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J. Biol. Chem., Vol. 277, Issue 21, 18658-18664, May 24, 2002

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Genes Essential to Sodium-dependent Bicarbonate Transport in Cyanobacteria

FUNCTION AND PHYLOGENETIC ANALYSIS^*

Mari Shibata, Hirokazu Katoh, Masatoshi Sonoda, Hiroshi Ohkawa, Masaya Shimoyama, Hideya Fukuzawa^§, Aaron Kaplan^¶, and Teruo Ogawa

From the  Bioscience Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan, the ^§ Graduate School of Biostudies, Kyoto University, Sakyo, Kyoto 606-8502, Japan, and the ^¶ Department of Plant Sciences, Hebrew University, 91904 Jerusalem, Israel

Received for publication, December 31, 2001, and in revised form, March 14, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The cyanobacterium Synechocystis sp. strain PCC 6803 possesses two CO₂ uptake systems and two HCO transporters. We transformed a mutant impaired in CO₂ uptake and in cmpA-D encoding a HCOtransporter with a transposon inactivation library, and we recovered mutants unable to take up HCO and grow in low CO₂ at pH 9.0. They are all tagged within slr1512 (designated sbtA). We show that SbtA-mediated transport is induced by low CO₂, requires Na⁺, and plays the major role in HCO uptake in Synechocystis. Inactivation of slr1509 (homologous to ntpJ encoding a Na⁺/K⁺-translocating protein) abolished the ability of cells to grow at [Na⁺] higher than 100 mM and severely depressed the activity of the SbtA-mediated HCO transport. We propose that the SbtA-mediated HCO transport is driven by µNa⁺ across the plasma membrane, which is disrupted by inactivating ntpJ. Phylogenetic analyses indicated that two types of sbtA exist in various cyanobacterial strains, all of which possess ntpJ. The sbtA gene is the first one identified as essential to Na⁺-dependent HCO transport in photosynthetic organisms and may play a crucial role in carbon acquisition when CO₂ supply is limited, or in Prochlorococcus strains that do not possess CO₂ uptake systems or Cmp-dependent HCO transport.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Growth of many photosynthetic microorganisms depends on the activity of a CO₂-concentrating mechanism (CCM),¹ which raises the [CO₂] in close proximity to ribulose-1,5-bisphosphate carboxylase/oxygenase and thereby enables efficient CO₂ fixation despite the low affinity of the enzyme for CO₂ (1, 2). In the cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter Synechocystis 6803), the CCM involves active CO₂ uptake and HCO transport. We have recently identified two systems for CO₂ uptake in Synechocystis 6803, one constitutive and the other inducible by low CO₂ (3). As deduced from phylogenetic analysis of proteins encoded by the genes involved, these CO₂ uptake systems are present in various cyanobacteria with the exception of Prochlorococcus marinus (3). The inducible system that depends on NdhD3/NdhF3/CupA shows higher maximal activity and higher affinity for CO₂ than the constitutive, NdhD4/NdhF4/CupB-dependent system. Inactivation of two different genes, one encoding a component of the constitutive system and the other a constituent of the inducible system, abolished CO₂ uptake activity. The double mutants were unable to grow at pH 7.0 under air level of CO₂ (3, 4). In contrast, because the mutants possessed HCO transport capability, they could grow like the wild type (WT) at pH 9.0 in air.

