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Originally published In Press as doi:10.1074/jbc.M310282200 on October 14, 2003

J. Biol. Chem., Vol. 279, Issue 6, 4330-4338, February 6, 2004
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Isolation and Functional Characterization of Ca2+/H+ Antiporters from Cyanobacteria*

Rungaroon Waditee{ddagger}, Gazi Sakir Hossain§, Yoshito Tanaka§, Tatsunosuke Nakamura¶, Masamitsu Shikata||, Jun Takano||, Tetsuko Takabe**, and Teruhiro Takabe{ddagger}§{ddagger}{ddagger}

From the {ddagger}Research Institute, and §Graduate School of Environmental and Human Sciences, Meijo University, Nagoya, 468-8502, Faculty of Pharmacy, Niigata University of Pharmacy and Applied Life Science, Niigata, 950-2081, ||Shimadzu Co. Nakagyou-ku, Kyoto, 604-8511, and **Graduate School of Agricultural Science, Nagoya University, Chikusa-ku, Nagoya, 464-8601, Japan

Received for publication, September 16, 2003 , and in revised form, October 13, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Modulation of cytosolic Ca2+ levels is essential for adapted physiological responses and is determined by two opposite fluxes, Ca2+ influx via channels and Ca2+ efflux via active transporters (1–3). For Ca2+ efflux, the primary pump Ca2+-ATPase and secondary transporter Ca2+ exchanger are believed to play important roles. When compared with other Ca2+ transporters, few studies have been focused on the molecular mechanisms of H+-coupled Ca2+ antiporter (4).

Ca2+/H+ antiporters (CAXs)1 have been cloned from bacteria, fungi, and plants, most of which are vacuolar CAXs (5–7). 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 (5–7). Information on the molecular properties of CAX has been emerging from the studies on plant CAXs, especially from Arabidopsis (4, 7–15). Four Arabidopsis CAXs (AtCAX1–4) were identified by their ability to sequester Ca2+ into yeast vacuoles in Saccharomyces cerevisiae mutants deleted of the vacuolar Ca2+-ATPase and Ca2+/H+ antiporter (ScVCX1) (7, 9, 12, 14). It was shown that AtCAX1, AtCAX3, and AtCAX4 specifically transport Ca2+, whereas AtCAX2 transports Ca2+, Mn2+, and Cd2+ (7, 9, 14). In these vacuolar type AtCAXs, the presence of an N-terminal autoinhibition domain and a 9-amino-acid region required for Ca2+ transport (Ca2+ domain) has been reported (9–14). By contrast, little is known about H+-coupled Ca2+ efflux antiporters. Hitherto, a plasma membrane Ca2+/H+ 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+/H+ and Ca2+/H+ exchange reactions at alkaline pH (16). Essentially nothing is known about molecular properties of Ca2+/H+ 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 Ca2+/H+ antiporter gene (17–19). Cyanobacteria are oxygen-evolving photosynthetic prokaryotes that can acclimate to a wide range of environmental changes (20, 21). Although the role of Ca2+ for stress responses in prokaryotic cells has not been clearly demonstrated, direct evidence of Ca2+ signaling in cyanobacteria has become available recently (22). Therefore, it was interesting to characterize the putative Ca2+/H+ 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 Ca2+, but not of Na+. The exchange activity between Ca2+ and H+ is essential for salt tolerance at alkaline pH in which the acidic amino acid residues in TM segments are involved.


   MATERIALS AND METHODS
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 described previously (23, 24). Synechocystis sp. PCC 6803 and Synechococcus sp. 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. The cells with the disrupted synCAX gene were grown in the same conditions as the wild type except for supplementation with chloramphenicol (150 µg·ml-1). E. coli TO114 cells were grown at 37 °C in LBK medium or Tris-E medium (25). Ampicillin, erythromycin, kanamycin, and chloramphenical were used at final concentrations of 50, 160, 30, and 30 µg ml-1, respectively.

