Isolation and functional characterization of Ca2+/H+ antiporters from cyanobacteria.

Genome sequences of cyanobacteria, Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and Thermosynechococcus elongatus BP-1 revealed the presence of a single Ca2+/H+ antiporter in these organisms. Here, we isolated the putative Ca2+/H+ antiporter gene from Synechocystis sp. PCC 6803 (synCAX) as well as a homologous gene from a halotolerant cyanobacterium Aphanothece halophytica (apCAX). In contrast to plant vacuolar CAXs, the full-length apCAX and synCAX genes complemented the Ca2+-sensitive phenotype of an Escherichia coli mutant. ApCAX and SynCAX proteins catalyzed specifically the Ca2+/H+ exchange reaction at alkaline pH. Immunological analysis suggested their localization in plasma membranes. The Synechocystis sp. PCC 6803 cells disrupted of synCAX exhibited lower Ca2+ efflux activity and a salt-sensitive phenotype. Overexpression of ApCAX and SynCAX enhanced the salt tolerance of Synechococcus sp. PCC 7942 cells. Mutagenesis analyses indicate the importance of two conserved acidic amino acid residues, Glu-74 and Glu-324, in the transmembrane segments for the exchange activity. These results clearly indicate that cyanobacteria contain a Ca2+/H+ antiporter in their plasma membranes, which plays an important role for salt tolerance.

Modulation of cytosolic Ca 2ϩ levels is essential for adapted physiological responses and is determined by two opposite fluxes, Ca 2ϩ influx via channels and Ca 2ϩ efflux via active transporters (1)(2)(3). For Ca 2ϩ efflux, the primary pump Ca 2ϩ -ATPase and secondary transporter Ca 2ϩ exchanger are believed to play important roles. When compared with other Ca 2ϩ transporters, few studies have been focused on the molecular mechanisms of H ϩ -coupled Ca 2ϩ antiporter (4).
Ca 2ϩ /H ϩ antiporters (CAXs) 1 have been cloned from bacte-ria, fungi, and plants, most of which are vacuolar CAXs (5)(6)(7). CAXs have in general 10 -14 transmembrane (TM)-spanning domain with about 400 amino acid residues (2-7). CAXs contain a central hydrophilic motif rich in acidic amino acid residues that bisect the polypeptide into two approximately equal segments (5)(6)(7). Information on the molecular properties of CAX has been emerging from the studies on plant CAXs, especially from Arabidopsis (4,(7)(8)(9)(10)(11)(12)(13)(14)(15). Four Arabidopsis CAXs (AtCAX1-4) were identified by their ability to sequester Ca 2ϩ into yeast vacuoles in Saccharomyces cerevisiae mutants deleted of the vacuolar Ca 2ϩ -ATPase and Ca 2ϩ /H ϩ antiporter (ScVCX1) (7,9,12,14). It was shown that AtCAX1, AtCAX3, and AtCAX4 specifically transport Ca 2ϩ , whereas AtCAX2 transports Ca 2ϩ , Mn 2ϩ , and Cd 2ϩ (7,9,14). In these vacuolar type AtCAXs, the presence of an N-terminal autoinhibition domain and a 9-amino-acid region required for Ca 2ϩ transport (Ca 2ϩ domain) has been reported (9 -14). By contrast, little is known about H ϩ -coupled Ca 2ϩ efflux antiporters. Hitherto, a plasma membrane Ca 2ϩ /H ϩ antiporter gene (chaA) has only been isolated from Escherichia coli (5). In ChaA, neither an N-terminal autoinhibition domain nor a 9-amino-acid region was reported. ChaA has been shown to catalyze both Na ϩ /H ϩ and Ca 2ϩ /H ϩ exchange reactions at alkaline pH (16). Essentially nothing is known about molecular properties of Ca 2ϩ /H ϩ antiporters from other organisms, especially those on plasma membranes. Recent genome sequences of cyanobacteria, Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and Thermosynechococcus elongatus BP-1 suggest the presence of a single putative Ca 2ϩ /H ϩ antiporter gene (17)(18)(19). Cyanobacteria are oxygenevolving photosynthetic prokaryotes that can acclimate to a wide range of environmental changes (20,21). Although the role of Ca 2ϩ for stress responses in prokaryotic cells has not been clearly demonstrated, direct evidence of Ca 2ϩ signaling in cyanobacteria has become available recently (22). Therefore, it was interesting to characterize the putative Ca 2ϩ /H ϩ antiporter of cyanobacteria. Here, we isolated the CAX genes from Synechocystis sp. PCC 6803 (synCAX) and from a halotolerant cyanobacterium Aphanothece halophytica (apCAX). It is shown that SynCAX and ApCAX are localized on plasma membranes and catalyze the efflux of Ca 2ϩ , but not of Na ϩ . The exchange activity between Ca 2ϩ and H ϩ is essential for salt tolerance at alkaline pH in which the acidic amino acid residues in TM segments are involved.  