An ABC-type HCO transporter encoded by cmpABCD has been identified in Synechococcus sp. strain PCC 7942 (thereafter Synechococcus 7942) (5). Inactivation of cmp genes in Synechocystis 6803, however, had little effect on the HCO transport activity. This indicated that another HCO transporter, as yet unidentified, plays a central role in HCO uptake. Sodium ions are essential for cyanobacterial growth, particularly at alkaline pH values (6), and they were implicated in HCO uptake (7). These results are consistent with the suggestion that a Na⁺-dependent HCO transporter might be functioning in cyanobacteria (7-10). In this paper we bring evidence that slr1512 (designated sbtA for sodium-bicarbonate transport A) and slr1509 (ntpJ) are essential for Na⁺-dependent HCO transport and that sbtA most likely encodes a novel HCO transporter, the first one identified in photosynthetic organisms. We suggest that SbtA-mediated HCO transport could be driven by the electrochemical gradient of Na⁺ across the plasma membrane, established by NtpJ.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Growth Conditions-- WT and mutant cells of Synechocystis 6803 were grown at 30 °C in BG11 medium (11) containing 20 mM CHES-KOH, pH 9.0, and bubbled with either 3% CO₂ in air (v/v) or air alone. Solid medium contained BG11 buffered at pH 9.0 and was supplemented with 1.5% agar and 5 mM sodium thiosulfate. Continuous illumination was provided by fluorescent lamps (50 µmol of photons m² s¹; 400-700 nm).

Construction and Isolation of Mutants-- B1 is the mutant where several nucleotides within ndhB were replaced, as previously described (12, 13). This mutant does not take up CO₂ but showed normal HCO transport activity. Construction of mutants ndhD3, ndhD4, cmpA, and ntpJ has been described previously (12) and/or deposited in the web site CyanoMutants (www.kazusa.or.jp/cyano/mutants/). Strains bearing multiple mutations were obtained following transformation of given Synechocystis 6803 mutants with the constructs used to generate other single mutants.

A Genomic Priming System (New England Biolabs) was used to mobilize a transposon containing chloramphenicol resistance (Cm^R) gene for random insertion into the DNA of 110 different cosmids, which contained DNA fragments of Synechocystis 6803 previously used for genomic sequencing (14). The B1/cmpA mutant, defective in active CO₂ uptake, was transformed with this transposon inactivation library. Colonies formed on plates containing chloramphenicol, kanamycin, and spectinomycin were transferred to duplicate plates buffered at pH 9.0 containing the same drugs. One plate was placed under 3% CO₂ in air (v/v) and the other in air alone. Mutants growing under 3% CO₂, but not in air, were recovered. The exact position of the Cm^R cassette in the mutant genome was determined as described previously (3).

Measurements of HCO Uptake and O₂ Evolution-- The rate of HCO uptake was measured using H¹⁴CO in an assay buffer (50 mM CHES-KOH for pH 9.0 or TES-KOH for pH 7.0 and 8.0 containing 15 mM NaCl, 0.3 mM MgSO₄, 0.26 mM CaCl₂, and 0.22 mM K₂HPO₄) as previously reported (5). HCO uptake was initiated by the addition of NaH¹⁴CO₃/KHCO₃. The sample was immediately illuminated with white light (400 µmol of photons m²s¹). Uptake was terminated by rapid filtration of the cells onto a glass filter (GF/B, Whatman) by suction, followed by immediate washing of the filter with 5 ml of the assay buffer. Oxygen evolution was measured with an O₂ electrode (Rank Brothers, Cambridge, United Kingdom) on cells suspended in BG11 medium (pH 9.0) containing 15 mM NaCl. Cell suspensions (corresponding to chlorophyll concentration of 10 µg/ml) were illuminated with white light (400 µmol of photons m² s¹), and, when O₂ evolution ceased, NaHCO was added stepwise to attain the final concentrations of 5, 15, 30, 100, and 400 µM, respectively.

RT-PCR Analysis of Expression-- The amount of transcripts was evaluated by the RT-PCR method (15). RNAs were extracted from Synechocystis 6803 cells grown under 3% CO₂ or after 2 and 6 h of bubbling with air, by the method of Aiba et al. (16), treated with RNase-free DNase I (Roche Molecular Biochemicals), and then purified by phenol/chloroform extraction and ethanol precipitation. Reverse transcription reaction was performed using Superscript II (Invitrogen) and reverse primers. The products were amplified by PCR and then analyzed by electrophoresis on 0.8% agarose gel. Primers were designed so that the amplified products would be internal to the coding region of the genes. All the forward primers were designed for the sequences downstream of the translation initiation codon and the reverse primers to obtain the PCR products of about 350 bp. RNaseP gene was used as a control template (17). Reverse transcriptase was omitted from the RT reaction mixture to confirm the absence of contamination of genomic DNA.

