Proc. Nati. Acad. Sci. USA Vol. 89, pp. 4437-4441, May 1992 Biochemistry A gene homologous to chloroplast carbonic anhydrase (icfA) is essential to photosynthetic carbon dioxide fixation by Synechococcus PCC7942 (cyanobacteria phylogeny/ribulose-1,5-bisphosphate carboxylase/oxygenase/CO2 transport/insertional mutagenesis) HIDEYA FUKUZAWA*, Eui SUZUKIt, YUTAKA KOMUKAIf, AND SHIGETOH MIYACHI§ Institute of Applied Microbiology, University of Tokyo, Tokyo 113, Japan Communicated by N. Edward Tolbert, January 24, 1992 (receivedfor review October 1, 1991)

ABSTRACT To understand the C02-concentrating mech- sensitive high-CO2-requiring mutant of Synechococcus anism in cyanobacteria, a genomic DNA firgment that com- PCC7942. This strain is defective in the utilization of intra- plements a temperature-sensitive high-CO2 (5%)-requiring cellularly accumulated Ci at nonpermissive temperatures mutant of Synechococcus PCC7942 has been isolated. An open (13). The genomic region complementing this mutation is reading frame (0RF272) encoding a polypeptide of 272 amino located .20 kilobases (kb) downstream of the structural acids (Mr, 30,184) was found within the genomic region located genes for RuBP carboxylase/oxygenase subunits (rbcLS). 20 kilobases downstream from the genes for ribulose-1,5- We describe here the physiological characterization of bisphosphate carboxylase/oxygenase (rbcLS). Insertion of a mutants in which an ORE within the genomic region was kanamycin-resistance gene cartridge within the 0RF272 in disrupted by insertional mutagenesis and identification of a wild-type cells led to a high-CO2-requiring phenotype. Strains gene that should be responsible for mediating CO2 fixation in carrying a gene disabled by insertional mutagenesis accumu- the cyanobacteria. Since the amino acid sequence ofthe ORF lated inorganic carbon in the cells, but they could not fix it deduced from the nucleotide sequence¶ showed significant efficiently, even though ribulose-1,5-bisphosphate carboxylase similarity to those of plant chloroplast CAs and Escherichia activity was comparable to that of the wild-type strain. There- coli CA, this ORF is assumed to be a gene encoding cyano- fore, the 0RF272 was designated as a gene iwfA, which is bacterial CA. essential to inorganic carbon fixation. Furthermore, the pre- dicted icfA gene product shared significant sequence similari- ties with plant chloroplast carbonic anhydrases (CAs) from pea MATERIALS AND METHODS (22%) and spinach (22%) and also with the Escherichia coli Strains and Growth Conditions. Wild-type (wt) and mutant cynT gene product (31%), which was recently identified to be cells of Synechococcus sp. PCC7942 were grown in BG-11 E. coli CA. These results indicate that the putative CA encoded medium (14) buffered with 30 mM Hepes-NaOH (pH 7.8) in by iwfA is essential to photosynthetic carbon dioxide fixation in an atmosphere of 5% C02/95% air under continuous illumi- cyanobacteria and that plant chloroplast CAs may have evolved nation at 3.5 W/m2. from a common ancestor of the prokaryotic CAs, which are Construction of Plasmids and Transformation. Genomic distinct from mammalian CAs and Chlamydomonas periplas- DNA fragments of Synechococcus PCC7942 in plasmid mic CAs. pBM3.8 (13) were subcloned into pUC19 and pBluescript II KS+ (Stratagene). DNA fragments encoding an aminoglyco- Cyanobacteria have a very high affinity for environmental side 3'-phosphotransferase of 1.3 kb, isolated from pUC4K CO2 as compared to most higher plants (1). This high affinity (15) and pUC4KISS (16), were inserted into HincII, Xho I, has been assumed to be due to the operation of a C02- and Sph I sites of the Synechococcus DNA to generate concentrating mechanism (2, 3), which can be divided into at