Proc. Nati. Acad. Sci. USAVol. 89, pp. 4437-4441, May 1992Biochemistry

A gene homologous to chloroplast carbonic anhydrase (icfA) isessential to photosynthetic carbon dioxide fixation bySynechococcus 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-anism in cyanobacteria, a genomic DNA firgment that com-plements a temperature-sensitive high-CO2 (5%)-requiringmutant of Synechococcus PCC7942 has been isolated. An openreading frame (0RF272) encoding a polypeptide of 272 aminoacids (Mr, 30,184) was found within the genomic region located20 kilobases downstream from the genes for ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcLS). Insertion of akanamycin-resistance gene cartridge within the 0RF272 inwild-type cells led to a high-CO2-requiring phenotype. Strainscarrying a gene disabled by insertional mutagenesis accumu-lated inorganic carbon in the cells, but they could not fix itefficiently, even though ribulose-1,5-bisphosphate carboxylaseactivity was comparable to that of the wild-type strain. There-fore, the 0RF272 was designated as a gene iwfA, which isessential to inorganic carbon fixation. Furthermore, the pre-dicted icfA gene product shared significant sequence similari-ties with plant chloroplast carbonic anhydrases (CAs) from pea(22%) and spinach (22%) and also with the Escherichia colicynT gene product (31%), which was recently identified to beE. coli CA. These results indicate that the putative CA encodedby iwfA is essential to photosynthetic carbon dioxide fixation incyanobacteria and that plant chloroplast CAs may have evolvedfrom a common ancestor of the prokaryotic CAs, which aredistinct from mammalian CAs and Chlamydomonas periplas-mic CAs.

Cyanobacteria have a very high affinity for environmentalCO2 as compared to most higher plants (1). This high affinityhas been assumed to be due to the operation of a C02-concentrating mechanism (2, 3), which can be divided into atleast two steps: (i) accumulation of inorganic carbon (C1) inthe cells by a putative energy-dependent transporter (2-4)and (ii) efficient CO2 fixation within the carboxysomes wherethe majority of active ribulose-1,5-bisphosphate (RuBP) car-boxylase is located (5). Since C, accumulation is inhibited byethoxzolamide, an inhibitor of carbonic anhydrase (CA),some component related to CA is assumed to operate in theCi transport step (6, 7). It has been suggested that thecarboxysome serves as the permeability barrier for CO2 toprevent its leakage out ofthe cells (8) and contains CA, whichcatalyzes dehydration of HCO- (9). This assumption issupported by the observation that Synechococcus PCC7942cells that express human CA in the cytoplasm require highCO2 (5%) for growth (8). However, the significance ofCA inthe C02-concentrating mechanism is not fully established.

Several high-CO2-requiring mutants have been isolated andopen reading frames (ORFs) responsible for their defectshave been identified (4, 10-12). We have reported (13) thecloning of a genomic region that complements a temperature-

sensitive high-CO2-requiring mutant of SynechococcusPCC7942. This strain is defective in the utilization of intra-cellularly accumulated Ci at nonpermissive temperatures(13). The genomic region complementing this mutation islocated .20 kilobases (kb) downstream of the structuralgenes for RuBP carboxylase/oxygenase subunits (rbcLS).We describe here the physiological characterization of

mutants in which an ORE within the genomic region wasdisrupted by insertional mutagenesis and identification of agene that should be responsible for mediating CO2 fixation inthe cyanobacteria. Since the amino acid sequence ofthe ORFdeduced from the nucleotide sequence¶ showed significantsimilarity to those of plant chloroplast CAs and Escherichiacoli CA, this ORF is assumed to be a gene encoding cyano-bacterial CA.

MATERIALS AND METHODSStrains and Growth Conditions. Wild-type (wt) and mutant

cells of Synechococcus sp. PCC7942 were grown in BG-11medium (14) buffered with 30 mM Hepes-NaOH (pH 7.8) inan atmosphere of 5% C02/95% air under continuous illumi-nation at 3.5 W/m2.