Table I. The synCAX (slr1336) was amplified from genomic DNA of Synechocystis sp. PCC 6803 by the primer set, SynCa/ H-F1 and SynCa/H-R1. PCR products for apCAX and synCAX, were ligated into the EcoRV restriction site of pBSKϩ (Stratagene, La Jolla, CA) and sequenced. The resulting plasmids were designated as pAp-CAX-SK and pSynCAX-SK, respectively. The plasmid pApCAX-SK was digested with NcoI and BamHI, whereas plasmid pSynCAX-SK was digested with BamHI. The resulting fragments were ligated into the corresponding sites of pTrcHis2C (Invitrogen). The generated plasmids, pApCAX and pSynCAX, fused in-frame to six histidines at the Cterminal, were transformed first to E. coli DH5␣ cells and then to E. coli T0114 cells in which the nhaA, nhaB, and chaA genes were deleted (16,26). The preparation of an expression vector for the Na ϩ /H ϩ antiporter from Synechocystis sp. PCC 6803 (SynNhaP1) was described previously (27).

Strains and
For the construction of an expression vector for ApCAX in cyanobacteria, the promoter region of ApCAX ϳ400 bp was amplified from the genomic DNA of A. halophytica using the primer sets, ApCa/HProF and ApCa/HproR, and ligated into the NcoI site of pApCAX. After checking the orientation of the promoter, the full length of ApCAX containing both the promoter and a His tag was amplified by the primer set, ApCa/HproF and HisBamHI, blunt-ended, and ligated into the BamHIdigested site of the E. coli/Synechococcus shuttle vector, pUC303-Bm (21). The resulting plasmid was used to transform Synechococcus sp. PCC7942 cells (21). Essentially, the same method was used for Syn-CAX. In this case, the promoter region was amplified using the primer set, SynCa/HProF and SynCa/HproR.
Site-directed Mutagenesis of apCAX-Changes of amino acids, Glu-74 and Glu-324, in ApCAX to Asp, Gln, and His were carried out by the PCR method as described previously (28). Briefly, for the case of Glu-74 mutants, two fragments were amplified by the primer sets, ApCa/H-F1 and E74DQH-R and E74DQH-F and ApCa/H-R, using the pApCAX-SK as the template. After removing the primer sets, two PCR-amplified fragments were mixed, heated, and annealed and were then used as the templates for amplification by the primer sets, ApCa/ H-F1 and ApCa/H-R1. PCR products were ligated into the EcoRV restriction site of pBSKϩ and sequenced. The mutants E74D, E74Q, and E74H were transferred to pTrcHis2C and used for the transformation of TO114. The mutants E324D, E324Q, and E324H were constructed essentially by the same method.
Ca 2ϩ /H ϩ Antiporter Activity-TO114 cells were grown in LBK medium containing ampicillin, erythromycin, kanamycin, and chloramphenical. When the A 620 reached 0.6 -0.8, 0.5 mM isopropyl-␤-D-thiogalactopyranoside was added. Cells were allowed to grow for a further 3 h and then harvested by centrifugation. Everted membranes were prepared using a French press as described previously (27,29). The anti-porter activity was assayed based upon the establishment of ⌬pH (TM pH gradient) by the addition of salt to the reaction mixture, which contained 10 mM Tris-Cl (titrated with HCl to the indicated pH), 140 mM choline chloride or 140 mM KCl, 1 M acridine orange, and membrane vesicles (50 -75 g of protein) in a volume of 2 ml. The ⌬pH was monitored with acridine orange fluorescence, which was obtained by excitation at 492 nm and emission at 525 nm. Before the addition of salt, Tris-DL-lactate (2 mM) was added to initiate fluorescence quenching (Q) due to respiration. Lactate energized the vesicles, which then accumulated H ϩ internally, causing the accumulation of dye whose fluorescence was thereby quenched. Upon the addition of salt, fluorescence increased due to the excretion of H ϩ by the antiporters and consequent leakage of the dye. The changes in fluorescence (⌬Q) were monitored. Then, NH 4 Cl was added to dissipate the ⌬pH.