recombinant plasmids for gene disruption and designated least two steps: (i) accumulation of inorganic carbon (C1) in pHC::Km, pXH::Km, and pSP::Km, respectively. These the cells by a putative energy-dependent transporter (2-4) plasmids (10 ug) were used for transformation of Synecho- and (ii) efficient CO2 fixation within the carboxysomes where coccus PCC7942 (17). Transformants were selected on agar the majority of active ribulose-1,5-bisphosphate (RuBP) car- plates containing kanamycin at 10 gg/ml in 5% C02/95% air. boxylase is located (5). Since C, accumulation is inhibited by Hybridization and Sequence Analysis. DNA fragments were ethoxzolamide, an inhibitor of carbonic anhydrase (CA), labeled with [32P]dCTP using a random-primer labeling kit some component related to CA is assumed to operate in the (Amersham) and used in Southern blot hybridizations as Ci transport step (6, 7). It has been suggested that the probes. The nucleotide sequences of DNA fragments sub- carboxysome serves as the permeability barrier for CO2 to cloned in M13mp18 and M13mpl9 were determined using a prevent its leakage out ofthe cells (8) and contains CA, which deazadideoxynucleotide sequencing kit (Takara Shuzo, Ky- catalyzes dehydration of HCO- (9). This assumption is supported by the observation that Synechococcus PCC7942 Abbreviations: CA, carbonic anhydrase; C;, inorganic carbon; Kmr, cells that express human CA in the cytoplasm require high kanamycin resistance; ORF, open reading frame; RuBP, ribulose CO2 (5%) for growth (8). However, the significance of CA in 1,5-bisphosphate; wt, wild type. the *To whom reprint requests should be sent at the present address: C02-concentrating mechanism is not fully established. Department of Agricultural Chemistry, Faculty of Agriculture, Several high-CO2-requiring mutants have been isolated and Kyoto University, Kyoto 606-01, Japan. open reading frames (ORFs) responsible for their defects tPresent address: Department of Biology, Faculty of Science, Ibar- have been identified (4, 10-12). We have reported (13) the aki University, Mito 310, Japan. cloning ofa genomic region that complements a temperature- tPresent address: Department of Biology, Faculty of Science, Nagoya University, Nagoya 464-01, Japan. §Present address: Marine Biotechnology Institute, Co. Ltd., 2-35-10 The publication costs of this article were defrayed in part by page charge Hongo, Tokyo 113, Japan. payment. This article must therefore be hereby marked "advertisement" 1The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. M77095).

4437 Downloaded by guest on September 25, 2021 4438 Biochemistry: Fukuzawa et al. Proc. Natl. Acad. Sci. USA 89 (1992) oto). The sequences obtained were compiled and analyzed by Sp Ba Sc P P Hc SpHd X PSp P Bg a software DNASIS (Hitachi, Tokyo) using protein data bases, 11 I I National Biomedical Research Foundation Protein Identifi- A A A cation Resource (release 26.0) and Swiss-Prot (release 15.0). For construction of phylogenetic tree, the software reported in ref. 18 was used. ATG Other Measurements. The cell density of a culture was determined by measuring the OD at 720 nm. Accumulation ictA (ORF272) and fixation of Ci were determined by the silicone oil cen- trifugation method (13) at 300C. NaH14CO3 was added to the FIG. 1. Restriction map of the 1.7-kb BamHI-Bgl II genomic assay mixture at 50 uM. RuBP carboxylase activity was region and location ofiefA (ORF272). Solid bar, the 0.43-kb HindIII- assayed by the incorporation of 14Co2 into the acid-stable Pst I region that complements a temperature-sensitive high-CO2- fraction at 300C (19). requiring mutant, C3P-O; stippled box, coding region of icfA (ORF272) with the translation initiation codon ATG; solid triangles, three