Construction of Plasmids and Transformation. GenomicDNA fragments of Synechococcus PCC7942 in plasmidpBM3.8 (13) were subcloned into pUC19 and pBluescript IIKS+ (Stratagene). DNA fragments encoding an aminoglyco-side 3'-phosphotransferase of 1.3 kb, isolated from pUC4K(15) and pUC4KISS (16), were inserted into HincII, Xho I,and Sph I sites of the Synechococcus DNA to generaterecombinant plasmids for gene disruption and designatedpHC::Km, pXH::Km, and pSP::Km, respectively. Theseplasmids (10 ug) were used for transformation of Synecho-coccus PCC7942 (17). Transformants were selected on agarplates containing kanamycin at 10 gg/ml in 5% C02/95% air.

Hybridization and Sequence Analysis. DNA fragments werelabeled with [32P]dCTP using a random-primer labeling kit(Amersham) and used in Southern blot hybridizations asprobes. The nucleotide sequences of DNA fragments sub-cloned in M13mp18 and M13mpl9 were determined using adeazadideoxynucleotide sequencing kit (Takara Shuzo, Ky-

Abbreviations: CA, carbonic anhydrase; C;, inorganic carbon; Kmr,kanamycin resistance; ORF, open reading frame; RuBP, ribulose1,5-bisphosphate; wt, wild type.*To whom reprint requests should be sent at the present address:Department of Agricultural Chemistry, Faculty of Agriculture,Kyoto University, Kyoto 606-01, Japan.

tPresent address: Department of Biology, Faculty of Science, Ibar-aki University, Mito 310, Japan.tPresent address: Department of Biology, Faculty of Science,Nagoya University, Nagoya 464-01, Japan.§Present address: Marine Biotechnology Institute, Co. Ltd., 2-35-10Hongo, Tokyo 113, Japan.1The sequence reported in this paper has been deposited in theGenBank data base (accession no. M77095).

4437

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Oct

ober

5, 2

020

4438 Biochemistry: Fukuzawa et al.

oto). The sequences obtained were compiled and analyzed bya software DNASIS (Hitachi, Tokyo) using protein data bases,National Biomedical Research Foundation Protein Identifi-cation Resource (release 26.0) and Swiss-Prot (release 15.0).For construction of phylogenetic tree, the software reportedin ref. 18 was used.Other Measurements. The cell density of a culture was

determined by measuring the OD at 720 nm. Accumulationand fixation of Ci were determined by the silicone oil cen-trifugation method (13) at 300C. NaH14CO3 was added to theassay mixture at 50 uM. RuBP carboxylase activity wasassayed by the incorporation of 14Co2 into the acid-stablefraction at 300C (19).

RESULTSNucleotide Sequence of the Genomic Region. We have

cloned (13) a genomic DNA fragment that complements thetemperature-sensitive high-C02-requiring mutant C3P-O.Complementation occurred with plasmids containing 36-kbEcoRI, 3.8-kb BamHI, 0.87-kb Pst I, and 15-kb HindIlIfragments, all of which overlapped in a 0.43-kb HindIII-PstI region as shown in Fig. 1. This region was located 20 kbdownstream from the coding region for subunits of RuBPcarboxylase/oxygenase.To characterize the genomic region that complements the

mutation, the nucleotide sequence was determined for the1662-base-pair (bp) Sac I-Bgl II region (Fig. 2) that containsthe 0.43-kb HindIII-Pst I region (see also Fig. 1). An ORF(0RF272) possibly encoding a polypeptide of272 amino acids(M, 30,184) was found in the HindIII-Bgl II region thatoverlapped the 0.43-kb HindIII-Pst I region. The codingregion of0RF272 begins 61 bp downstream from HindIll siteand ends 28 bp upstream from Bgl II site. A possibleribosome-binding sequence (GAG) was found 10 bp upstreamfrom the translation initiation codon.