Construction of Expression Vectors for ApCAX and SynCAX—The apCAX gene was amplified from genomic DNA of A. halophytica by the primer set, ApCa/H-F1 and ApCa/H-R1. The sequences of all the primers are shown in 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 pBSK+ (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 C-terminal, were transformed first to E. coli DH5{alpha} 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+/H+ antiporter from Synechocystis sp. PCC 6803 (SynNhaP1) was described previously (27).


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  		TABLE I
Primers used for isolation and expression of Ca2+/H+ antiporter genes

 
For the construction of an expression vector for ApCAX in cyanobacteria, the promoter region of ApCAX ~400 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 BamHI-digested 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 pBSK+ 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.

Ca2+/H+ Antiporter Activity—TO114 cells were grown in LBK medium containing ampicillin, erythromycin, kanamycin, and chloramphenical. When the A620 reached 0.6–0.8, 0.5 mM isopropyl-{beta}-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 antiporter activity was assayed based upon the establishment of {Delta}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 µM acridine orange, and membrane vesicles (50–75 µg of protein) in a volume of 2 ml. The {Delta}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+ internally, causing the accumulation of dye whose fluorescence was thereby quenched. Upon the addition of salt, fluorescence increased due to the excretion of H+ by the antiporters and consequent leakage of the dye. The changes in fluorescence ({Delta}Q) were monitored. Then, NH4Cl was added to dissipate the {Delta}pH.

Disruption of synCAX—The synCAX in Synechocystis sp. PCC6803 was disrupted by insertion of the chloramphenical resistance gene (cmlr). The DNA fragment, ~0.8 kbp, covering cmlr 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 pBSK+ and then digested with HincII and SmaI of pBSK+. The blunt-ended fragment of cmlr was ligated into the NcoI site at position 543 bp of synCAX in pSynCAXSK+, which was prepared by partial digestion with NcoI and blunting. Insertion of the cmlr cassette into the correct site was confirmed by DNA sequencing. The cmlr-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 µg/ml.

Detection of Ca2+ Efflux and Membrane Potential—To prepare the Ca2+-loading cells, both the wild type and disrupted cells were incubated for 1 h with 50 mM Ca2+ in buffer A (20 mM HEPES and 140 mM KCl) at various pH values (30, 31). Ca2+-loaded cells were diluted 10-fold in buffer A and then transferred to the assay medium containing Ca2+ indicator (2 ml) (20 mM HEPES, pH 7.2, 3 mM MgSO4, 27 µM arsenazo III) (31). Ca2+ 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-C3-(5) (31). The assay medium (1 ml) had the same composition as that used for the detection of Ca2+ efflux, but it was supplemented with 1 µM diS-C3-(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 His6 (His6-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).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning of Putative Ca2+/H+ 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- 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 N-terminal 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).



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  		FIG. 1.
Comparison of the deduced amino acid sequences of Ca+/H+ antiporters. A, alignment of the deduced amino acid sequences of Ca+/H+ 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 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 Ca2+/H+ 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 GenBankTM D90912 [GenBank] from Synechocystis sp. PCC 6803 (SynCAX), U36603 [GenBank] from S. cerevisiae vacuolar antiporter (ScVCX1), AB012932 [GenBank] from V. radiata (VrCAX), AF053229 [GenBank] from N. crassa (NcCAX), AF461691 [GenBank] from A. thaliana (AtCAX1), and L28709 [GenBank] from E. coli (ChaA).

 
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).



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  		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 His6-His tag.

 
Complementation Test in E. coli TO114 —It has been reported that plant vacuolar CAXs such as AtCAX1–4 could not complement the Ca2+-sensitive phenotype of a yeast vacuolar mutant deleted of the vacuolar Ca2+-ATPase and Ca2+/H+ 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, 12–14). 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 CaCl2 (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 CaCl2, 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 Ca2+-sensitive phenotype of E. coli mutant.