restriction sites at which a kanamycin-resistance (Kmi) gene RESULTS cartridge was inserted; Ba, BamHI; Bg, Bgi II; Hc, HinclI, Hd, HindIII; P, Pst I; Sc, Sac I; Sp, Sph I; X, Xho I. Nucleotide Sequence of the Genomic Region. We have cloned (13) a genomic DNA fragment that complements the the left portion of the 3.8-kb BamHI fragment in the plasmid temperature-sensitive high-C02-requiring mutant C3P-O. pBM3.8 (13), was subcloned into BamHI-Sal I-digested Complementation occurred with plasmids containing 36-kb pUC19. This plasmid designated as pBX1.0 was linearized by EcoRI, 3.8-kb BamHI, 0.87-kb Pst I, and 15-kb HindIlI cutting at the unique HinclI site and ligated with the 1.3-kb fragments, all of which overlapped in a 0.43-kb HindIII-Pst HincHI Kmr gene cartridge. The recombinant plasmid I region as shown in Fig. 1. This region was located 20 kb pHC::Km was used in the transformation ofwt cells to obtain downstream from the coding region for subunits of RuBP insertional mutants at the HinclI site. (ii) For insertional carboxylase/oxygenase. at the Xho I site located inside the ORF272, a To characterize the genomic region that complements the mutagenesis mutation, the nucleotide sequence was determined for the 1.7-kb BamHI-Bgl II fragment (Fig. 1) was subcloned into 1662-base-pair (bp) Sac I-Bgl II region (Fig. 2) that contains pUC19 and named pBB1.7. Plasmid pBB1.7 was linearized the 0.43-kb HindIII-Pst I region (see also Fig. 1). An ORF with Xho I and ligated with the 1.3-kb Sal I Kmr gene (0RF272) possibly encoding a polypeptide of272 amino acids cartridge to obtain the plasmid pXH: :Km. (iii) For insertional (M, 30,184) was found in the HindIII-Bgl II region that mutagenesis at the Sph I site located also in 0RF272, a 1.7-kb overlapped the 0.43-kb HindIII-Pst I region. The coding Xho I fragment (whose left end is shown in Fig. 1) in the region of0RF272 begins 61 bp downstream from HindIll site plasmid pBM3.8 (13) was inserted into pBluescript II KS+. and ends 28 bp upstream from Bgl II site. A possible The recombinant plasmid designated pXH1.7 was linearized ribosome-binding sequence (GAG) was found 10 bp upstream by Sph I and ligated with the 1.3-kb Sph I Kmrgene cartridge. from the translation initiation codon. The generated plasmid was designated pSP::Km. Construction of Insertional Mutants of Cyanobacteria. The The kanamycin-resistant derivatives of Synechococcus significance of ORF272 was examined by insertion of a Kmr PCC7942 were selected on kanamycin-containing agar plates gene cartridge at HincII, Xho I, and Sph I sites inside or after transformation of wt cells with plasmids pHC::Km, outside the coding region of the ORF272 in wt cells (Fig. 1). pXH::Km, and pSP::Km. Fifty colonies of each type of the (i) For insertional mutagenesis at the HincII site, which is kanamycin-resistant transformants were replica-plated and outside ORF272, a 1.0-kb BamHI-Xho I fragment, which is incubated in 5% C02/95% air (high CO2 condition) and in

GAGCTCGCCGTTATCMATTTCGGTGGTGGTGCCTAGGCCMATCACCTCCCCTTGCAGAGGAATCTGACGMATAGMATAGTGTTCGCTGATGTCACCTTGAATCAGTGCCGTCACAGTGTT 120 AGCGACAGTAAGTTTCAACATCTCTTCGCCTAGATAGGGGCTGTTCTCCAAATCCTGTGGGTAGTGATCCAAGCTGTGGAGTAGCCTGCAGCGATCGCGCCTAGTGCTTTGCTGTATGGC 240 GTCTCGCTAAAAAGGAGCGGGTGTAAAAGTTGTATTGAAAATCGAGTTAATTTGGTTGATAGTTTCAACCGACTGCAGATCAAMGCGCTCCTTGTAAGTAGGGCTGGCTTTGGAGTTG 360 TTGAGCMACATCCTGATTGATTTGCCTGCTTTCCTGCTTGAGTCGAGTCTGGATTGCCACCGTTTCTTCAGGCGTAATCTTGCCATCTTGTAGAGAGTTACAMATTGGCAGCAGCGCATT 480 HincII GTATTGGCGATCGCTCAGACTGGAGAGATCCTGACGGGTMATCGTGCCGTTATTGAGGTTGACGCAAGTGAGATAGATCAGCGTTGTGCCGTAGAGACTGCTAGGGGCTGGCTGAGCCAT 600 GACTGCGACAGGGACTCCGATCACGCTGATGGCGCTGACCACTGCTGCCCCACTCAAAACTCGCATGCTGCACTCCTCAGGGMATCTCTGTGGCCTTAGCCTACCCAGCTGAGGGATTGT 720 