A periplasmic, {alpha}-type carbonic anhydrase from Rhodopseudomonas palustris is essential for bicarbonate uptake

László G. Puskása,1, Masayuki Inui1, Kenneth Zahn1 and Hideaki Yukawa1

Research Institute of Innovative Technology for the Earth (RITE), 9-2 Kizugawadai, Kizu, Soraku, Kyoto 619-0292, Japan1

Author for correspondence: Hideaki Yukawa. Tel: +81 774 75 2308. Fax: +81 774 75 2321. e-mail: yukawa{at}rite.or.jp


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Intact cells of the purple non-sulfur bacterium Rhodopseudomonas palustris growing anaerobically, but not aerobically, contain carbonic anhydrase (CA) activity. The native enzyme was purified >2000-fold to apparent homogeneity and found to be a dimer with an estimated molecular mass of 54 kDa and a subunit molecular mass of 27 kDa. The CA gene (acaP) was cloned and its sequence revealed that it was homologous to {alpha}-type CAs. The upstream region of acaP was fused to the lacZ gene and ß-galactosidase activity was measured under different growth conditions. Acetazolamide inhibited purified CA with an IC50 in the range of 10-8 M, and in the culture media concentrations as low as 30 µM inhibited phototrophic growth under anaerobic, light conditions when bicarbonate was used. An acaP::Kanr mutant strain was constructed by insertion of a kanamycin-resistance cassette and showed a growth pattern similar to wild-type cells grown in the presence of CA inhibitor. CO2 gas supplied as an inorganic carbon source reversed the effect of mutation or acetazolamide. CA activity measurements, fusion and Western blot experiments confirmed that CA is expressed under different anaerobic conditions independently of bicarbonate or CO2 and that there is no expression under aerobic conditions.

Keywords: carbonic anhydrase, inorganic carbon uptake, Rhodopseudomonas palustris, periplasmic enzyme

Abbreviations: AZ, acetazolamide; CA, carbonic anhydrase; Ci, inorganic carbon; PNSB, purple non-sulfur bacterium; RubisCo, ribulose-bisphosphate carboxylase-oxygenase

a Present address: Biological Research Center, Hungarian Academy of Sciences, DNA-Chip Laboratory, Szeged, PO Box 521, H-6701, Hungary.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Carbonic anhydrase (CA; EC 4 . 2 . 1 . 1) is a zinc-containing metalloenzyme catalysing the reversible hydration of CO2. CA is important in biological systems because the uncatalysed interconversion between CO2 and HC is slow around neutral pH. Its high efficiency catalysis is fundamental to many biological processes, such as photosynthesis, respiration, pH homeostasis and ion transport (Badger & Price, 1992 ; Tashian, 1989 ; Vandenberg et al., 1996 ). Presently, three distinct classes of CAs are recognized based on amino acid sequence comparisons (Hewett-Emmett & Tashian, 1996 ). Among these gene families, significant sequence homologies cannot be recognized. CA has been described in photosynthetic organisms, including higher plants, micro- and macroalgae, and cyanobacteria (Burnell et al., 1990 ; Fukuzawa et al., 1992 ; Hatch & Burnell, 1990 ; Kimpel et al., 1983 ; Price et al., 1992 ). Some photosynthetic organisms have multiple types of CA enzyme found in different cellular locations that serve different functions in the photosynthetic process. In cyanobacteria and algae, different types of CA have been found. The role of extracellular CA is to improve the efficiency of inorganic carbon (Ci) transport and it is located in the periplasm or in the cell membrane (Price et al., 1992 ). The function of the intracellular CA in these organisms is to convert HC to CO2 for fixation by ribulose-bisphosphate carboxylase-oxygenase (RubisCo) (McKay et al., 1993 ; Rawat & Moroney, 1995 ). In plants, intracellular CA enhances the rate of CO2 to HC conversion, for fixation by phosphoenolpyruvate carboxylase (Hatch & Burnell, 1990 ). CA has been proposed to be involved in Ci utilization in most anaerobic bacteria and archaea (Alber & Ferry, 1994 ; Kaplan et al., 1991 ). CA function in processes other than Ci utilization has also been verified in several bacteria. In Escherichia coli the cynT gene, encoding CA, is part of the cyanate degradation operon. Its physiological role is to prevent depletion of cellular HC during cyanate decomposition due to loss of CO2 (Guilloton et al., 1992 ). In acetogenic bacteria the function of CA might be to participate in increasing the intracellular CO2 concentration and to regulate intracellular pH during acetate production (Braus-Stromeyer et al., 1997 ).