Other Methods-- Procedures previously described were used for the measurement of comparative cell growth on agar plates buffered at pH 9.0 (4, 12).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A Gene Involved in a Novel HCO Transport System-- To isolate novel mutants impaired in HCO uptake in Synechocystis 6803 and identify the relevant genes, it was essential to use strains defective in both CO₂ uptake and in the cmp operon that encodes an ABC-type HCO transporter (5). The B1 strain, impaired in ndhB, was selected as a proper host because it is unable to take up CO₂ and does not grow at pH 7.0 under air level of CO₂ (12, 13). On the other hand, this mutant exhibited normal HCO transport activity and could grow like the WT in air at pH 9.0 (12), conditions where inorganic carbon (C_i) is mainly supplied by HCO transport. Inactivation of the cmp operon in the B1 mutant did not change its growth characteristics at pH 9.0, under either high or low levels of CO₂ (data not shown), suggesting that HCO uptake capability was not impaired. We transformed the double mutant B1/cmpA with a transposon-bearing inactivation library (3) and isolated four mutants defective in their ability to grow at pH 9.0 under air level of CO₂ and unable to take up HCO. All these mutants (NB-3, -9, -10, and -48) had Cm^R cassettes at various sites within a single gene, slr1512 (designated sbtA, Fig. 1A). WT Synechocystis 6803 and the ndhD3, ndhD4, ndhD3/ndhD4 (hereafter ndhD3/D4), and ndhD3/D4/cmpA mutants were transformed with the genomic DNA from strain NB-3 to interrupt their sbtA. As shown in Fig. 1B, all the mutants obtained, with the exception of ndhD3/D4/sbtA and ndhD3/D4/cmpA/sbtA (data not shown), grew like the WT at pH 9.0 in air and in 3% CO₂. Inactivation of sbtA and/or cmpA in WT cells had no effect on their growth (data not shown), presumably because the mutants were able to take up sufficient CO₂ to support their growth. Similarly, disruption of sbtA in the single ndhD3 or ndhD4 mutants, which are able to take up CO₂ either by the constitutive or by the inducible systems (3, 4, 12), had no effect on their growth (Fig. 1B, upper panel). It is most likely that the ability to take up HCO enabled growth of the ndhD3/D4 mutant at alkaline pH and air level of CO₂. However, inactivation of sbtA in this double mutant resulted in the loss of its ability to grow under low CO₂ even at pH 9 (Fig. 1B). These results suggested that the gene product of sbtA is involved in HCO transport and that its activity could support growth of the ndhD3/D4 mutant, particularly at pH 9.0. In contrast to the ndhD3/D4/sbtA mutant, inactivation of cmpA in the ndhD3/D4 strain scarcely affected its growth (Fig. 1B). These results indicated that the contribution of the Cmp-dependent HCO transport to the growth of Synechocystis 6803 is negligible. All mutants examined, with the exception of ndhD3/D4/sbtA (Fig. 1B, lower panel) and ndhD3/D4/sbtA/cmpA (data not shown) grew like the WT on agar plates under 3% CO₂. The latter mutants could grow like the WT in liquid medium at pH 9.0 in 3% CO₂ in air (v/v) but not in air alone (Fig. 1C).

View larger version (21K): [in this window] [in a new window]   Fig. 1.   The structure of the sbtA (slr1512) region and the CmR cassette tags and HygR cassette interrupting the genes (A), the growth of WT and mutants on agar plates (B), and the growth of WT and ndhD3/D4/cmpA/sbtA mutant in liquid (C) at pH 9. 0 under air or air enriched with 3% CO2 (v/v). A, the positions of the CmR cassette in sbtA are 109, 155, 441, and 1056 base pairs downstream of the initiation codon of sbtA for NB-10, NB-48, NB-3, and NB-9, respectively. A fragment between 98 and 142 base pairs downstream of the initiation codon of slr1513 was replaced with a hygromycin resistance cassette (HygR). The horizontal arrows indicate the direction of the cassettes. B, 2 µl of cell suspensions with densities corresponding to OD730 nm values of 0.1 (upper rows of panels in B), 0.01 (middle rows), and 0.001 (lower rows) were spotted on agar plates containing medium BG11 buffered at pH 9.0. The plates were incubated under 3% CO2 in air (v/v) or air alone for 5 days at 50 µmol of photons m2s1. C, the growth of WT (triangles) and ndhD3/D4/cmpA/sbtA mutant (circles) in BG11 (pH 9.0) under 3% CO2 in air (v/v) (H, open symbols) or air (L, closed symbols).