Disruption of synCAX-The synCAX in Synechocystis sp. PCC6803 was disrupted by insertion of the chloramphenical resistance gene (cml r ). The DNA fragment, ϳ0.8 kbp, covering cml r was amplified by the primer set, pACYCcml-F and pACYCcml-R, using pACYC184 (New England Biolabs, Beverly, MA) as a template. The PCR product was subcloned into the EcoRV restriction site of pBSKϩ and then digested with HincII and SmaI of pBSKϩ. The blunt-ended fragment of cml r was ligated into the NcoI site at position 543 bp of synCAX in pSynCAX-SKϩ, which was prepared by partial digestion with NcoI and blunting. Insertion of the cml r cassette into the correct site was confirmed by DNA sequencing. The cml r -containing synCAX was transferred to Synechocystis sp. PCC6803 by electroporation (500 V, 48 ohms, and 125 microfarads) using an Electrocell Manipulator (model 600 M; BTX). The disrupted mutants were selected on BG11 medium containing 0.5% agar supplemented with chloramphenical at a final concentration of 150 g/ml.
Detection of Ca 2ϩ Efflux and Membrane Potential-To prepare the Ca 2ϩ -loading cells, both the wild type and disrupted cells were incubated for 1 h with 50 mM Ca 2ϩ in buffer A (20 mM HEPES and 140 mM KCl) at various pH values (30,31). Ca 2ϩ -loaded cells were diluted 10-fold in buffer A and then transferred to the assay medium containing Ca 2ϩ indicator (2 ml) (20 mM HEPES, pH 7.2, 3 mM MgSO 4 , 27 M arsenazo III) (31). Ca 2ϩ efflux from the Synechocystis sp. PCC 6803 cells was monitored by the absorbance change of arsenazo III with a Hitachi 557 spectrophotometer in double wavelength mode (650 -720 nm). The plasma membrane potential of Synechocystis cells was assayed with the potential-sensitive cyanide dye diS-C 3 -(5) (31). The assay medium (1 ml) had the same composition as that used for the detection of Ca 2ϩ efflux, but it was supplemented with 1 M diS-C 3 -(5). The fluorescence was measured by excitation at 620 nm and emission at 670 nm with a Shimadzu RF-5300PC spectrofluorophotometer.
Other Methods-The nucleotide sequences were determined using an ABI310 genetic analyzer (Applied Biosystems, Foster City, CA). Cellular ions were determined by Shimadzu Personal Ion Analyzer PIA-1000. SDS-PAGE and Western blotting analysis were carried out as described previously (27,28). An antibody raised against His 6 (His 6 -His tag) was obtained from R&D systems (Minneapolis, MN). Protein was determined by Lowry's method as described (27). Chlorophyll a was extracted by 90% methanol in dim light and calculated from the absorbance at 665 nm (21,32). For the preparation of phycobiliproteins, Synechocystis sp. PCC6803 cells were sonicated, streptomycin sulfate (1%) was added, and homogenate was centrifuged (32). Using the resulting supernatant, the phycobiliprotein content was determined spectrophotometrically. Plasma membranes were prepared by a discontinuous sucrose density gradient centrifugation method (33).
Computer Analysis-The hydropathy profile of proteins was predicted by the computer-assisted procedure according to the method of Kyte and Doolittle (34). The possible TM structure of ApCAX was predicted by the computer program TopPredII (35).

Cloning of Putative Ca 2ϩ /H ϩ Antiporter Genes from A. halophytica and Synechocystis Cells-From the shotgun clones of A.