Purple non-sulfur bacteria (PNSB) can grow either heterotrophically under aerobic conditions or phototrophically under anaerobic light conditions using bicarbonate or CO2 as Ci source and hydrogen or organic compounds as electron donors (Imhoff, 1995 ). They exhibit remarkable versatility in aromatic compound transformations and their use as biocatalysts for bioremediation has been suggested (Harwood & Gibson, 1988 ). Recently, the use of PNSB for waste treatment and subsequent fixation of CO2 released during industrial processes was proposed (Brenner et al., 1998 ). Little information exists with regard to the mechanism of CO2 utilization in these bacteria, and their Ci uptake mechanism is unexplored. Because CA is known to play an important role in Ci transport and in photosynthetic processes in other organisms, in the present study we intended to verify the presence and the possible function of this enzyme in PNSB. Intracellular, but not periplasmic, CA has been detected in Rhodospirillum rubrum (Gill et al., 1984 ); however, the biological function of this enzyme was not investigated.

Here we report the identification of a periplasmic, {alpha}-type CA from Rhodopseudomonas palustris. This is the only type found in animals, but it can also be found in the periplasm and the thylakoid membranes of the eukaryotic unicellular green alga Chlamydomonas reinhardtii (Coleman et al., 1984 ; Karlsson et al., 1998 ) and in the eubacterium Neisseria gonorrhoeae (Chirica et al., 1997 ). In the PNSB Rhodobacter sphaeroides, Rhodobacter capsulatus and Rsp. rubrum ß- and {gamma}-type, but not {alpha}-type, CAs have been detected by immunoblotting with polyclonal antisera and based on sequence searches of the incomplete genome sequences (Smith et al., 1999 ). We describe the cloning of CA gene, designated acaP, encoding CA protein (AcaP) in Rps. palustris.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cultivation of bacteria.
Rps. palustris No. 7, Rba. sphaeroides and Rsp. rubrum were cultivated at 30 °C in a minimal medium (Fujii et al., 1983 ) containing 5 mM 1-propanol and 20 mM NaHCO3 (pH 7·5) under anaerobic conditions using a light incubator when the activity of the strains were compared, when the effect of different concentrations of acetazolamide (AZ; Sigma) was determined and for purification of CA from Rps. palustris No. 7. When comparing CA activities using different organic compounds, the same minimal media was used but instead of 1-propanol, 2 mM hexanoic acid or butylamine was added. When comparing CA activities in different Ci concentrations, 1, 5, 10 and 20 mM sodium bicarbonate and 2 mM 1-propanol was used in minimal media (pH 7·5). When the effect of CO2 was studied instead of NaHCO3, CO2 was bubbled into the media (3% CO2 in argon). Photoautotrophic growth was studied by growing cells on agar plates anaerobically using CO2 and H2 gas. Comparison of growth of mutant and wild-type cells was also performed on Petri plates set in a small chamber and filled with CO2 with or without H2 at the concentration mentioned above. Aerobic cultivation was performed in van Niel medium (van Niel, 1944 ) containing 5 mM 1-propanol on a rotary shaker (150 r.p.m.) with or without 20 mM sodium bicarbonate. Fermentative growth of cells was carried out anaerobically in the dark using van Niels minimal medium with 4 mM pyruvate on agar plates.

Sample preparation and enzyme assays.
Cells were collected by centrifugation, washed and suspended in 50 mM sodium phosphate buffer (pH 7·2) containing 1 mM zinc sulfate. For measurement of CA of intact cells, 200 ml of this suspension (15 OD660 U ml-1) was used directly. Cells were treated to obtain the periplasmic and cytoplasmic fractions as previously published (Brandner et al., 1989 ; Tai & Kaplan, 1985 ). For total-cell CA activity and for purification of CA from Rps. palustris No. 7, cell suspension (0·4 g wet weight ml-1) was passed through a French pressure cell (20000 p.s.i.; 138 MPa) in the presence of DNase I. The cell debris was centrifuged at 16000 r.p.m. for 20 min at 4 °C and the supernatant was used.

CA activity was measured from the pH decrease after the addition of 1 ml CO2-saturated water to 20 mM Veronal buffer (pH 8·5) (Wako Chemicals) containing the test material in a total volume of 2·8 ml. Units of enzyme activity (U) were calculated using the equation U=(t0-t)/t, where t and t0 represent the time for a pH change from 8·5 to 7·0 with and without sample (Wilbur & Anderson, 1948 ). Protein concentrations were determined colorimetrically by the method of Bradford (1976 ) using Bio-Rad dye reagents and standards. ß-Galactosidase activity was determined by measuring the initial rate of hydrolysis of ONPG (Malamy & Horecker, 1964 ). Cytochrome c2 levels were determined by measuring the difference in the A550 between the fully reduced and oxidized forms (Brandner et al., 1989 ).