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  		FIG. 3.
Effects of CaCl2, NaCl, and LiCl on the growth rates of E. coli cells expressing ApCAX, SynCAX, and SynNhaP1. A, effects of CaCl2. B, effects of NaCl. C, effects of LiCl. The control TO114 cells and transformant TO114 cells expressing ApCAX, SynCAX, and SynNhaP1 at logarithmic phase were transferred to the agar plates containing Tris-E medium (pH 8.0) and 0.1 mM CaCl2 (A), LBK medium (pH 7.0) and 0.2 M NaCl (B), and LBK medium (pH 8.0) and 4 mM LiCl (C). Photographs were taken after 2 days. 10-fold different concentrations of cells (x1 and x1/10) were tested. As shown in D, the control and transformant TO114 cells at logarithmic phase in Tris-E medium were transferred to the Tris-E medium containing 0.1 mM CaCl2 at pH 8.0. Each value shows the average of three independent measurements.

 
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 Ca2+/H+ exchange but not Na+/H+ or Li+/H+ exchange reactions.

Ca2+/H+ Antiporter Activity—Next, as shown in Fig. 4, we examined exchange activities using everted membrane vesicles of TO114 (26, 27). Exchange activity between Ca2+ and H+ 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+ on Ca2+ binding and/or transport. Exchange activity between Na+, Li+, or Mg2+ with H+ could not be detected in all conditions tested (data not shown).



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  		FIG. 4.
The activities of CAXs measured by the acridine orange fluorescence quenching method. The control TO114 cells and transformed TO114 cells expressing ApCAX and SynCAX were grown in LBK medium from which the everted membrane vesicles were prepared. The antiporter activity was measured as described under "Materials and Methods." A–D show the Ca2+/H+-antiporter activities at pH 8.8, 8.5, 8.0, and 7.5, respectively. The final concentration of CaCl2 was 1 mM. Each value shows the average of three independent measurements.

 
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+ in the gene-disrupted cells was about 3-fold higher than that of the wild type cells, whereas the cellular K+ contents were similar (Fig. 5, F and G). These results indicate that SynCAX plays an important role for stabilization in pigments at alkaline pH.



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  		FIG. 5.
Effects of synCAX disruption on the cellular contents. The wild type and synCAX-disrupted ({Delta}SynCAX) Synechocystis sp. PCC 6803 cells were grown in BG11 medium at pH 7.5 and 8.8. After 14 days, cellular contents were measured. A, photograph. B, cell number. C, contents of soluble proteins. D, chlorophyll contents. E, contents of phycobiliproteins. PE, phycoerythrin; PB, phycobiliprotein; PC, phycocyanin. F, contents of cellular Na+. G, contents of cellular K+. White bars, wild type cells; black bars, synCAX-disrupted cells. Each value in B–G shows the average of three independent measurements.

 
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.



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  		FIG. 6.
Effects of NaCl on the growth of wild type and synCAX-disrupted 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.

 
Effects of synCAX Disruption on the Efflux Activity—Next, we examined whether the SynCAX could catalyze the efflux of Ca2+ from Synechocystis sp. PCC 6803 cells. After Ca2+ loading, both the wild type and gene-disrupted cells were transferred to a Ca2+-free assay medium containing the Ca2+ indicator arsenazo III as described under "Materials and Methods." Fig. 7A shows that the effluxed Ca2+ 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 Ca2+/H+ antiporter with H+:Ca2+ 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-C3-(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 Ca2+ in exchange for H+ with H+:Ca2+ stoichiometry higher than 2 and diminishes the membrane potentials.



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  		FIG. 7.
Efflux activities and membrane potential of wild type and synCAX-disrupted cells. The wild type and synCAX-disrupted Synechocystis sp. PCC 6803 cells were grown in BG11 medium at pH 8.8. A, efflux activity of Ca2+-loaded wild type and synCAX-disrupted ({Delta}synCAX) cells. B, membrane potential of wild type and synCAX-disrupted ({Delta}synCAX) cells. The same cell number was used in these experiments. Efflux activities and membrane potential of Ca2+-loaded cells were measured as described under "Materials and Methods." In A, each value shows the average of three independent measurements.