halophytica, one open reading frame homologous to the syn-CAX was found. Its gene was isolated by a PCR method and sequenced as described under "Materials and Methods." The predicted gene product ApCAX consists of 373 amino acids with a molecular mass of 39,710 Da (Fig. 1A). The ClustalW analysis of seven kinds of CAXs (Fig. 1B) showed highest homology to the CAX from Synechocystis sp. PCC 6803 (SynCAX) (69%) and then to vacuolar antiporters from Saccharomyces cerevisiae (ScVCX1) (43%), Neurospora crassa (NcCAX) (40%), Vigna ra- A, alignment of the deduced amino acid sequences of Ca ϩ /H ϩ antiporters from seven organisms. The sequences were aligned by the program ClustalW. The amino acid residues conserved in all sequences are shown by a star, and conservative substitutions are shown by a dot. Predicted membrane-spanning regions are marked above the Ca 2ϩ /H ϩ Antiporter from Cyanobacteria 4332 diata (VrCAX) (39%), and Arabidopsis thaliana (AtCAX1) (38%), and lowest homology to E. coli ChaA (35%). These facts indicate that ApCAX shows high homology to the cyanobacterial antiporters and considerable homology to those from vacuolar CAXs and ChaA. It would be noted that ApCAX showed lower homology to E. coli ChaA than it showed to vacuolar CAXs. It is also evident that vacuolar CAXs have longer Nterminal sequences than those of prokaryotic CAXs (Fig. 1A). Analysis of the hydropathy plot and the TM prediction program predicted 11 putative TM-spanning segments in these CAXs (Fig. 1, A and C). All CAXs contain a central hydrophilic motif, rich in acidic amino acid residues (Fig. 1, A and C).
Construction of Expression Vectors for apCAX and synCAX, and Their Expression in E. coli-Expression vectors for ApCAX and SynCAX were constructed as described under "Materials and Methods" (Fig. 2A). Western blotting analysis indicated that ApCAX and SynCAX could be expressed in E. coli with reasonable size and similar expression levels, which would provide the basis for functional comparison of these CAXs (Fig. 2B).
Complementation Test in E. coli TO114 -It has been reported that plant vacuolar CAXs such as AtCAX1-4 could not complement the Ca 2ϩ -sensitive phenotype of a yeast vacuolar mutant deleted of the vacuolar Ca 2ϩ -ATPase and Ca 2ϩ /H ϩ antiporter (ScVCX1) when expressed at full length (7,9,12,14) but did complement the yeast mutant when expressed with an N-terminal deletion (7,9,(12)(13)(14). In AtCAX1, the autoinhibition domain contains 36 amino acid residues. Therefore, we examined whether ApCAX and SynCAX could complement the salt-sensitive phenotype of E. coli mutant TO114 cells. Due to the absence of the nhaA, nhaB, and chaA genes, TO114 cells could not grow if the medium contained 100 mM CaCl 2 (Fig.  3A), 200 mM NaCl (Fig. 3B), or 4 mM LiCl (Fig. 3C) (16,26,27). However, the TO114 cells expressing ApCAX and SynCAX could grow on solid medium containing 100 mM CaCl 2 , whereas the control cells and SynNhaP1-expressing cells could not grow (Fig. 3A). The growth rates of the cells transformed with ApCAX, SynCAX, and empty vector in the liquid medium were in this order (Fig. 3D). These results indicate that ApCAX and SynCAX complemented the Ca 2ϩ -sensitive phenotype of E. coli mutant. Fig. 3B shows that the cells expressing ApCAX and SynCAX could not grow in the presence of 200 mM NaCl, whereas the cells expressing SynNhaP1 could grow under the same conditions, which is consistent with the results of a previous report (27). Essentially similar results were obtained in the case of LiCl (Fig. 3C). These data indicate that ApCAX and SynCAX catalyze Ca 2ϩ /H ϩ exchange but not Na ϩ /H ϩ or Li ϩ /H ϩ exchange reactions.
Ca 2ϩ /H ϩ Antiporter Activity-Next, as shown in Fig. 4, we examined exchange activities using everted membrane vesicles of TO114 (26,27). Exchange activity between Ca 2ϩ and H ϩ was observed in the cells expressing ApCAX and SynCAX with ApCAX exhibiting higher exchange activity than SynCAX. The pH optimum of exchange activity for ApCAX was 8.8, whereas that of SynCAX was 8.0. These results are consistent with the complementation tests (Fig. 3). When choline was replaced with KCl in the reaction buffer, the exchange activity increased, especially at alkaline pHs, suggesting an effect of K ϩ on Ca 2ϩ binding and/or transport. Exchange activity between alignment. The central hydrophilic motif rich in acidic amino acid residues in AtCAX1 is boxed. The N-terminal autoinhibition domain in AtCAX1 is underlined. B, phylogenetic analysis of seven Ca 2ϩ /H ϩ antiporters. Multiple sequence alignment and generation of a phylogenetic tree were performed with ClustalW and TreeView software, respectively. C, hydropathy plots of ApCAX, SynCAX, AtCAX1, and ChaA. Hydropathy was computed, and the central hydrophilic motif rich in acidic residues is underlined. The accession numbers for CAXs are GenBank TM D90912 from Synechocystis sp. PCC 6803 (SynCAX), U36603 from S. cerevisiae vacuolar antiporter (ScVCX1), AB012932 from V. radiata (VrCAX), AF053229 from N. crassa (NcCAX), AF461691 from A. thaliana (AtCAX1), and L28709 from E. coli (ChaA).