Construction of Insertional Mutants of Cyanobacteria. Thesignificance of ORF272 was examined by insertion of a Kmrgene cartridge at HincII, Xho I, and Sph I sites inside oroutside the coding region of the ORF272 in wt cells (Fig. 1).(i) For insertional mutagenesis at the HincII site, which isoutside ORF272, a 1.0-kb BamHI-Xho I fragment, which is

SpBa Sc P P Hc SpHd X PSp P Bg

11 I I

A A A

ATG

ictA (ORF272)

FIG. 1. Restriction map of the 1.7-kb BamHI-Bgl II genomicregion and location of iefA (ORF272). Solid bar, the 0.43-kb HindIII-Pst I region that complements a temperature-sensitive high-CO2-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) genecartridge was inserted; Ba, BamHI; Bg, Bgi II; Hc, HinclI, Hd,HindIII; P, Pst I; Sc, Sac I; Sp, Sph I; X, Xho I.

the left portion of the 3.8-kb BamHI fragment in the plasmidpBM3.8 (13), was subcloned into BamHI-Sal I-digestedpUC19. This plasmid designated as pBX1.0 was linearized bycutting at the unique HinclI site and ligated with the 1.3-kbHincHI Kmr gene cartridge. The recombinant plasmidpHC::Km was used in the transformation ofwt cells to obtaininsertional mutants at the HinclI site. (ii) For insertionalmutagenesis at the Xho I site located inside the ORF272, a1.7-kb BamHI-Bgl II fragment (Fig. 1) was subcloned intopUC19 and named pBB1.7. Plasmid pBB1.7 was linearizedwith Xho I and ligated with the 1.3-kb Sal I Kmr genecartridge to obtain the plasmid pXH: :Km. (iii) For insertionalmutagenesis at the Sph I site located also in 0RF272, a 1.7-kbXho I fragment (whose left end is shown in Fig. 1) in theplasmid pBM3.8 (13) was inserted into pBluescript II KS+.The recombinant plasmid designated pXH1.7 was linearizedby Sph I and ligated with the 1.3-kb Sph I Kmrgene cartridge.The generated plasmid was designated pSP::Km.The kanamycin-resistant derivatives of Synechococcus

PCC7942 were selected on kanamycin-containing agar platesafter transformation of wt cells with plasmids pHC::Km,pXH::Km, and pSP::Km. Fifty colonies of each type of thekanamycin-resistant transformants were replica-plated andincubated in 5% C02/95% air (high CO2 condition) and in

GAGCTCGCCGTTATCMATTTCGGTGGTGGTGCCTAGGCCMATCACCTCCCCTTGCAGAGGAATCTGACGMATAGMATAGTGTTCGCTGATGTCACCTTGAATCAGTGCCGTCACAGTGTTAGCGACAGTAAGTTTCAACATCTCTTCGCCTAGATAGGGGCTGTTCTCCAAATCCTGTGGGTAGTGATCCAAGCTGTGGAGTAGCCTGCAGCGATCGCGCCTAGTGCTTTGCTGTATGGCGTCTCGCTAAAAAGGAGCGGGTGTAAAAGTTGTATTGAAAATCGAGTTAATTTGGTTGATAGTTTCAACCGACTGCAGATCAAMGCGCTCCTTGTAAGTAGGGCTGGCTTTGGAGTTGTTGAGCMACATCCTGATTGATTTGCCTGCTTTCCTGCTTGAGTCGAGTCTGGATTGCCACCGTTTCTTCAGGCGTAATCTTGCCATCTTGTAGAGAGTTACAMATTGGCAGCAGCGCATT

HincIIGTATTGGCGATCGCTCAGACTGGAGAGATCCTGACGGGTMATCGTGCCGTTATTGAGGTTGACGCAAGTGAGATAGATCAGCGTTGTGCCGTAGAGACTGCTAGGGGCTGGCTGAGCCATGACTGCGACAGGGACTCCGATCACGCTGATGGCGCTGACCACTGCTGCCCCACTCAAAACTCGCATGCTGCACTCCTCAGGGMATCTCTGTGGCCTTAGCCTACCCAGCTGAGGGATTGTCTGCACTTATC6ACTCCATCGGATTTTGCAAAGCTTGTAGGCGGGCGATCGCTTCTCCGCGATAATGCTTCTCAGTCCCGAGTATCACTGGCATGCGCAAGCTCATCGAGGGGTTACGGC

gag N R K L I E G L R HXhoI

ATTTCCGTACGTCCTACTACCCGTCTCATCGGGACCTGTTCGAGCAGTTTGCCAAAGGTCAGCACCCTCGAGTCCTGTTCATTACCTGCTCAGACTCGCGCATTGACCCTMACCTCATTAF 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