Enzyme purification and N-terminal amino acid sequence determination.
Cells from 2 l total culture volume were collected and disrupted with a French press. Centrifugation of the cell extract was then performed for 20 min at 16000 g. About 90% of the total CA activity was obtained in the supernatant fraction. Ammonium sulfate was added to the supernatant (15 g 100 ml-1) and the mixture stirred for 1 h at 4 °C. After centrifugation for 20 min at 16000 g, the same amount of ammonium sulfate was added and the extract treated as above. The precipitate was collected by centrifugation, dissolved in sodium phosphate buffer (50 mM, pH 7·2 containing 1 mM zinc sulfate) and stored on ice for 2 h. The mixture was loaded onto a p-aminomethylbenzenesulfonamide-agarose (Sigma) (3 ml bed volume) column equilibrated with the same buffer. The column was washed with buffer (20 ml), buffer containing 0·7 M NaCl (30 ml) and 0·3 M sodium azide in the same buffer (30 ml). CA activity was eluted with 0·6 M sodium azide. The eluate was concentrated using a 50 kDa cut-off Amicon column. The concentrated eluate was diluted with phosphate buffer, reconcentrated with the same column and rediluted. The diluted enzyme solution was loaded onto Q-Sepharose (Pharmacia) and the flow-through was collected. This step was repeated using a cation-exchange column, CM-Sepharose (Pharmacia). Under various pH conditions CA activity would not bind to anion or cation exchange resins. Although CA could not bind to these columns, collections of the flow-through served to purify CA an additional 1·2-fold. Ammonium sulfate was added to 1·7 M and the sample was loaded onto a Phenyl-Sepharose (Pharmacia) column equilibrated with phosphate buffer (50 mM, pH 7·2) containing 1·7 M ammonium sulfate. After a 50 ml washing step (in the same buffer as used in equilibration), the activity was eluted with 1·6 M ammonium sulfate solution. The fractions containing CA activities were dialysed against phosphate buffer and concentrated as described in the affinity chromatography step.

After purification, 8 µg CA enzyme was transferred onto a nitrocellulose membrane (Millipore) and the N-terminal sequence of the protein was determined commercially by the automated Edman degradation procedure (Takara).

Immunoblot analysis of Rps. palustris CA.
The soluble cell fractions were analysed by immunoblotting for CA content after separating the proteins on polyacrylamide gels under denaturing (using SDS) or native conditions. Proteins were electrophoretically transferred to nitrocellulose (Millipore). Immunoblotting was performed with mouse antiserum against purified CA and goat anti-mouse antibody–alkaline phosphates conjugate as a detection method, as described previously (Blake et al., 1984 ).

Cloning and nucleotide sequence determination of acaP.
Based on the amino acid sequence of the N terminus of CA from Rps. palustris (AEGAYHW) the following degenerate primer was synthesized: acap1, 5'-GC(CG)GA(AG)GG(CT)GC(CG)TGGGG-3'. The second primer for PCR was designed according to the highly conserved amino acid motif GSLTTPP among {alpha}-type CAs: acap2, 5'-CGGCGG(CG)GT(CG)GT(CG)AG(CG)GA(CG)CC-3'. PCR was carried out in a total volume of 100 µl with 10 ng Rps. palustris genomic DNA, 0·2 mM dNTPs, 2% DMSO and 1xTaq polymerase buffer with MgCl2 and 4 U Taq DNA polymerase (Takara) for 30 cycles at temperatures of 95 °C for denaturation (1 min), 57 oC for annealing (1 min) and 72 oC for extension (1 min). After agarose gel electrophoresis of the PCR mixture, the desired fragment (516 nt long) was purified with a PCR purification kit (Pharmacia) and cloned into the pGEMT vector (Promega) and sequenced by the dye-terminator method using ABI reagents and protocol (Perkin Elmer). Using the same primers and PCR conditions, a full-length genomic clone was identified using PCR from a NaeI genomic library of Rps. palustris DNA in the pBluescript SK(II) vector (Stratagene) by screening serial dilutions of pooled plasmids as template. The nucleotide sequence of the whole NaeI fragment was determined as described below. The nucleotide sequence and the 255 aa ORF encoding Rps. palustris CA, designated acaP, is available in the GenBank nucleotide sequence database with the accession number AB022175.

