhaS genes in Synechocystis. We created single and double mutants of Synechocystis in which individual nhaS genes were disrupted by insertion of an antibiotic resistance gene cartridge. We verified the disruption of the nhaS1, nhaS2, nhaS4, and nhaS5 genes on all copies of the chromosomal DNA by PCR. We failed to disrupt the nhaS3 gene under any conditions tested. In our efforts to disrupt the nhaS3 gene we used the following media: BG11 medium that contained 18 mM Na⁺ ions (pH 7.5), a low-sodium medium in which all the sodium salts of BG11 medium had been replaced by potassium salts (this medium was estimated to contain 50 µM Na⁺ ions from the extent of contamination by Na⁺ ions of the potassium salts [Wako Pure Chemical Industries, Ltd., Oaska, Japan] that we used), and media prepared by adding different concentrations of NaCl (100 µM to 100 mM) to the low-salt medium. The single mutants that we did obtain did not show any phenotypic changes in terms of sensitivity to high concentrations of NaCl (data not shown). nhaS1nhaS2 cells grew more slowly than wild-type cells both in BG11 medium and in a high-salt medium prepared by adding NaCl to 0.5 M to BG11 medium (Fig. 5A). The retardation of growth of nhaS1nhaS2 cells, compared to the growth of wild-type cells, appeared to be greater in the presence of 0.5 M NaCl than in its absence. In contrast, nhaS4nhaS5 cells grew as rapidly as wild-type cells regardless of the presence or absence of 0.5 M NaCl (Fig. 5B).

View larger version (15K): [in this window] [in a new window]   FIG. 5.   Growth curves for Synechocystis in BG11 medium that contained 18 mM Na+ ions (open symbols) or in high-salt medium prepared by increasing the concentration of NaCl in BG11 medium to 0.5 M (closed symbols). (A) Wild-type cells (circles) and mutant cells with disrupted nhaS1 and nhaS2 genes (nhaS1nhaS2) (squares). (B) Wild-type cells (circles) and mutant cells with disrupted nhaS4 and nhaS5 genes (nhaS4nhaS5) (triangles). The results were obtained from three independent determinations for each line of cells.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Homologs of eukaryotic and prokaryotic Na⁺/H⁺ antiporters in Synechocystis. Phylogenetic analysis (Fig. 6A) revealed that NhaS1 and NhaS2 are related to isoforms of NHE found in vertebrates (NHE1 to -6 and NHE) and to NHE-like Na⁺/H⁺ antiporters in plant, fungal, and bacterial cells. NhaS1 and NhaS2 appeared to be most similar to NhaP, an Na⁺/H⁺ antiporter of Pseudomonas aeruginosa (46), and to SOS1, a putative Na⁺/H⁺ antiporter in A. thaliana (40). NhaS3, NhaS4, and NhaS5 resembled NapA, an Na⁺/H⁺ antiporter in Enterococcus hirae (42, 51), as well as KefC, a putative K⁺/H⁺ antiporter in E. coli (27). Genes for homologs of both NHE-like ("eukaryotic") and NapA-like ("prokaryotic") Na⁺/H⁺ antiporters have been found in many eubacteria, archaea, and eukaryotes, suggesting that the two types of Na⁺/H⁺ antiporter might have been selected early in evolution. NHE-like and NapA-like Na⁺/H⁺ antiporters appear to have distinct properties. The isoforms of NHE catalyze the electroneutral exchange of Na⁺ ions for protons, being activated by internal protons (50). It has been proposed that Nhx1 of S. cerevisiae, an NHE-like Na⁺/H⁺ antiporter, might be activated by decreases in cytoplasmic pH (28). The isoforms of NHE have rather high K_m values for Na⁺ ions, which range from 4.7 to 59 mM (32). A high K_m (7 mM) for Na⁺ ions was also reported for vacuoles of A. thaliana that overexpressed the AtNHX1 gene (1). In contrast, it was reported that NapA has a relatively low K_m (1.0 mM) for Na⁺ ions (42). The present study of the expression in E. coli of cyanobacterial genes from Synechocystis provides the first example, to our knowledge, of the functional identification of the two types of Na⁺/H⁺ antiporter in a single organism. A. thaliana has a number of genes for putative NHE-like and NapA-like Na⁺/H⁺ antiporters. They might be localized in different tissues and membranes.