CTGCACTTATC6ACTCCATCGGATTTTGCAAAGCTTGTAGGCGGGCGATCGCTTCTCCGCGATAATGCTTCTCAGTCCCGAGTATCACTGGCATGCGCAAGCTCATCGAGGGGTTACGGC 840 gag N R K L I E G L R H XhoI ATTTCCGTACGTCCTACTACCCGTCTCATCGGGACCTGTTCGAGCAGTTTGCCAAAGGTCAGCACCCTCGAGTCCTGTTCATTACCTGCTCAGACTCGCGCATTGACCCTMACCTCATTA 960 F R T S Y Y P S H R D L F E Q F A K 6 Q H P R V L F I T C S D S R I D P N L I T CCCAGTCGGGCATGGGTGAGCTGTTCGTCATTCGCAACGCTGGCAATCTGATCCCGCCCTTCGGTGCCGCCMACGGTGGTGMAGGGGCATCGATCGMATACGCGATCGCAGCTTTGAACA 1080 Q S 6 N 6 E L F V I R N A 6 N L I PP F 6 A A N 6 6 E 6 A S I E Y A I A A L N I SphI TTGAGCATGTTGTGGTCTGCGGTCACTCGCACTGCGGTGCGATGAAAGGGCTGCTCAAGCTCAATCAGCTGCMGAGGACATGCCGCTGGTCTATGACTGGCTGCAGCATGCCCAAGCCA 1200 E H VV V C G H S H C G A M K G LL K L N Q L Q E D M P L V Y D WI L Q H A Q A T CCCGCCGCCTAGTCTTGGATACTACAGCGGTTATGAGACTGSACGACTTGGTAGAGATTCTGGTCGCCGAGAATGTGCTGACGCAGATCGAGAACCTTAAGACCTACCCGATCGTGCGAT 1320 R R L V L D N Y S G Y E T DD L V E I L V A E N V L T Q I E N L K T Y P I V R S CGCGCCTTTTCCAAGGCAGCTGCAGATTTTTGGCTGGATTTATGAAGTTGAAAGCGGCGAGGTCTTGCAGATTAGCCGTACCAGCAGTGATGACACAGGCATTGATGAATGTCCAGTGC 1440 R L F Q 6 K L Q I F 6 W I Y E V E S 6 E V L Q I S R T S S D D T 6 I D E C P V R GTTTGCCCGGCAGCCAGGAGAAAGCCATTCTCGGTCGTTGTGTCGTCCCCCTGACCGAAGAAGTGGCCGTTGCTCCACCAGAGCCGGAGCCTGTGATCGCGGCTGTGGCGGCTCCACCCG 1560 L P G S Q E K A I L G R C V V P L T E E V A V A PP E P E P V I A A V A A P P A CCAACTACTCCAGTCGCGGTTGGTTGGCCCCTGAACAACAACAGCGGATTTATCGCGGCAATGCTAGCTAGGATCGAAGCATCTTCGACCCTGCTGAGATCT 1662 NY S S R G W L A P E Q Q Q R I Y R G N A S * FIG. 2. Nucleotide sequence of the 1662-bp Sac I-Bgl II region of Synechococcus sp. PCC7942 genome. The deduced amino acid sequence ofthe gene icfA (ORF272) is shown below the nucleotide sequence. An asterisk indicates translation termination codon. Restriction sites (HincII, Xho I, and Sph I) at which Kmr gene cartridges were inserted are indicated. The putative ribosome-binding site is shown as gag. Downloaded by guest on September 25, 2021 Biochemistry: Fukuzawa et al. Proc. Natl. Acad. Sci. USA 89 (1992) 4439 ordinary air (low CO2 condition). Transformants obtained amount offixed carbon in strains XH-5 and SP-2 at 80 sec was with pHC::Km grew both in high and low CO2 conditions. In 0.163 and 0.079 nmol/,ul of cell volume, which were only contrast, those obtained with pXH::Km and pSP::Km could 5.0%o and 2.4% of that in the wt strain, respectively. The grow in high CO2 conditions but not in low CO2 conditions. activity of CO2 fixation in XH-5 and SP-2 cells was signifi- In one exception, a colony transformed with pXH::Km grew cantly decreased, although the activity of Ci accumulation in low CO2 conditions, probably due to an insertion of the was as high as in the wt strain. Strains XH-5 and SP-2, but not Kmr gene outside the chromosomal region of interest, and HC-2, therefore, were defective in utilization of intracellu- this clone was not examined further. One colony was selected larly accumulated CQ. Activities of RuBP carboxylase in cell from each of the three types of the transformants. These extracts ofthe kanamycin-resistant transformants cultured in strains were designated HC-2, XH-5, and SP-2. low CO2 conditions were comparable to that in wt cells as In each case the mutagenized genomic fragment on the shown (13). donor plasmids was substituted for wt genomic region on the chromosome through homologous recombination, resulting DISCUSSION in the targeted insertional mutagenesis. The insertion of the Kmr gene at the target sites was confirmed by Southern blot An ORF that could code for a polypeptide of 272 amino acids analysis of the total DNA from the transformants with the was found in the genomic region and was shown to comple- 1.3-kb Kmr gene cartridge or the 3.8-kb BamHI fragment of ment a temperature-sensitive