FIG. 2. Expression of ApCAX and SynCAX in E. coli. ApCAX and
SynCAX were expressed in TO114, and everted membranes were prepared. ApCAX and SynCAX were detected with the antibody raised against the His 6 -His tag. Na ϩ , Li ϩ , or Mg 2ϩ with H ϩ could not be detected in all conditions tested (data not shown).
Disruption of synCAX Caused the Rapid Degradation of Pigments at Alkaline pH-To study the physiological function of CAX, the synCAX gene of Synechocystis sp. PCC 6803 was disrupted as described under "Materials and Methods." Complete segregation of synCAX was checked by PCR (data not shown). When the wild type and synCAX-disrupted cells were grown in BG11 medium at pH 7.5, the growth rates and their phenotypes were similar (see Fig. 5, B-G). However, clear evidence of the difference in phenotypes was observed after long term incubation at alkaline pH. Cell cultures with the disrupted gene became pale green after 14 days of incubation, whereas the wild type cells retained their green color (Fig. 5A). Under these conditions, the cell number and total soluble proteins in the wild type and disrupted cells were similar (Fig. 5,   B and C). However, the levels of chlorophyll, phycocyanin, and phycobiliprotein in the disrupted cells were only about 30, 20, and 10% of the wild type cells, respectively (Fig. 5, D and E). The level of cellular Na ϩ in the gene-disrupted cells was about 3-fold higher than that of the wild type cells, whereas the cellular K ϩ contents were similar (Fig. 5, F and G). These results indicate that SynCAX plays an important role for stabilization in pigments at alkaline pH.
Disruption of synCAX Caused Salt-sensitive Phenotype at Alkaline pH-Next, we examined the stress tolerance of synCAX-disrupted Synechocystis sp. PCC 6803 cells. Fig. 6A shows that both the wild type and disrupted cells could grow at pH 7.5 with similar rates when the concentration of NaCl in BG11 medium was as high as 0.3 M. However, when the concentration of NaCl was increased to 0.5 M, only the wild type cells could survive, although their growth rate was reduced. The difference became more clear when the cells were grown at alkaline pH. At pH 8.8, the gene-disrupted cells could not grow in BG11 medium containing 0.3 and 0.5 M NaCl, whereas the wild type cells could grow under these conditions (Fig. 6B). These results clearly indicate that the disruption of synCAX caused a salt-sensitive phenotype at alkaline pH.
Effects of synCAX Disruption on the Efflux Activity-Next, we examined whether the SynCAX could catalyze the efflux of Ca 2ϩ from Synechocystis sp. PCC 6803 cells. After Ca 2ϩ loading, both the wild type and gene-disrupted cells were transferred to a Ca 2ϩ -free assay medium containing the Ca 2ϩ indicator arsenazo III as described under "Materials and Methods." Fig. 7A shows that the effluxed Ca 2ϩ by the wild type cells was higher than that by the gene-disrupted cells. The differences between wild type and disrupted cells were larger at alkaline pH, which is consistent with the results of exchange activities (Fig. 4). We also examined the effects of synCAX disruption on the membrane potential because the electrogenic properties of a plant vacuolar Ca 2ϩ /H ϩ antiporter with H ϩ :Ca 2ϩ stoichiometry of 3 has been reported (36). Membrane potentials of wild type and synCAX-disrupted cells were measured by using the potential-sensitive probe diS-C 3 -(5) (31). Fig. 7B shows that membrane potentials of the gene-disrupted cells were more negative than those of wild type cells, which is consistent with the electrogenic properties of SynCAX. These data support the view point that SynCAX catalyzes the efflux of Ca 2ϩ in exchange for H ϩ with H ϩ :Ca 2ϩ stoichiometry higher than 2 and diminishes the membrane potentials.