Construction of acaP mutant strain.
A kanamycin-resistance gene cassette was excised from vector pUC4K (Pharmacia) by PstI restriction enzyme cleavage and gel purification, and inserted into the PstI site within the coding region of acaP (into the 225th nt position). The recombinant fragment was amplified with two specific primers complementary to regions flanking the genomic NaeI sites, which also contained BamHI sites at their 5' ends. The resulting fragment was digested with BamHI and cloned into the pGP-704 vector that had been digested with BglII. Procedures used to obtain the Rps. palustris double crossover acaP::kanR insertion mutant strain were as described by Inui et al. (1999 ).

Construction of acaP–lacZ fusion vector.
The coding region of the lacZ gene was excised from the plasmid pMC1871 (Pharmacia) with PstI/BamHI and cloned into the pBluescript SK (II) multicloning site resulting in the plasmid pBlac. A 500 bp upstream region of acaP (from the first Ala codon of the mature CA enzyme) was amplified with primers having EcoRI and PstI sites, cut with the same enzymes and cloned into pBlac upstream of the lacZ gene. The EcoRI–BamHI fragment was cut out and cloned into the corresponding sites of pMG103 to produce plasmid pMlaca. pMlaca was electroporated into Rps. palustris cells or transformed into E. coli cells and the transformants were selected with kanamycin and used in ß-galactosidase fusion studies.

DNA sequencing.
DNA sequencing was performed using Thermo Sequenase dye-terminators and samples were run on an ABI 373 fluorescent sequencer. All plasmid inserts produced by PCR in this study were confirmed by sequencing.

Materials.
All chemicals were purchased from Wako Chemicals unless otherwise mentioned. Goat anti-mouse antibody–alkaline phosphatase conjugate was purchased from Boehringer Mannheim Biochemicals. Restriction endonucleases were obtained from Takara Biochemicals and were used according to the manufacturer’s specifications.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
CA activity in Rps. palustris cells
Protein preparations of Rps. palustris cells cultivated under anaerobic conditions exhibited total CA activities from total cell protein [2·95–3·20 U (mg protein)-1], similar to those of other CA-containing bacteria (Alber & Ferry, 1994 ; Braus-Stromeyer et al., 1997 ), whereas cells grown aerobically showed little or no activity (<0·1 U) (Table 1). Intact cells showed CA activity [0·6 U (OD600 U bacteria)-1] due to periplasmic or membrane-bound CA enzyme. Activity was measured from cells that were cultivated under different anaerobic conditions: fermentative dark conditions using pyruvate as carbon source, photoheterotrophic light conditions using different organic compounds and CO2 or bicarbonate as the Ci source, photoautotrophic light condition using H2 and CO2. Cells that were cultivated under aerobic heterotrophic conditions with or without bicarbonate in the media showed no activity. This result did not depend on the applied organic carbon source (Table 1).


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Table 1. CA activities of Rps. palustris

 
Enzyme purification
Under a variety of conditions, CA did not bind to cation- or anion-exchange columns. However, these steps were useful to remove certain contaminants. Major purification was achieved with affinity chromatography. Strong binding of the enzyme to the p-aminomethylbenzenesulfonamide-agarose column was observed and this is consistent with the high affinity of CA for AZ. The enzyme was purified more than 2000-fold, with an overall recovery of 38% (Table 2). The specific activity of the purified enzyme (6269 U mg-1) is comparable to that of commercially available human erythrocyte isozyme II (7870 U mg-1).


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Table 2. Purification of CA from Rps. palustris

 
Properties of the purified enzyme
The enzyme appeared as a single band of about 27 kDa on a 4–20% gradient SDS polyacrylamide gel after gel electrophoresis and Coomassie brilliant blue staining (Fig. 1a). On a native gel (acrylamide gradient 4–20%) CA also gave a single band (Fig. 1b). To examine the presence of CA enzyme in cells cultivated under different conditions and for localization studies, mouse polyclonal antisera were raised against the purified CA from Rps. palustris. As shown in Fig. 2, the antibodies detected a polypeptide of the same size as CA in a Western blot analysis and could detect very small amounts of purified CA protein. When antiserum was used against whole-cell extracts, no other protein showed reactivity, thus it seems that the antiserum is specific to CA from Rps. palustris.



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Fig. 1. Denaturing (a) and native (b) PAGE of CA from Rps. palustris. The molecular mass of the markers is indicated. One microgramme protein from the Phenyl-Sepharose chromatography step was applied.