high-CO2-requiring mutant of wt genome as probes (data not shown). Synechococcus PCC7942. Since, in our initial sequence de- termination, we missed a cytidine residue at position 1019 in Characterization of the Kanamycin-Resistant Transform- Fig. 2, no significant ORFs were predicted in the genomic ants. Growth of the kanamycin-resistant transformants in region. Revision of the sequence data by correcting this liquid culture was examined at various concentrations ofCO2 residue led us to identify the ORF272. When the Kmr gene by monitoring cell density. At first kanamycin-resistant was inserted within the coding region of ORF272, transform- transformants were divided into two culture bottles at the ants XH-5 and SP-2 required 5% CO2 for growth in liquid same cell density, and cultures were started by bubbling with medium and on agar plates. On the other hand, insertion at 5% C02/95% air. After the OD720 reached 0.1 unit, the the HincII site, which is 275 bp upstream from the coding bubbling gas in one of the two bottles was changed to air region ofthe 0RF272 in the transformant HC-2, did not affect (0.04% C02) for -24 hr. The concentration of CO2 was then the growth rate in low CO2 conditions. In insertional mutants changed back to 5%. Transformant HC-2 showed growth XH-5 and SP-2, the activity of CO2 fixation was <5% of that rates in 5% and in 0.04% CO2 (ordinary air) comparable to in wt cells whereas that of C, accumulation was as high as in those of wt cells. On the other hand, growth ofXH-5 and SP-2 wt cells (Fig. 3). These results indicate that a gene involved stopped when the CO2 concentration was lowered from 5% in efficient fixation of C, was disrupted by the insertion at the to 0.04%. Their growth was restored wherrthe CO2 level was Xho I and the Sph I sites. Therefore, the ORF272 containing changed back to 5% (data not shown). These results dem- both of the restriction sites should play a pivotal role in C, onstrate that the two strains XH-5 and SP-2 require high CO2 fixation and was designated as the gene icfA. concentration (5%) for growth but that HC-2 does not. Activities of RuBP carboxylase in extracts of the kanamy- The accumulation and fixation of Ci in the kanamycin- cin-resistant transformants XH-5 and SP-2 were comparable resistant transformants and wt cells were determined by the to that in wt cells, as if the icfA gene product is not involved silicone oil centrifugation method by adding 50 ,uM in the assembly or activation of the CO2 fixing enzyme. The NaH14CO3 as a carbon source (Fig. 3). The three gene- function of the icfA gene product as a CA would be to disrupted strains ofcells and wt cells were able to accumulate replenish CO2 from HCO for RuBP carboxylase. C, to high levels. The amount of fixed carbon measured as A search for data bases indicated that the deduced amino acid-stable 14C after 80 sec was 3.28 and 2.04 nmol/,l of cell acid sequence of the icfA gene product showed significant volume in the wt and HC-2 strains. On the other hand, the similarities to sequences of chloroplast CAs from pea (22% identity; ref. 20) and spinach (22%; ref. 21) and of the E. coli AlI 4 - HC-2 cynT product (31%; ref. 22; Fig. 4). The cynT gene product,

.-% previously considered to be a cyanate permease (23), is a CA 3F whose function is to catalyze the formation of HCO from CO2 formed in the HCO--dependent decomposition of cy- En 2 anate by cyanase (M. Guilloton and P. M. Anderson, per- sonal communication). These sequence similarities suggest