Inactivation of the slr1513 gene, located downstream of sbtA (Fig. 1A), within the ndhD3/D4 mutant had no effect on growth performance (data not shown). This result ruled out a possible pleiotropic effect because of interruption of sbtA. The possibility that sbtA encodes a novel HCO transporter was examined further by measuring the activity of HCO transport and the expression of sbtA in the WT and the mutants (Figs. 2 and 3).

View larger version (16K): [in this window] [in a new window]   Fig. 2.   The uptake of HCO by the WT and various mutants (A) and by the ndhD3/D4/cmpA mutant (B). Unless otherwise stated, cells grown at 3% CO2 in air (v/v) were aerated with air overnight and were suspended in the assay buffer of pH 9 containing 15 mM NaCl and 400 µM HCO. Cells were suspended in the assay buffer of pH 8 and 7 for columns j and k, respectively, and in the assay buffer of pH 9.0 in which NaCl was replaced with KCl for column g. H-cells were used for column h. Cells were incubated for 15 s either in light (columns a-k) or in darkness (columns c'-e'). Vertical bars indicate standard deviations (n = 5).

View larger version (53K): [in this window] [in a new window]   Fig. 3.   The transcript levels of sbtA and cmpA in the WT (lanes a-c) and ndhD3/D4 mutant (lanes d-f). Transcript abundance in H-cells (lanes a and d) or H-cells adapted to air for 2 (lanes b and e) and 6 h (lanes c and f) was determined by the RT-PCR method (15). The transcript levels of RNase P (17) in each sample are shown as a control. The absence of contamination of DNA was confirmed by PCR without reverse transcriptase reaction.

A Low CO₂-inducible, Na⁺-dependent HCO Transport Is Mediated by SbtA-- Fig. 2A shows the amounts of HCO taken up by WT and various mutants during a 15-s incubation with 400 µM HCO. There was no significant difference between the amounts of HCO taken up by the WT and by mutants ndhD3/D4, ndhD3/D4/cmpA (columns a, b, and c). HCO uptake by ndhD3/D4/cmpA was about 6 times higher in light than in darkness (columns c and c'). Inactivation of sbtA in ndhD3/D4 severely depressed the rate of HCO uptake (columns d and d'); disruption of cmpA in ndhD3/D4/sbtA reduced the HCO transport activity somewhat further (columns e and e'). The low level of HCO uptake observed in ndhD3/D4/sbtA/cmpA most likely reflected nonspecific adherence of ¹⁴C_i to the cells because light did not stimulate this apparent uptake. These data indicated that the SbtA-mediated system plays the major role in HCO uptake in Synechocystis 6803 and that the contribution of the Cmp-mediated HCO transport was very small. This is in agreement with the ability of ndhD3/D4/cmpA, but not ndhD3/D4/sbtA and ndhD3/D4/cmpA/sbtA, to grow under low [CO₂] (Fig. 1B).