CCCAGTCGGGCATGGGTGAGCTGTTCGTCATTCGCAACGCTGGCAATCTGATCCCGCCCTTCGGTGCCGCCMACGGTGGTGMAGGGGCATCGATCGMATACGCGATCGCAGCTTTGAACAQ S 6 N 6 E L F V I R N A 6 N L I P P F 6 A A N 6 6 E 6 A S I E Y A I A A L N I

SphITTGAGCATGTTGTGGTCTGCGGTCACTCGCACTGCGGTGCGATGAAAGGGCTGCTCAAGCTCAATCAGCTGCMGAGGACATGCCGCTGGTCTATGACTGGCTGCAGCATGCCCAAGCCA

E H V V V C G H S H C G A M K G L L K L N Q L Q E D M P L V Y D WI L Q H A Q A T

120240360480

600720840

960

1080

1200

CCCGCCGCCTAGTCTTGGATACTACAGCGGTTATGAGACTGSACGACTTGGTAGAGATTCTGGTCGCCGAGAATGTGCTGACGCAGATCGAGAACCTTAAGACCTACCCGATCGTGCGAT 1320R R L V L D N Y S G Y E T D D 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

CGCGCCTTTTCCAAGGCAGCTGCAGATTTTTGGCTGGATTTATGAAGTTGAAAGCGGCGAGGTCTTGCAGATTAGCCGTACCAGCAGTGATGACACAGGCATTGATGAATGTCCAGTGCR 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

1440

GTTTGCCCGGCAGCCAGGAGAAAGCCATTCTCGGTCGTTGTGTCGTCCCCCTGACCGAAGAAGTGGCCGTTGCTCCACCAGAGCCGGAGCCTGTGATCGCGGCTGTGGCGGCTCCACCCG 1560L P G S Q E K A I L G R C V V P L T E E V A V A P P E P E P V I A A V A A P P A

CCAACTACTCCAGTCGCGGTTGGTTGGCCCCTGAACAACAACAGCGGATTTATCGCGGCAATGCTAGCTAGGATCGAAGCATCTTCGACCCTGCTGAGATCTN Y S S R G W L A P E Q Q Q R I Y R G N A S *

1662

FIG. 2. Nucleotide sequence of the 1662-bp Sac I-Bgl II region of Synechococcus sp. PCC7942 genome. The deduced amino acid sequenceofthe 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.

Proc. Natl. Acad. Sci. USA 89 (1992)

Dow

nloa

ded

by g

uest

on

Oct

ober

5, 2

020

Proc. Natl. Acad. Sci. USA 89 (1992) 4439

ordinary air (low CO2 condition). Transformants obtainedwith pHC::Km grew both in high and low CO2 conditions. Incontrast, those obtained with pXH::Km and pSP::Km couldgrow in high CO2 conditions but not in low CO2 conditions.In one exception, a colony transformed with pXH::Km grewin low CO2 conditions, probably due to an insertion of theKmr gene outside the chromosomal region of interest, andthis clone was not examined further. One colony was selectedfrom each of the three types of the transformants. Thesestrains were designated HC-2, XH-5, and SP-2.

In each case the mutagenized genomic fragment on thedonor plasmids was substituted for wt genomic region on thechromosome through homologous recombination, resultingin the targeted insertional mutagenesis. The insertion of theKmr gene at the target sites was confirmed by Southern blotanalysis of the total DNA from the transformants with the1.3-kb Kmr gene cartridge or the 3.8-kb BamHI fragment ofwt genome as probes (data not shown).