 


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Fig. 2. Immunoblot analysis of protein extracts from Rps. palustris cultivated under different conditions. Lane 1 contains purified CA as a control. Under photoautotrophic (lane 2) and photoheterotrophic (lanes 3 and 4) conditions, cells were cultivated in minimal media at 35 °C in the light using bicarbonate or CO2 as the Ci source as indicated. Under fermentative conditions (lane 5) cells were grown in the dark in an anaerobic chamber using pyruvate in rich media at 35 °C. When cells were cultivated under heterotrophic conditions in the presence of oxygen (lanes 6, 7 and 8), the same media, and organic and inorganic carbon compounds were used as in the case of the corresponding photoheterotrophic conditions (lanes 3, 4 and 5). Aerobic growth in the dark was at 35 °C with shaking. Blotting was performed with mouse antiserum against purified CA and goat anti-mouse antibody–alkaline phosphatase conjugate as a detection method.

 
The migration of the purified enzyme was analysed on a series of denaturing and native gels containing different polyacrylamide concentrations and the apparent size of the enzyme was determined by the method of Ferguson (1964 ). Under denaturing conditions the estimated molecular mass of CA is about 27 kDa while under native conditions the molecular mass is approximately 54 kDa, suggesting that the native enzyme is a dimer. The NH2 terminus of the purified CA protein was determined by the automated Edman degradation procedure using a band isolated from a native gel. NH2-terminal analysis of the first 12 aa revealed a single homogeneous sequence (AEGAYHWGYEGE), so the CA dimer is probably composed of identical subunits.

We determined the IC50 values (concentration required for 50% inhibition of activity) of several inhibitors of CA reported from studies in other organisms (Maren & Sanyal, 1983 ; Sugrue, 1996 ) (, 6·0x10-3 M; Cl-, >10-2 M; AZ, 3·1x10-8 M). AZ was a more effective inhibitor of this CA than of other bacterial CAs (Braus-Stromeyer et al., 1997 ; Yu et al., 1992 ). The values obtained here are more characteristic of human CAs and other CAs from the {alpha} family (Armstrong et al., 1966 ; Lindskog et al., 1971 ; Price et al., 1992 ). Monovalent ions were significantly less effective than for some bacterial CAs (Braus-Stromeyer et al., 1997 ; Yu et al., 1992 ). The enzyme also has an esterase activity detected with p-nitrophenylacetate as the substrate (data not shown).

Cellular localization of CA
Rps. palustris is a Gram-negative bacterium and contains two cellular compartments, the cytoplasm and the periplasm. CA activity was found only in the periplasmic space (the cytoplasmic fraction exhibited 0·49 U, while the periplasmic fraction showed 11·5 U). The purity of the periplasmic extract was evaluated by using marker enzymes with known localizations. We chose cytochrome c2 as the periplasmic, and ß-galactosidase as the cytoplasmic marker. Fractionation and measurements of marker enzyme activities were performed as published elsewhere (Brandner et al., 1989 ; Tai & Kaplan, 1985 ). In the cells grown under photosynthetic conditions, cytochrome c2 and CA were found only in the periplasmic fraction (997 pmol Cyt c in the periplasm, and 173 pmol Cyt c in the cytoplasmic fraction), while ß-galactosidase activity appeared only in the cytoplasmic fraction (260 Miller U in the cytoplasm and 38 Miller U in the periplasmic fraction). Since wild-type Rps. palustris No. 7 strain does not show ß-galactosidase activity, we constructed a plasmid which contains the E. coli lacZ gene using an Rps. palustris shuttle vector (Inui et al., 2000 ). To examine the localization of CA by another method we used immunoblot analysis. We used specific antisera against the purified enzyme and checked the protein fractions from the periplasm and the cytosol. We were able to confirm the periplasmic location of CA as observed with enzymic assays (data not shown).