_ 1 that the gene icfA encodes CA in cyanobacteria. A transit E peptide found in the plant chloroplast CAs that is necessary L---j- 04 for transport of the protein into chloroplasts does not occur in icfA and cynTproducts. Five amino acids, Cys-39, Glu-82, o 4 8 SP-2 His-98, Cys-101, and Glu-153 (in icfA numbering), which are ' ' candidates for zinc binding, were found to be conserved in all a'6 . of the prokaryotic-type CAs. Since the icfA gene product has *- no membrane-spanning domain, this putative CA may not be

x located in the lipid membrane. The COOH-terminal region of 14- the icfA product was not conserved in other CAs, suggesting 0 1 2l that this COOH-terminal domain may be necessary to be UL assembled in the carboxysome. The occurrence of CA activity has also been detected in 0 40 80 0 40 80 Synechococcus PCC7942 by Badger and Price (24). They Time (sec) suggested that CA is located in the carboxysomes because of FIG. 3. Time courses of intracellular C, concentration and the observation that expression of a high level of CA from amount of fixed "4C-labeled carbon in the wt strain and mutants human in the cytoplasm of PCC7942 resulted in a high CO2 HC-2, XH-5, and SP-2, as determined by the silicone oil centrifu- requirement in the transformant (8). Carboxysomes are poly- gation method at 30'C. hedral protein complexes consisting mainly of RuBP carbox- Downloaded by guest on September 25, 2021 4440 Biochemistry: Fukuzawa et al. Proc. Natl. Acad. Sci. USA 89 (1992) 0 IcfA MRKLIEGLRHFRTSYYPSHRDLFEQFAKGQHPRVLFITCSDSRIDPNLITQSGNGELFVI 60 CynT :KEI:D:FLK:QREAF:KREA::K:L:TQ:S::T:::S:::::LV:E:V::REP:D:::: 60 S-CA 98-ELADGGTPSASYPVQRIK::FIK:KKEK:EKNPA:YGELS:::A:KFMVFA:::::VC:SHVLDFQP::AFMV 73 P-CA 104-QLGTTSSSDGIPKSEASERIKT:FL::KKEK:DKNPA:YGEL::::S:PFMVFA:::::VC:SHVLDFQP::AFVV 76

IcfA RNAGNLIPPFGAAN-GGEGASIEYAIAALNIEHVVVCGHSHCGAMKGLLKLNQLQED-MPLVYDWLQHAQATRRLVLDNY 138 CynT :::::IV:SY:PEP-::VS::V:::V:::RVSDI:I::::N::::TAIASC-:C-M:H::A:SH::RY:DSA-:V:NEAR 136 S-CA ::IA:MV:V:DKDKYA:V::A::::VLH:KV:NI::I:::A::6I:::MSFPDAGPTTTDFIE::VKICLPAKHK::AEH 153 P-CA ::VA::V::YDQRKYA:T::A::::VLH:KVSNI::I:::A::GI::::SFPFDGTYSTDFIEE:VKIGLPAKAK:KAQH 156 0 IcfA SGYETDDLVEILVAENVLTQIENLKTYPIVRSRLFQGKLQIFGWIYEVESGEVLQISRTSSDDTGIDECPVRLPGSQEKA 218 CynT PHSDLPSKAAAM:R:::IA:LA::Q:H:S::LA:EE:G-SLH::V:DI:::SIAAFDGATRQFVPLAAN:RVCAIRLRQP 215 S-CA GNATFAEQCTHCEK:A:NVSLG::L:::F::DG:VKKT:ALQ:GY:DFVN:SFELWGLEYGLSPSQSV...... 221 P-CA GDAPFAE:CTHCEK:A:NASLG::L:::F::EG:VNKT:ALKVGY:DFVK:SFELWGLEFGLSSTFSV...... 224

IcfA ILGRCVVPLTEEVAVAPPEPEPVIAAVAAPPANYSSRGWLAPEQQQRIYRGNAS 272 CynT TAA...... 218 S-CA P-CA FIG. 4. Amino acid sequence comparison among the icfA gene product (IcfA), E. coli CA (CynT), and mature plant chloroplast CAs from spinach (S-CA) and pea (P-CA). Amino acid residues identical to those ofthe icfA gene product are indicated by colons. Length oftransit peptides in the precursor chloroplast CAs is indicated as numbers of amino acids at the NHrterminal residues of mature chloroplast CAs. The IcfA sequence extends beyond the other sequences at the COOH terminus. Possible zinc-binding amino acid residues (cysteine, histidine, and glutamic acid) are marked by solid circles. ylase/oxygenase found in some autotrophic prokaryotes (5). II mutants in that they cannot fix intracellularly accumulated It has been proposed that the conversion of intracellularly C, yet they have normal-shaped carboxysomes as examined accumulated HCO- into CO2 is catalyzed by CA located by electron microscopy (data not shown). In addition, the within the carboxysomes (9, 12). However, no direct evi- type II mutants were complemented with 3.5-kb BamHI dence for CA in the carboxysomes has been obtained in genomic DNA fragments from wt cells. The restriction map Synechococcus. Since the activity of C, accumulation is of the 3.5-kb BamHI genomic region that complements the inhibited with ethoxzolamide, a potent CA inhibitor, some type II mutants is almost identical to that ofthe 3.8-kb BamHI components related to CA have been proposed to operate that we characterized (J.