Characterization of the Kanamycin-Resistant Transform-ants. Growth of the kanamycin-resistant transformants inliquid culture was examined at various concentrations ofCO2by monitoring cell density. At first kanamycin-resistanttransformants were divided into two culture bottles at thesame cell density, and cultures were started by bubbling with5% C02/95% air. After the OD720 reached 0.1 unit, thebubbling gas in one of the two bottles was changed to air(0.04% C02) for -24 hr. The concentration of CO2 was thenchanged back to 5%. Transformant HC-2 showed growthrates in 5% and in 0.04% CO2 (ordinary air) comparable tothose of wt cells. On the other hand, growth ofXH-5 and SP-2stopped when the CO2 concentration was lowered from 5%to 0.04%. Their growth was restored wherrthe CO2 level waschanged back to 5% (data not shown). These results dem-onstrate that the two strains XH-5 and SP-2 require high CO2concentration (5%) for growth but that HC-2 does not.The accumulation and fixation of Ci in the kanamycin-

resistant transformants and wt cells were determined by thesilicone oil centrifugation method by adding 50 ,uMNaH14CO3 as a carbon source (Fig. 3). The three gene-disrupted strains of cells and wt cells were able to accumulateC, to high levels. The amount of fixed carbon measured asacid-stable 14C after 80 sec was 3.28 and 2.04 nmol/,l of cellvolume in the wt and HC-2 strains. On the other hand, the

AlI

.-%

En

E_

o 4' '

a'*-

x

14-

0UL

4

2

1

04

8

1 2l

0 40 80 0

Time (sec)

- HC-2

SP-2

40 80

FIG. 3. Time courses of intracellular C, concentration andamount of fixed "4C-labeled carbon in the wt strain and mutantsHC-2, XH-5, and SP-2, as determined by the silicone oil centrifu-gation method at 30'C.

amount offixed carbon in strains XH-5 and SP-2 at 80 sec was0.163 and 0.079 nmol/,ul of cell volume, which were only5.0%o and 2.4% of that in the wt strain, respectively. Theactivity of CO2 fixation in XH-5 and SP-2 cells was signifi-cantly decreased, although the activity of Ci accumulationwas as high as in the wt strain. Strains XH-5 and SP-2, but notHC-2, therefore, were defective in utilization of intracellu-larly accumulated CQ. Activities of RuBP carboxylase in cellextracts of the kanamycin-resistant transformants cultured inlow CO2 conditions were comparable to that in wt cells asshown (13).

DISCUSSIONAn ORF that could code for a polypeptide of 272 amino acidswas found in the genomic region and was shown to comple-ment a temperature-sensitive high-CO2-requiring mutant ofSynechococcus PCC7942. Since, in our initial sequence de-termination, we missed a cytidine residue at position 1019 inFig. 2, no significant ORFs were predicted in the genomicregion. Revision of the sequence data by correcting thisresidue led us to identify the ORF272. When the Kmr genewas inserted within the coding region of ORF272, transform-ants XH-5 and SP-2 required 5% CO2 for growth in liquidmedium and on agar plates. On the other hand, insertion atthe HincII site, which is 275 bp upstream from the codingregion ofthe 0RF272 in the transformant HC-2, did not affectthe growth rate in low CO2 conditions. In insertional mutantsXH-5 and SP-2, the activity of CO2 fixation was <5% of thatin wt cells whereas that of C, accumulation was as high as inwt cells (Fig. 3). These results indicate that a gene involvedin efficient fixation of C, was disrupted by the insertion at theXho I and the Sph I sites. Therefore, the ORF272 containingboth of the restriction sites should play a pivotal role in C,fixation and was designated as the gene icfA.

Activities of RuBP carboxylase in extracts of the kanamy-cin-resistant transformants XH-5 and SP-2 were comparableto that in wt cells, as if the icfA gene product is not involvedin the assembly or activation of the CO2 fixing enzyme. Thefunction of the icfA gene product as a CA would be toreplenish CO2 from HCO for RuBP carboxylase.A search for data bases indicated that the deduced amino

acid sequence of the icfA gene product showed significantsimilarities to sequences of chloroplast CAs from pea (22%identity; ref. 20) and spinach (22%; ref. 21) and of the E. colicynT product (31%; ref. 22; Fig. 4). The cynT gene product,previously considered to be a cyanate permease (23), is a CAwhose function is to catalyze the formation of HCO fromCO2 formed in the HCO--dependent decomposition of cy-anate by cyanase (M. Guilloton and P. M. Anderson, per-sonal communication). These sequence similarities suggestthat the gene icfA encodes CA in cyanobacteria. A transitpeptide found in the plant chloroplast CAs that is necessaryfor transport of the protein into chloroplasts does not occurin icfA and cynT products. Five amino acids, Cys-39, Glu-82,His-98, Cys-101, and Glu-153 (in icfA numbering), which arecandidates for zinc binding, were found to be conserved in allof the prokaryotic-type CAs. Since the icfA gene product hasno membrane-spanning domain, this putative CA may not belocated in the lipid membrane. The COOH-terminal region ofthe icfA product was not conserved in other CAs, suggestingthat this COOH-terminal domain may be necessary to beassembled in the carboxysome.The occurrence of CA activity has also been detected in