Cloning and sequence data comparison of CA gene, acaP
Based on the sequence of the first 12 NH2-terminal amino acid residues of the purified CA and from studies of inhibitor specificity, we assumed that Rps. palustris CA belongs to the {alpha} class. Two degenerate primers were designed for PCR based on the first NH2-terminal 8 aa and on one internal conserved sequence region present in all {alpha}-CAs (Hewett-Emmett & Tashian, 1996 ) (see Methods). PCR was used to amplify a DNA fragment of 516 bp which was cloned and sequenced. Nucleotide sequence similarity comparisons showed that this fragment had significant homology to other {alpha}-CAs in the database. To identify an E. coli clone having the full-length Rps. palustris CA gene, PCR amplification was used to screen a NaeI genomic library from Rps. palustris in pBluescript SK II. A 1443 bp NaeI fragment was found to contain the full-length CA gene. From the DNA sequence and the NH2-terminal amino acid determination we assume that there is a 25 aa signal sequence which is responsible for the periplasmic export of the enzyme. A positively charged part in the amino terminus of the possible signal motif and a following hydrophobic core are recognized characteristics of bacterial signal peptides (von Heijne, 1984 ). The 18 residue hydrophobic region ends in the +1, -1 AXA motif characteristic of the recognition site of signal peptidase (Perlman & Halvorson, 1983 ). The protein is thus predicted to be made as a 27,302 Da precursor which is cleaved between Ala-25 and Ala-26 to produce a mature 230 residue protein of 24772 Da. The putative initiation codon (ATG) is preceded by a possible ribosome-binding site sequence (GGAGA), 5 nt upstream. Downstream from the ORF is a possible rho-independent transcription terminator. The nucleotide sequence and the ORF, designated acaP, encodes the 255 aa Rps. palustris CA. The deduced amino acid sequence of acaP was aligned with the sequences of CA from Anabaena, Synechococcus, Erwinia carotovora, Klebsiella pneumoniae, N. gonorrhoeae and human CA isozymes I and II. Rps. palustris CA showed the highest homology to bacterial CAs (35·7–45·7% identities). Comparing Rps. palustris CA with the two human CA isozymes I and II, the identities were 33% and 34·6%, respectively. The highest identity was found with the ß-Proteobacterium Neisseria (42·9%) and the {gamma}-Proteobacteria Erwinia (45·7%) and Klebsiella (42·9%).

Regulation of acaP expression
CA activity was measured and was the same when cells were grown under different anaerobic conditions: photoheterotrophic light, photoautotrophic light or fermentative dark conditions. In the presence of oxygen, CA activity could not be detected using the media listed in Table 1. The presence of a CA band as demonstrated by immunoblot analysis under each condition (Fig. 2) was in agreement with the activity data. The enzyme was detected under different anaerobic conditions. No difference was detected in the amount of CA enzyme in cells grown in low and high concentrations of bicarbonate under different anaerobic conditions (data not shown).

To investigate the transcriptional regulation of acaP, an acaP::lacZ fusion was constructed. The non-coding, upstream region with the proposed translation initiation signals of acaP was fused translationally in-frame to the first codon of lacZ. In E. coli the ß-galactosidase activity remained unchanged when cells were shifted from aerobic to anaerobic conditions, while a dramatic change was observed in the case of Rps. palustris, where activity was more than 20-fold higher when cells were incubated in the absence of oxygen (0·9 compared to 19·7 Miller U). Expression of ß-galactosidase occurred within several hours and cells plated in the presence of X-Gal became blue when examined on LB plates containing the indicator. In E. coli the expression of acaP gene seemed to be constitutive compared to expression from the native lac promoter, and the strength of the acaP promoter was approximately 13-fold weaker than the lac promoter both in E. coli and in Rps. palustris. The immunoblotting and ß-galactosidase fusion studies suggest that CA expression is regulated at the transcriptional level.

Inhibition of phototrophic growth by CA inhibitors
Since AZ was an effective in vitro inhibitor of CA, we studied the effect of AZ on the growth of Rps. palustris under anaerobic light conditions. Preliminary studies showed that CA activity could be completely inhibited in intact cells using 0·2 mM AZ. Lower concentrations of AZ inhibited photosynthetic growth in a concentration-dependent manner when bicarbonate was used as the Ci source (Fig. 3a). Even higher concentrations of AZ than these had no effect on aerobic growth (data not shown). These results are in good agreement with the observation that extracts from cells harvested under aerobic conditions exhibited no CA activity. Cells showed CA activity when grown under photoautotrophic and fermentative conditions. When CO2 was used instead of bicarbonate under photoheterotrophic conditions, AZ had no inhibitory effect on growth (data not shown).



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Fig. 3. (a) Effects of different concentrations of AZ on photoheterotrophic growth of wild-type Rps. palustris cells. Without AZ, {bullet}; with 60 µM AZ, {blacksquare}; 30 µM AZ, {blacktriangleup}. The growth pattern of the acaP mutant ({circ}) without the inhibitor under the same conditions using bicarbonate as the Ci source and ethanol as the organic carbon compound is also shown. Cells were incubated at 35 °C in the light in minimal medium. (b) Photoheterotrophic growth of the wild-type ({blacktriangleup}) and mutant ({triangleup}) cells in the presence of CO2 and of the wild-type ({bullet}) and mutant cells ({circ}) in the presence of bicarbonate. (c) The effects of pH on photoheterotrophic growth; wild-type ({bullet}) and mutant cells ({circ}) at pH 7; wild-type ({blacktriangleup}) and mutant cells ({triangleup}) at pH 6 using the same conditions as in (a).