A small amount of transcript originating from sbtA was detected in the WT and ndhD3/D4 mutant cells of Synechocystis 6803 grown under 3% CO₂ (H-cells; Fig. 3, lanes a and d for sbtA), but the transcript abundance increased significantly within 2-6 h of exposure to air level of CO₂ (lanes b, c, e, and f for sbtA). These data indicated that expression of sbtA was induced by low CO₂ in the WT and ndhD3/D4 mutant, in agreement with the large rise in HCO transport activity in cells acclimated to air level of CO₂ (Fig. 2, columns c, f, and i for L-cells versus column h for H-cells). A transcript of cmpA was not detectable in H-cells of the WT and ndhD3/D4 mutant (Fig. 3, lanes a and d for cmpA) but was detected in the WT cells acclimated to air for 6 h (lane c for cmpA). The cmpA transcript was barely detectable in the mutant even after 6 h of acclimation to air (lane f for cmpA). This may explain the very low activity of the Cmp-dependent HCO transport in the ndhD3/D4/sbtA mutant (Fig. 2, column d).

The SbtA-dependent HCO uptake was strongly affected by the ambient pH level. At pH levels 8.0 (Fig. 2, column j) and 7.0 (column k), HCO uptake was approximately 50 and 20%, respectively, that observed at pH 9.0 (column i). SbtA-mediated HCO transport was almost completely abolished when NaCl in the medium was replaced with KCl (column g), indicating that HCO transport is specifically dependent on the presence of Na⁺ ions. Fig. 4 (A and B) shows the dependence of the SbtA-mediated HCO transport in the ndhD3/D4/cmpA mutant to HCO and Na⁺ concentrations, respectively. Maximal rate of HCO uptake was reached at 100 µM HCO, and the K_1/2(HCO) value was ~16 µM (Fig. 4A, open circles). Photosynthetic O₂ evolution displayed a similar dependence on external [HCO] (closed circles), suggesting that in this mutant photosynthesis was rate-limited by the SbtA-mediated HCO transport. Dependence of the SbtA-mediated HCO transport on ambient [Na⁺] was further supported by the nature of the curve relating HCO uptake to [Na⁺] (Fig. 4B). Maximal HCO uptake was attained at 6 mM Na⁺, and the concentration of Na⁺ essential to support half-maximal HCO transport was ~1 mM (Fig. 4B). These results are in general agreement with an earlier report (10) on the response of HCO uptake in Synechocystis 6803 to the presence of Na⁺. The higher maximal rate of HCO uptake observed before was, most likely, the result of simultaneous uptake of CO₂ in WT where both the constitutive and the inducible CO₂ uptake systems (3) are functional. Furthermore, analysis of CO₂ uptake by mutant cmpA/sbtA (unable to take up HCO; Fig. 4C) showed that it increased linearly with the ambient [HCO] well above the amount of CO₂ that could be produced spontaneously from HCO at pH 9.0 (broken line). These data clearly indicated that conversion of HCO to CO₂ at the cell surface is faster than expected from physicochemical considerations based on the concentration of HCO and pH in the bulk medium. Formation of CO₂ may be catalyzed by a periplasmically located carbonic anhydrase (18) or accelerated by light-dependent proton extrusion that could acidify the periplasmic space (8, 19, 20).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   The uptake of HCO by the ndhD3/D4/cmpA (A and B) and cmpA/sbtA (C) strains as a function of HCO (A and C) and Na+ (B) concentrations. HCO uptake was measured in the medium of pH 9.0 containing 15 mM NaCl for A and C and 15 mM KCl/400 µM HCO for B, and various concentrations of HCO for A and C and NaCl for B. The closed triangles in C indicate the values obtained for the ndhD3/D4/cmpA/sbtA mutant. Vertical bars indicate standard deviations (n = 5). O2 evolution was measured with cells suspended in BG-11 medium buffered at pH 9.0 containing 15 mM NaCl.