Synechococcus PCC7942 by Badger and Price (24). Theysuggested that CA is located in the carboxysomes because ofthe observation that expression of a high level of CA fromhuman in the cytoplasm of PCC7942 resulted in a high CO2requirement in the transformant (8). Carboxysomes are poly-hedral protein complexes consisting mainly ofRuBP carbox-

Biochemistry: Fukuzawa et al.

L---j-

3F

6 .

Dow

nloa

ded

by g

uest

on

Oct

ober

5, 2

020

4440 Biochemistry: Fukuzawa et al.

0IcfA MRKLIEGLRHFRTSYYPSHRDLFEQFAKGQHPRVLFITCSDSRIDPNLITQSGNGELFVICynT :KEI:D:FLK:QREAF:KREA::K:L:TQ:S::T:::S:::::LV:E:V::REP:D::::S-CA 98-ELADGGTPSASYPVQRIK::FIK:KKEK:EKNPA:YGELS:::A:KFMVFA:::::VC:SHVLDFQP::AFMVP-CA 104-QLGTTSSSDGIPKSEASERIKT:FL::KKEK:DKNPA:YGEL::::S:PFMVFA:::::VC:SHVLDFQP::AFVV

IcfA RNAGNLIPPFGAAN-GGEGASIEYAIAALNIEHVVVCGHSHCGAMKGLLKLNQLQED-MPLVYDWLQHAQATRRLVLDNYCynT :::::IV:SY:PEP-::VS::V:::V:::RVSDI:I::::N::::TAIASC-:C-M:H::A:SH::RY:DSA-:V:NEARS-CA ::IA:MV:V:DKDKYA:V::A::::VLH:KV:NI::I:::A::6I:::MSFPDAGPTTTDFIE::VKICLPAKHK::AEHP-CA ::VA::V::YDQRKYA:T::A::::VLH:KVSNI::I:::A::GI::::SFPFDGTYSTDFIEE:VKIGLPAKAK:KAQH

0IcfA SGYETDDLVEILVAENVLTQIENLKTYPIVRSRLFQGKLQIFGWIYEVESGEVLQISRTSSDDTGIDECPVRLPGSQEKACynT PHSDLPSKAAAM:R:::IA:LA::Q:H:S::LA:EE:G-SLH::V:DI:::SIAAFDGATRQFVPLAAN:RVCAIRLRQPS-CA GNATFAEQCTHCEK:A:NVSLG::L:::F::DG:VKKT:ALQ:GY:DFVN:SFELWGLEYGLSPSQSV............P-CA GDAPFAE:CTHCEK:A:NASLG::L:::F::EG:VNKT:ALKVGY:DFVK:SFELWGLEFGLSSTFSV............

60607376

138136153156

218215221224

IcfA ILGRCVVPLTEEVAVAPPEPEPVIAAVAAPPANYSSRGWLAPEQQQRIYRGNAS 272CynT TAA................................................... 218S-CAP-CA

FIG. 4. Amino acid sequence comparison among the icfA gene product (IcfA), E. coli CA (CynT), and mature plant chloroplast CAs fromspinach (S-CA) and pea (P-CA). Amino acid residues identical to those ofthe icfA gene product are indicated by colons. Length oftransit peptidesin the precursor chloroplast CAs is indicated as numbers of amino acids at the NHrterminal residues of mature chloroplast CAs. The IcfAsequence extends beyond the other sequences at the COOH terminus. Possible zinc-binding amino acid residues (cysteine, histidine, andglutamic acid) are marked by solid circles.

ylase/oxygenase found in some autotrophic prokaryotes (5).It has been proposed that the conversion of intracellularlyaccumulated HCO- into CO2 is catalyzed by CA locatedwithin the carboxysomes (9, 12). However, no direct evi-dence for CA in the carboxysomes has been obtained inSynechococcus. Since the activity of C, accumulation isinhibited with ethoxzolamide, a potent CA inhibitor, somecomponents related to CA have been proposed to operatealso in the Ci transport step. However, the cells carrying adisruption of icfA that encodes the putative CA constructedin this study accumulated CQ. Therefore, another CA isozymeor CA-related protein may be operating in the transportsystems.