 
Insertion mutation of acaP and characterization of the mutant strain
To obtain more information regarding the physiological role of CA, we made a mutant strain of Rps. palustris by insertion of a kanamycin-resistance cassette into the coding region of acaP. The single-crossover mutants showed resistance to another antibiotic, gentamicin, while strains that displayed only kanamycin resistance were double-crossover mutants and these were selected for further analysis. The homologous recombination event was confirmed by PCR and Southern blotting (data not shown). The mutant strain showed a similar growth pattern to wild-type cells cultivated in the presence of 60 µM AZ in minimal medium containing bicarbonate as the Ci source (Fig. 3a). Phototrophic growth was inhibited for several days when bicarbonate was used. The very slow growth can be explained by the slow conversion of bicarbonate to CO2 without catalysis at close-to-neutral pH in liquid media. Using CO2 with different organic compounds or with H2, no difference in growth was observed between the wild-type and the mutant cells using solid media in small anaerobic chambers (data not shown).

Effect of CO2 and pH on acaP mutant and wild-type cells
When cell growth was arrested by AZ under phototrophic conditions using bicarbonate, the inhibitory effect could be diminished by bubbling CO2 gas into the media (data not shown). An acaP mutant strain could also grow when the media was supplemented with CO2 (Fig. 3b). The same effect could be seen when the pH of the media was lowered with buffers. Although wild-type cells grew more slowly in lower pH (pH 5–6) media, mutant bacteria showed increased growth, probably due to the increased CO2 conversion from bicarbonate (Fig. 3c).


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
One report on CA from the PNSB has so far appeared where a cytoplasmic CA from Rsp. rubrum was purified and some features of the enzyme were studied (Gill et al., 1984 ). The physiological role of this enzyme was not studied, nor was a periplasmic location established. In this report, CA from Rps. palustris is studied in detail. We have found that intact cells exhibit CA activity when cultivated under photosynthetic growth conditions. Enzyme activity could be localized mainly to the periplasmic space and this was confirmed by using cytochrome c2 and ß-galactosidase enzymes as control markers localized to the periplasm and cytoplasm, respectively. CA activity was unchanged when cells were grown under different anaerobic conditions: photoheterotrophic light, photoautotrophic light or fermentative dark conditions. Under photoheterotrophic conditions, the levels of CA are not regulated in response to the growth substrate, and enzymic activities are similar in alcohol, organic acid or alkylamine-grown cells. No correlation was established between CA activity and the concentration of Ci in the media. Little [<0·1 U (mg protein)-1] or no activity was detected when cells were cultured under aerobic conditions.

CA protein structural relationships
Cloning of the acaP gene and determination of the amino acid sequence demonstrates that Rps. palustris CA is homologous to {alpha}-type CAs. Stronger homology and other distinctive features characteristic of bacterial CAs of the same family were recognized, which suggest a common molecular structure and also a role in the localization of the enzyme. The NH2 terminus of all bacterial CAs contains a possible signal sequence and the AXA signal peptidase recognition motif can be seen in all of the cases except in the Klebsiella enzyme. Four distinct deletions in the sequence can be seen when compared to human CA isozymes I and II (data not shown). Three of them are located in the regions that connect ß strands D, E and F, which are central characteristics of the dominating structural element, the 10-stranded ß sheet of all {alpha}-CAs (Eriksson et al., 1988 ). A fourth gap is located close to the C terminus and might result in a shorter corner loop which is longer in the human isoenzymes molecular periphery region (Eriksson et al., 1988 ). Thirty-six amino acids are found to be highly conserved among all {alpha}-CAs. Three histidines (His-94, His-96, His-119), which are the three zinc ligands in the enzyme, as well as other conserved amino acids (Gln-92, Glu-117, Ala/Val-121, Val-143) are unchanged. Interestingly, there are two positions where all bacterial CAs have cysteine residues (Cys-53 and Cys-200; Rps. palustris numbering) while human isozymes have alanine and leucine in those positions. In the case of the newly discovered {alpha}-type CA from the thylakoid membrane of C. reinhardtii, cysteines are also found in these positions (Karlsson et al., 1998 ); this may suggest a close evolutionary relationship to bacterial CAs.