View larger version (56K): [in this window] [in a new window]   FIG. 6.   Relationships of NhaS proteins to other Na+/H+ antiporters and related proteins. (A) Phylogenetic relationships as determined with the CLUSTAL W multiple sequence alignment algorithm (45). (B) Secondary structures predicted from hydropathy profiles, as determined by the algorithm of Kyte and Doolittle (24). Putative transmembrane segments are boxed, and segments that exhibit the strongest homology are shaded. (C) Alignment of amino acid sequences of two strongly homologous segmentsthe putative fifth (V) and sixth (VI) segmentsof NhaS proteins, which correspond, respectively, to the sixth (M5a) and seventh (M5b) segments of NHE1. Identical residues are shaded, and conserved Glu and Asp residues are indicated by dots. NHE1, human P19634; AtNhx1, A. thaliana AAD16946; Nhx1, S. cerevisiae NP  010744; NhaP, P. aeruginosa BAA31695; SOS1, A. thaliana AAF76139; NhaA, NhaB, and KefC, E. coli C64722, G64864, and QQECRD, respectively; NapA, Enterococcus hirae A42111.

Functional expression of the nhaS1, nhaS3, and nhaS4 genes in E. coli under the control of the nhaAp promoter. The nhaS genes were expressed at very low levels in wild-type Synechocystis (unpublished results). This observation suggested that expression of each nhaS gene in E. coli from its own promoter would not result in a sufficient level of product. However, overproduction of proteins that contain several transmembrane segments might be expected to have detrimental effects on host cells. In wild-type E. coli, NhaA is a membrane-bound protein that is present at a low level (less than 0.2% of the total membrane proteins [43]). When this protein was overexpressed under the control of the strongly inducible tac promoter, cell growth ceased (43). Therefore, we chose to use the nhaAp promoter for expression of the various nhaS genes in E. coli at appropriate levels.

NhaS1 and NhaS3 are low-affinity and high-affinity Na⁺/H⁺ antiporters, respectively. The kinetic properties of the Na⁺/H⁺ antiport system in IMVs prepared from nhaS1⁺ cells (Table 1) indicated that the expressed protein, NhaS1, had low affinity for Na⁺ ions (K_m, 7.7 mM) and for Li⁺ ions (K_m, 2.5 mM). The K_m for Na⁺ ions is close to that reported for AtNhx1 of A. thaliana (1). The lower K_m of NhaS1 for Li⁺ ions than for Na⁺ ions suggests that Li⁺ ions might be a better substrate than Na⁺ ions. However, transformation with the nhaS1 gene had only a minimal effect on the tolerance of the host cells to Li⁺ ions, while it dramatically increased the tolerance of host cells to Na⁺ ions (Table 2). This result suggests that the Li⁺/H⁺ antiport activity of NhaS1 might not have any physiological relevance.

Possible roles of NhaS1 and NhaS3 in Synechocystis. The existence of high-affinity and low-affinity Na⁺/H⁺ antiporters in Synechocystis is consistent with the ability of this organism to acclimate to a wide range of extracellular concentrations of Na⁺ ions. The low affinity of NhaS1 for Na⁺ ions suggests that this Na⁺/H⁺ antiporter might be able to function at relatively high concentrations of Na⁺ ions. However, disruption of the nhaS1 gene did not cause any phenotypic changes in the tolerance to high salt, suggesting that other Na⁺/H⁺ antiporters might complement the function of NhaS1. Disruption of both the nhaS1 and the nhaS2 genes resulted in retardation of growth in the standard BG11 medium. Moreover, retardation of the growth of nhaS1nhaS2 cells appeared to be enhanced by high salt. These results suggest that the functions of NhaS1 and NhaS2, homologs of eukaryotic Na⁺/H⁺ antiporters, might complement one another and that both might be involved in the tolerance of Synechocystis to high-salt stress.