NtpJ Is Involved in HCO Transport-- The specific dependence of the SbtA-mediated HCO transport on [Na⁺] (Figs. 2B and 4B) recalls earlier studies (7, 9, 10, 21) where various possibilities were raised to explain the role of Na⁺. If the µNa⁺ across the cytoplasmic membrane is essential for the operation of the SbtA-mediated HCO transport, inactivation of components involved in Na⁺ extrusion (primary Na⁺ or Na⁺/H⁺ pumps) should affect the HCO uptake and growth of a mutant unable to utilize CO₂ such as ndhD3/D4 (Fig. 5). Synechocystis 6803 can grow under a relatively high [NaCl] even exceeding 0.5 M (22). Inactivation of slr1509 (ntpJ), encoding a protein that belongs to a Na⁺-transporter family (motif.genome.ad.jp/), barely affected the growth of Synechocystis 6803 in BG11 medium at pH 9.0 in air, but growth was severely depressed when [NaCl] was raised above 100 mM (Fig. 5A). These results suggested that NtpJ could be involved in Na⁺ extrusion and that failure of the mutant to extrude Na⁺ abolished its growth at elevated [NaCl]. In contrast to the WT, inactivation of ntpJ completely abolished growth of the ndhD3/D4 mutant even in BG11 medium in air (Fig. 5B). On the other hand, under 3% CO₂, the ndhD3/D4/ntpJ mutant grew almost like the WT (Fig. 5B). These results suggested involvement of NtpJ in the supply of C_i for growth. This was confirmed by measuring the uptake of HCO uptake by this mutant (Fig. 5C). The HCO transport activity in the ndhD3/D4/ntpJ mutant was only about one third of that in the ndhD3/D4 mutant (Fig. 5C) and became much lower during longer exposure of the mutant to light. These results support the notion that NtpJ is a subunit of a Na⁺ extrusion pump essential for the SbtA-mediated HCO transport.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   Effect of inactivation of ntpJ in WT and in the ndhD3/D4 mutant on their growth and HCO uptake activity. A, growth rates of the WT and ntpJ strains in BG-11 medium, pH 8.0, containing various concentrations of NaCl under aeration with 3% CO2 in air (v/v). B, growth of the WT, ntpJ, and ndhD3/D4/ntpJ strains on agar plates buffered at pH 9.0 under the conditions described in the legend for Fig. 1. C, the HCO transport activity of low CO2-adapted cells of the ndhD3/D4 and ndhD3/D4/ntpJ mutants suspended in the assay buffer of pH 9 containing 15 mM NaCl and 400 µM HCO. Vertical bars indicate standard deviations (n = 5).

Phylogenetic Analysis of SbtA and NtpJ in Cyanobacteria-- Homologues of SbtA have been identified in Synechococcus sp. PCC 6301,² Synechococcus sp. PCC 7002,³ Anabaena PCC 7120 (www.kazusa.or.jp/cyano/), Nostoc punctiforme, P. marinus strains MED4 and MIT9313 (www.jgi.doe.gov/tempweb/JGI_microbial/html/index.html) and in the non-photosynthetic bacteria Mycobacterium tuberculosis (23), Caulobacter crescentus (24), and Bacillus halodurans (25). The phylogenetic tree (Fig. 6A) pointed to two types of SbtA in cyanobacteria, one consisting of 370-374 and the other of 324-339 amino acids. Anabaena possesses both types of SbtA. The sequence homology between the two types of SbtA is relatively weak, but analyses of hydrophobicity profiles indicated that both types contain 10 membrane-spanning domains that are structurally highly conserved (Fig. 7). Search for specific motifs with the aid of TargetP program (26) identified a signal polypeptide sequence in the N-terminal region of both types of SbtA, likely to target them to the cell exterior and/or the thylakoid lumen. Presently, the exact location of the SbtA is not known, but based on the data presented here and its involvement in Na⁺ exchange, it is most likely located on the cytoplasmic membrane.

View larger version (43K): [in this window] [in a new window]   Fig. 6.   Phylogenetic trees of SbtA (A) and NtpJ (B). Multiple sequence alignment was performed using the CLUSTAL program (34). Syn6803, Synechocystis 6803; Syn6301, Synechococcus sp. strain PCC 6301; Syn7002, Synechococcus sp. strain PCC 7002; Ana, Anabaena sp. strain PCC 7120; Nos, N. punctiforme; ProMED, P. marinus MED4; ProMIT, P. marinus MIT9313; Bacillus, B. halodurans; Caulo, C. crescentus; Myco, M. tuberculosis.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   The hydropathy profiles of two types of SbtA. The profiles were determined by the method of Kyte and Doolittle (35) using a window size of 17 amino acids.