Site-directed Mutagenesis of Glu-74 and Glu-324 in ApCAX-The analyses of hydropathy plot and TM prediction program suggest the presence of several conserved amino acid residues in TM segments (Fig. 1A). A topological model of ApCAX is shown in Fig. 8, which indicates that Glu-74 and Glu-324 are the only conserved charged amino acid residues in TM segments. The function of acidic amino acid residues in TM has never been reported in any CAXs. Therefore, we examined the mutation of Glu-74 and Glu-324 to Asn, Gln, and His. It was found that the E74D and E74H mutants did not exhibit the Ca 2ϩ /H ϩ antiporter activity at all pHs tested (Fig.  9A) and could not complement the Ca 2ϩ -sensitive phenotype of TO114 (data not shown), whereas the E74Q mutant exhibited partial activity, 50% of the wild type. The mutants E342D, E324H, and E324Q did not exhibit Ca 2ϩ /H ϩ antiporter activity at all pHs (Fig. 9B) and did not complement the Ca 2ϩ -sensitive phenotype of TO114 (data not shown). These results indicate that not only the negative charges on Glu-74 and Glu-324, but also their side chains, are crucial for the Ca 2ϩ /H ϩ exchange activity.

ApCAX and SynCAX Are Localized in Plasma Membranes and Are Involved in Salt Tolerance of a Freshwater Synechococcus sp. PCC 7942 Cells-To examine the localization of
ApCAX and SynCAX and their potential ability for abiotic tolerance, we overexpressed ApCAX and SynCAX in a freshwater Synechococcus sp. PCC 7942 cells. The plasma membranes and thylakoid membranes were isolated from the overexpressing cells. Separation of these two membranes was confirmed by two methods. One was pigment analysis. If the same amount of membrane proteins was compared, the absorbance at 665 nm due to chlorophyll in plasma membrane fraction was less than 10% of that in thylakoid membrane fraction (data not shown). The other was the SDS-PAGE pattern. The 37-and 42-kDa proteins known as plasma membrane proteins (33) were only observed in our plasma membrane fraction (data not shown). Immunoblotting analysis revealed the cross-reaction band of ApCAX and SynCAX only in the plasma membrane fraction (Fig. 10A). Next, we examined the salt tolerance of transformed cells. As shown in Fig. 10B, both the wild type and transformed cells could grow almost with the same rate in BG11 medium. However, in the BG11 medium containing 0.4 M NaCl, the wild type cells could not grow, whereas the cells expressing ApCAX and SynCAX could grow. These results clearly indicate that the overexpression of ApCAX and SynCAX could confer the salt tolerance to a freshwater cyanobacterium.  6. Effects of NaCl on the growth of wild type and synCAXdisrupted cells. The wild type and synCAX-disrupted Synechocystis sp. PCC 6803 cells at logarithmic phase were transferred to the BG11 medium containing the indicated concentrations of NaCl. Growth rates were monitored by absorbance change at 730 nm. Open symbols, wild type cells; closed symbols, synCAX-disrupted cells. Each value shows the average of three independent measurements.

DISCUSSION
We isolated putative Ca 2ϩ /H ϩ antiporter genes homologous to plant vacuolar ones from a halotolerant cyanobacterium A. halophytica (apCAX) and Synechocystis sp. PCC 6803 (syn-CAX). Based on the findings that the antiporter-deficient E. coli TO114 mutant cells became Ca 2ϩ -tolerant by transformation with the apCAX and synCAX genes (Fig. 3), by the direct observation of Ca 2ϩ /H ϩ antiporter activity in the membrane vesicles of transformants (Fig. 4), by the reduced Ca 2ϩ efflux activity of synCAX-disrupted cells (Fig. 7), and by immunoblotting analysis (Fig. 10), it was concluded that the putative genes, apCAX and synCAX, encode the plasma membrane Ca 2ϩ /H ϩ antiporters. The most striking physiological function of ApCAX and SynCAX is in their roles for salt tolerance (Figs. 6 and 10).
Hydropathy profiles, polypeptide size (about 400 amino acid residues), and hydrophilic acidic motif in the central region are conserved among CAXs from plant, yeast, cyanobacteria, and E. coli (Fig. 1, A and C), suggesting that the transport mechanisms of these CAXs might be similar. The facts that ApCAX and SynCAX are more homologous to the plant CAXs than to E. coli ChaA (Fig. 1B) suggest that plant CAXs were evolved from cyanobacteria CAX and that the N-terminal regulatory domain and the Ca 2ϩ domain were evolved later.