-W. Yu, G. D. Price, and M. R. also in the Ci transport step. However, the cells carrying a Badger, personal communication). Therefore, it is highly disruption of icfA that encodes the putative CA constructed possible that the icfA encodes carboxysomal CA and that this in this study accumulated CQ. Therefore, another CA isozyme gene is contained in the 3.5-kb BamHI genomic DNA that or CA-related protein may be operating in the transport complements the type II mutants. systems. In terrestrial plants, CA is located in chloroplasts of C3 Price and Badger (25) reported two types of high-CO2- plants and in the cytosol of mesophyll cells of C4 plants (26). requiring Synechococcus mutants, type I and type II, that are In C4 plants, CA is assumed to catalyze the hydration of CO2 defective in utilization of intracellularly accumulated CQ. to HCO-, which is the substrate for phosphoenolpyruvate Type I mutants have long rod-like carboxysomes and the type carboxylase (27). Chloroplast CAs from C3 plants show a high II mutants have normal polyhedral carboxysomes. They degree of sequence similarity (-76% identity) to each other. speculated that in the type I mutants the fast CO2 efflux to the Cytosolic CA from maize also has sequence similarity to cytosol could be caused by a mislocation of CA, which is chloroplast CAs at -60% identity (J. Burnell, personal com- normally located in the carboxysomes. On the other hand, munication). Since plant chloroplast CAs have sequence the type II mutants may be lacking carboxysomal CA, similarities with the cyanobacterial icfA product and E. coli although a normal level of CA activity was detected in the CAs, they have probably evolved from a common ancestor extract of type II mutants. Characteristics of the icfA- (protoprokaryote CA, Fig. SB). In contrast, the icfA gene defective mutants XH-5 and SP-2 seem to be similar to type product and plant chloroplast CAs have no significant se- with or the CA I quence similarity mammalian CA isozymes (28) CA1l j Amniotes Chlamydomonas periplasmic CAs (29-31). These findings CA III (consensus) suggest that CAs can be divided into two groups and that they Red Cell CA (Shark) have evolved from two distinct ancestors, a protoeukaryote CA VII (Human) CA (Fig. SA) and a protoprokaryote CA (Fig. SB). CA "Y" (Mouse) A CA IV (Human) This work was supported by Grant-in-Aid for Scientific Research A CA VI (Human, Sheep) from the Japanese Ministry of Education, Science and Culture and CAI (Chiamydomonas) a grant from the Japanese Ministry of Agriculture, Forestry and CA2 (Chiamydomonas) Fisheries. Chloroplast CA (Spinach) 1. Aizawa, K. & Miyachi, S. (1986) FEMS Microbiol. Rev. 39, B n | Chioroplast CA (Pea) 215-233. IcfA (Synechococcus) 2. Kaplan, A., Badger, M. R. & Berry, J. A. (1980) Planta 149, CynT (E. coil) 219-226. 3. Miller, A. G. & Colman, B. (1980) J. Bacteriol. 143, 1253-1259. FIG. 5. Generalized phylogenetic branching scheme for CAs 4. Ogawa, T. (1991) Proc. Natl. Acad. Sci. USA 88, 4275-4279. from mammals, alga, plants, and bacteria based on amino acid 5. Codd, G. A. & Marsden, W. J. N. (1984) Biol. Rev. 59, 389- sequence comparison. Relationship of mammalian CAs and Chlam- 422. ydomonas CA1 is from the ref. 28. The amino acid sequence of 6. Price, G. D. & Badger, M. R. (1989) Plant Physiol. 89, 37-43. Chlamydomonas CA2 is from the ref. 29. Eukaryotic and prokaryotic 7. Abe, T., Tsuzuki, M. & Miyachi, S. (1987) Plant Cell Physiol. ancestors of CAs are indicated as A and B, respectively. 28, 867-874. Downloaded by guest on September 25, 2021 Biochemistry: Fukuzawa et al. Proc. Nati. Acad. Sci. USA 89 (1992) 4441

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