All the cyanobacterial strains investigated possess the NtpJ essential for the operation of the SbtA-mediated HCO transport. The phylogenetic tree of NtpJ indicated two types of proteins, one present in both strains of P. marinus and the other in the other organisms (Fig. 6B).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Four Systems for C_i Acquisition in Synechocystis 6803-- Synechocystis 6803 appears to possess four different systems for C_i acquisition. Two of them, recently identified, are engaged in CO₂ uptake (3). The other two, involved in HCO transport, are the ABC-type transporter encoded by cmpA-D (5) and the SbtA-mediated system identified here. It was essential to inactivate both CO₂ uptake systems to recover the sbtA mutants because presence of either of them enabled photoautotrophic growth even at pH 9.0 in air (Fig. 1B). Measurements of growth and of HCO uptake (Figs. 1 and 2) indicated that SbtA plays the central role in HCO transport in Synechocystis 6803 and that the contribution of the CmpABCD-mediated HCO transport is negligible, also in mutant ndhD3/D4. Furthermore, lack of HCO uptake in the ndhD3/D4/sbtA/cmpA mutant ruled out the possibility that Slr1515 (homolog of IctB from Synechococcus 7942; Ref. 27) is an independent HCO transporter in Synechocystis 6803. The role of Slr1515 (IctB) in intracellular HCO accumulation in cyanobacteria is not known, and we were unable to inactivate slr1515 in Synechocystis 6803. The inability to inactivate ictB (or its homologue, slr1515) suggests that its gene product plays a very important role. Based upon the observations presented here, one might expect that this protein act downstream from SbtA/CmpA/NdhD3/NdhD4. Enhancement of the expression of sbtA by low CO₂ (Fig. 3) was in agreement with the considerable rise in HCO transport capability in cells grown under these conditions (Fig. 2).

The Nature and Mode of Energization of the SbtA-mediated HCO Transport-- Data presented here may help to identify the primary pump involved in the SbtA-mediated active HCO transport. SbtA does not possess an ATP-binding domain. It is therefore unlikely that ATP directly fuels it. SbtA-mediated HCO transport was strongly and specifically dependent on the presence of Na⁺ ions (Figs. 2B and 4B), and NtpJ was essential for both the growth of Synechocystis 6803 in the presence of elevated [Na⁺] and for HCO transport (Fig. 5). These data are consistent with the suggestion that SbtA is a component of a Na⁺/HCO symporter that drives the HCO transport secondary to a primary Na⁺ pump (7, 9, 10). The latter is essential to establish the µNa⁺ for active HCO accumulation. The nature of this primary sodium extrusion pump (28) is not known, but NtpJ is likely to be involved. Measurements of the µNa⁺ value and of the Na⁺ flux across the cytoplasmic membrane of Synechocystis 6803, as affected by [Na⁺], [HCO], and pH, are not available. In a detailed study, Ritchie et al. (21) measured some of these parameters in Synechococcus 7942. They concluded that µNa⁺ would be large enough to drive HCO uptake if the stoichiometry of Na⁺:HCO is 2:1 or 3:1. Because the internal HCO pool in Synechocystis 6803 is 8-10-fold smaller than in Synechococcus 7942 (10), a smaller µNa⁺ would suffice. Measurements of ²²Na⁺ uptake in Synechococcus 7942 showed large enhancement by the presence of HCO (8). On the other hand, Ritchie et al. (21) concluded that the Na⁺ flux was not sufficient to support the rate of photosynthesis (thought to be supported solely by HCO transport). However, photosynthesis in both Synechocystis 6803 and Synechococcus 7942 is largely supported by CO₂ uptake, even at high external pH.