The results of Figs. 3 and 4 indicate that both full-length ApCAX and full-length SynCAX could complement the Ca 2ϩsensitive phenotype of TO114 and exhibited the Ca 2ϩ /H ϩ exchange activity, which is different from that of plant CAXs. Since a stop codon appeared just before the first Met of ApCAX, it is unlikely that the Ca 2ϩ /H ϩ antiporter from A. halophytica encodes a longer polypeptide (data not shown). These facts indicate that ApCAX and SynCAX are both active, at least when expressed in E. coli. Results on the plant vacuolar AtCAX1 indicated that the exchange activity of AtCAX1 is regulated by protein factors (14). From the present data, the regulatory mechanisms of cyanobacterial CAXs are unknown. One possibility is that of regulation by non-protein factors, such as membrane potentials, since the Ca 2ϩ efflux by SynCAX affected the membrane potential (Fig. 7). Unlike Arabidopsis with its 11 putative CAX genes (2), cyanobacteria have only a single CAX gene (17)(18)(19), which might be more suitable for studying the physiological function of CAX. The role of H ϩcoupled Ca 2ϩ efflux by CAX in Ca 2ϩ homeostasis in cyanobacteria is an important subject to be clarified. FIG. 9. The activities of ApCAX mutants. The E. coli TO114 cells expressing ApCAX mutants were grown in LBK medium without the addition of extra salts. From these cells, the everted membrane vesicles were prepared. The antiporter activity was measured as described under "Materials and Methods." Each value shows the average of three independent measurements. Hitherto, the N-terminal regulatory domain (1-36 amino acid residues) and the Ca 2ϩ domain (89 -95 amino acid residues in the loop connecting TM5 and TM6) have been reported in AtCAXs (9 -14). However, these domains seem to be absent in ApCAX and SynCAX, and no information was available previously on the specific amino acid residues in the TM region needed for exchange activity. Here, we have shown that two acidic amino acid residues, Glu-74 and Glu-324, in TM segments (Fig. 8) are essential for the exchange activity (Fig. 9). These acidic amino acid residues might play important roles for binding and/or transport of the cations, Ca 2ϩ and H ϩ . Putative TM segments of Glu-74 and Glu-324 are TM3 and TM10, respectively, and are distributed symmetrically around the central TM segments, TM6 and TM7 (Fig. 8). Recently, it has been proposed that the Na ϩ /Ca 2ϩ exchanger has a two-domain structure, each with five TM segments with opposite membrane topologies (37). How these acidic amino acid residues, Glu-74 and Glu-324, are involved in cation binding and/or transport is an interesting question to be tested.
Figs. 6 and10 indicate that cyanobacteria CAXs are involved in salt tolerance at alkaline pH. This conclusion was obtained from the facts that disruption of synCAX caused a salt-sensitive phenotype at alkaline pH (Fig. 6) and overexpression of ApCAX and SynCAX enhanced the salt tolerance (Fig. 10). A simplistic explanation for these facts is that circulation of Ca 2ϩ via Ca 2ϩ efflux is important. Its inhibition causes the salt-sensitive phenotype. In addition to the efflux of Ca 2ϩ , maintaining a neutral pH inside cells would be important for salt tolerance at alkaline pH. Due to the electrogenic properties of the Ca 2ϩ /H ϩ antiporter, overexpression of the Ca 2ϩ /H ϩ antiporter would confer the salt tolerance to cyanobacteria.
The data of Fig. 3 show that the complementation ability of ApCAX is more effective than that of SynCAX. This finding would suggest an interesting application of ApCAX for the genetic engineering of salt-tolerant plants. Previously, we showed that the DnaK (38) and NhaP Na ϩ /H ϩ antiporter (26) from A. halophytica exhibited unique properties absent in the homologous genes from freshwater cyanobacteria. Transfer of these genes to plants and a cyanobacterium significantly improved their stress tolerance (21,39). Enhanced salt tolerance by introducing the vacuolar Na ϩ /H ϩ antiporter into plants has also been reported (40). Further studies on transgenic plants using ApCAX and NhaP Na ϩ /H ϩ antiporter from A. halophytica would enable us to obtain improved salt-tolerant plants.