 
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 Ca2+/H+ antiporter activity at all pHs tested (Fig. 9A) and could not complement the Ca2+-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 Ca2+/H+ antiporter activity at all pHs (Fig. 9B) and did not complement the Ca2+-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 Ca2+/H+ exchange activity.



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  		FIG. 8.
The schematic secondary structure model of ApCAX. The number of positive and negative charges on the loop regions is indicated. The conserved Glu-74 and Glu-324 in ApCAX are shown.

 



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  		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.

 
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 over-expressing 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.



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  		FIG. 10.
Expression of ApCAX and SynCAX in Synechococcus sp. PCC 7942 cells. A, localization of ApCAX and SynCAX. Freshwater Synechococcus sp. PCC 7942 cells transformed with vector only, ApCAX, and SynCAX were grown in BG11 medium. From these cells, plasma membranes (PM) and thylakoid membranes (TM) were isolated. An equal amount of membrane proteins (50 µg) was used for immunoblotting analysis. B and C, salt tolerance of Synechococcus sp. PCC 7942 cells expressing ApCAX and SynCAX. Synechococcus sp. PCC 7942 cells transformed with vector only, Ap-CAX, and SynCAX were grown in BG11 medium at pH 7.5 (B) or in BG11 medium containing 0.4 M NaCl (C). Each value shows the average of three independent measurements.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We isolated putative Ca2+/H+ 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 Ca2+-tolerant by transformation with the apCAX and synCAX genes (Fig. 3), by the direct observation of Ca2+/H+ antiporter activity in the membrane vesicles of transformants (Fig. 4), by the reduced Ca2+ 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 Ca2+/H+ 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 Ca2+ domain were evolved later.

The results of Figs. 3 and 4 indicate that both full-length ApCAX and full-length SynCAX could complement the Ca2+-sensitive phenotype of TO114 and exhibited the Ca2+/H+ 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 Ca2+/H+ 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 Ca2+ 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 (17–19), which might be more suitable for studying the physiological function of CAX. The role of H+-coupled Ca2+ efflux by CAX in Ca2+ homeostasis in cyanobacteria is an important subject to be clarified.

Hitherto, the N-terminal regulatory domain (1–36 amino acid residues) and the Ca2+ 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, Ca2+ and H+. 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+/Ca2+ 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 and 10 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 Ca2+ via Ca2+ efflux is important. Its inhibition causes the salt-sensitive phenotype. In addition to the efflux of Ca2+, maintaining a neutral pH inside cells would be important for salt tolerance at alkaline pH. Due to the electrogenic properties of the Ca2+/H+ antiporter, overexpression of the Ca2+/H+ 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+/H+ 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+/H+ antiporter into plants has also been reported (40). Further studies on transgenic plants using ApCAX and NhaP Na+/H+ antiporter from A. halophytica would enable us to obtain improved salt-tolerant plants.


   FOOTNOTES
 
The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB120300 [GenBank] .

* This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education and Science and Culture of Japan, the High-Tech Research Center of Meijo University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Research Institute of Meijo University, Tenpaku-ku, Nagoya, Aichi 468-8502, Japan. Tel.: 81-52-832-1151; Fax: 81-52-832-1545; E-mail: takabe{at}ccmfs.meijo-u.ac.jp.

1 The abbreviations used are: CAX, Ca2+/H+ antiporter; ApCAX, CAX from A. halophytica; AtCAX, CAX from Arabidopsis; SynCAX, CAX from Synechocystis sp. PCC 6803; SynNhaP1, Na+/H+ antiporter from Synechocystis PCC 6803; ScVCX1, S. cerevisiae vacuolar antiporter; TM, transmembrane. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Andre T. Jagendorf for critical reading of manuscript. We thank Eiko Tsunekawa for expert technical assistance.



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