The alternative possibility that HCO transport is energized by the µNa⁺ generated by a Na⁺/H⁺ antiporter, secondary to H⁺-ATPase (29), is unlikely. SbtA-mediated HCO transport activity was highest at pH 9.0 and lowest at pH 7.0, whereas the µH⁺ in cyanobacteria declines with rising pH. At alkaline pH such as 9.0, µH⁺ would not suffice to drive HCO uptake (8, 21). We cannot dismiss the possibility that Na⁺ binds to the HCO carrier and alters its kinetic parameters (7, 10). However, the fact that a ntpJ mutant was impaired in both the ability to grow under high Na⁺ and take up HCO lends support to the possibility that NtpJ is involved in Na⁺ extrusion rather than in the affinity of the HCO carrier for its substrate. This is further supported by the suggestion that NtpJ belongs to a Na⁺ transporter family (motif.genome.ad.jp/) and it is homologous to a subunit of HKT1 in Arabidopsis thaliana that mediates Na⁺ transport (30). We suggest that it is most plausible that the SbtA-mediated HCO transport is energized by a primary Na⁺ pump. Detailed studies on NtpJ and homologues of other subunits of HKT1 are being performed to assess their role in Na⁺ extrusion.

Comparative Sequence Analysis of SbtA-- All the cyanobacterial strains examined, with the exception of P. marinus strains, possess genes involved in CO₂ uptake (3). Phylogenetic analysis indicated that two types of SbtA exist in cyanobacteria; one in Synechocystis 6803, Synechococcus sp. PCC 6301, and Synechococcus sp. PCC 7002, and the other in N. punctiforme and P. marinus strains MED4 and MIT9313 (Fig. 5A). Anabaena sp. strain PCC 7120 possesses both types of SbtA. N. punctiforme is evolutionary very close to Anabaena PCC 7120. Therefore, it is likely that the second type of SbtA is located in the genomic regions of Nostoc yet to be revealed. We may conclude that P. marinus strains acquire C_i by HCO transport and that the SbtA-mediated HCO transport plays a crucial role in the acquisition of C_i either when the supply of CO₂ is limited or in organisms such as P. marinus strains that do not possess a CO₂ uptake system. The Prochlorococcus group is thought to be the most abundant photosynthetic organism on the planet (31), and is responsible for a significant fraction of CO₂ fixation in the oceans. The present study suggests a crucial role of the SbtA-mediated HCO transport in the acquisition of C_i by P. marinus and, therefore, for carbon fixation in the oceans.

Bicarbonate transporters are the principal regulators of pH in animal cells and have a vital role in acid-base movement. The functional family of HCO transporters includes Cl/HCO exchangers, three Na⁺/HCO co-transporters, and K⁺/HCO co-transporter (32, 33). These transporters are much larger than SbtA, and there was no similarity in amino acid sequences between SbtA and mammalian-type HCO transporters.

	FOOTNOTES

^* This work was supported by Grant-in-aid for Scientific Research B 2-12440228, by Human Frontier Science Program Grant RG0051/1997M (to T. O.), by Grant-in-aid for Scientific Research 12660300 (to H. F.), by Research for the Future Grant JSPS-RFTF97R16001 (to T.O. and H. F.), from the Japan Society for the Promotion of Science, by a grant from the USA-Israel Binational Science Foundation (to A. K.), and by a grant from Program MARS2 (a cooperation of the German Ministerium für Bildung, Wissenschaft, Forschung und Technologie and the Israeli Ministry of Science and Technology) (to A. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-52-789-5215; Fax: 81-52-789-5214; E-mail: ogawater@agr.nagoya-u.ac.jp.

Published, JBC Papers in Press, March 19, 2002, DOI 10.1074/jbc.M112468200

² M. Sugita, personal communication.

³ J. Zhao and D. Bryant, personal communication.

	ABBREVIATIONS

The abbreviations used are: CCM, CO₂-concentrating mechanism; C_i, inorganic carbon; H-cell, cell grown under 3% (v/v) CO₂ in air; L-cell, cell acclimated to air for 18 h in the light; WT, wild type; CHES, N-cyclohexyl-2-aminoethanesulfonic acid; TES, N-tris(hydroxymethyl)- methyl-2-aminoethanesulfonic acid; RT, reverse transcription.

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