Localization and expression of CA
CA and RubisCo are associated in the carboxysomes of cyanobacteria and possibly in the pyrenoids of algae (Price et al., 1992 ). The carboxysomes are postulated to contain a specialized CA at the active site to provide CO2. It is also possible that intracellular CA aids in the conversion of CO2 to HC for fixation by phosphoenolpyruvate carboxylase (Hatch & Burnell, 1990 ). Both biochemical pathways are found in photosynthetic bacteria (Buchanan et al., 1967 ; Inui et al., 1997 ; Tabita, 1995 ) although carboxysomes are not found in PNSB and our results suggest that CA enzyme is not associated with RubisCo, because the main CA activity was found in the periplasmic fraction.

Periplasmic localization and the presence of the enzyme only under anaerobic conditions suggests that CA participates in Ci utilization in this bacterium. We also tested two other PNSB, Rba. sphaeroides and Rsp. rubrum, and found the same phenomenon (data not shown). This suggests that an {alpha}-type CA exists in these PNSB as well. We cannot exclude the presence of other intracellular accumulation systems for Ci, because when different Ci concentrations were used under phototrophic conditions, the expression and activity of the CA studied here did not change. Other Ci transport proteins probably act under phototrophic conditions, either in cooperation with CA or independently. These aspects of the uptake mechanism await clarification.

Anaerobic induction and transcriptional regulation of the acaP gene is suggested by the analysis of the acaP::lacZ fusion. A 20-fold change in ß-galactosidase activity was observed in the case of Rps. palustris, when cells were incubated anaerobically. Induction occurred within several hours and was also detected on plates where the cells became blue in the presence of X-Gal. In E. coli, the ß-galactosidase activity remained unchanged when cells were shifted from aerobic to anaerobic conditions, suggesting that the anaerobic regulation of CA expression is specific for Rps. palustris. In E. coli, ß-galactosidase expression from the acaPlacZ fusion gene was much weaker than in the case of the full length lacZ gene with its own promoter. These results suggest that the promoter of acaP is quite weak and may be regulated at the transcriptional level by oxygen via an oxygen-responsive transcriptional regulatory system as found in other bacteria (Monson et al., 1995 ; Unden & Schirawski, 1997 ). However, FixL-like and Fnr-like recognition sequences found in other oxygen regulatory systems could not be discerned. It is also possible that the expression of CA is controlled by a global regulatory system, like the one recognized to control aerobic and anaerobic CO2 metabolism in Rsp. rubrum and Rba. sphaeroides. In these bacteria the RegA/RegB system was found to regulate the aerobic and anaerobic expression of genes encoding enzymes of primary and alternative CO2 fixation and integrates photosynthesis and nitrogen fixation (Qian & Tabita, 1996 ; Joshi & Tabita, 1996 ). However, a RegA-like consensus recognition motif (Du et al., 1998 ) was not found in the sequence of acaP, nor upstream of the coding region, which suggests that a different regulatory system is involved.

Physiological role of CA
In PNSB, CA has an important role under anaerobic growth conditions when bicarbonate is the Ci source. Growth inhibition could be detected by inhibiting the extracellular CA of Rps. palustris by the specific CA inhibitor AZ, and a growth defect was observed in the acaP mutant. Growth of wild-type cells in the presence of AZ or the acaP mutant was slowed by several days but this inhibitory effect could be largely reversed using CO2, or partially reversed when the pH of the media was lowered and the non-enzymic conversion of bicarbonate to CO2 is much faster. From these results we assume that the cell membrane is a barrier to the bicarbonate ion, but that CO2 can penetrate by diffusion into the cytoplasm. The function of CA in Rps. palustris, and probably in other PNSB, is to accelerate the bicarbonate–CO2 conversion in the periplasmic space. This process might be analogous to CA function in cyanobacteria and algae; however, intact cells of cyanobacteria exhibit very low CA activity and inactivation of the cyanobacterial ecaA gene, which encodes extracellular CA, did not give a dramatic change in photosynthetic growth (Soltes-Rak et al., 1997 ). Although there are slight differences in periplasmic CA activities, CA seems to function universally in Ci utilization in photosynthetic micro-organisms.

We believe that this work contributes to a basic understanding of the key reactions in Ci utilization and of the nature of anaerobic/aerobic gene regulation in Rps. palustris. We also hope that this knowledge will prove useful in efforts to improve CO2 fixation and photosynthetic ability in this species for a variety of biotechnological applications.


   ACKNOWLEDGEMENTS
 
We thank H. Yamagata for helpful discussions. This work was supported by the New Energy and Industrial Technology Development Organization (NEDO). L. G. Puskás was supported by a NEDO fellowship.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 9 May 2000; revised 19 July 2000; accepted 1 August 2000.