Price and Badger (25) reported two types of high-CO2-requiring Synechococcus mutants, type I and type II, that aredefective in utilization of intracellularly accumulated CQ.Type I mutants have long rod-like carboxysomes and the typeII mutants have normal polyhedral carboxysomes. Theyspeculated that in the type I mutants the fast CO2 efflux to thecytosol could be caused by a mislocation of CA, which isnormally located in the carboxysomes. On the other hand,the type II mutants may be lacking carboxysomal CA,although a normal level of CA activity was detected in theextract of type II mutants. Characteristics of the icfA-defective mutants XH-5 and SP-2 seem to be similar to type

CA ICA1l j AmniotesCA III (consensus)Red Cell CA (Shark)CA VII (Human)CA "Y" (Mouse)

A CA IV (Human)A CA VI (Human, Sheep)

CAI (Chiamydomonas)CA2 (Chiamydomonas)

Chloroplast CA (Spinach)B n | Chioroplast CA (Pea)

IcfA (Synechococcus)CynT (E. coil)

FIG. 5. Generalized phylogenetic branching scheme for CAsfrom mammals, alga, plants, and bacteria based on amino acidsequence comparison. Relationship of mammalian CAs and Chlam-ydomonas CA1 is from the ref. 28. The amino acid sequence ofChlamydomonas CA2 is from the ref. 29. Eukaryotic and prokaryoticancestors of CAs are indicated as A and B, respectively.

II mutants in that they cannot fix intracellularly accumulatedC, yet they have normal-shaped carboxysomes as examinedby electron microscopy (data not shown). In addition, thetype II mutants were complemented with 3.5-kb BamHIgenomic DNA fragments from wt cells. The restriction mapof the 3.5-kb BamHI genomic region that complements thetype II mutants is almost identical to that ofthe 3.8-kbBamHIthat we characterized (J.-W. Yu, G. D. Price, and M. R.Badger, personal communication). Therefore, it is highlypossible that the icfA encodes carboxysomal CA and that thisgene is contained in the 3.5-kb BamHI genomic DNA thatcomplements the type II mutants.

In terrestrial plants, CA is located in chloroplasts of C3plants and in the cytosol of mesophyll cells of C4 plants (26).In C4 plants, CA is assumed to catalyze the hydration ofCO2to HCO-, which is the substrate for phosphoenolpyruvatecarboxylase (27). Chloroplast CAs from C3 plants show a highdegree of sequence similarity (-76% identity) to each other.Cytosolic CA from maize also has sequence similarity tochloroplast CAs at -60% identity (J. Burnell, personal com-munication). Since plant chloroplast CAs have sequencesimilarities with the cyanobacterial icfA product and E. coliCAs, they have probably evolved from a common ancestor(protoprokaryote CA, Fig. SB). In contrast, the icfA geneproduct and plant chloroplast CAs have no significant se-quence similarity with mammalian CA isozymes (28) or theChlamydomonas periplasmic CAs (29-31). These findingssuggest that CAs can be divided into two groups and that theyhave evolved from two distinct ancestors, a protoeukaryoteCA (Fig. SA) and a protoprokaryote CA (Fig. SB).

This work was supported by Grant-in-Aid for Scientific Researchfrom the Japanese Ministry of Education, Science and Culture anda grant from the Japanese Ministry of Agriculture, Forestry andFisheries.

1. Aizawa, K. & Miyachi, S. (1986) FEMS Microbiol. Rev. 39,215-233.

2. Kaplan, A., Badger, M. R. & Berry, J. A. (1980) Planta 149,219-226.

3. Miller, A. G. & Colman, B. (1980) J. Bacteriol. 143, 1253-1259.4. Ogawa, T. (1991) Proc. Natl. Acad. Sci. USA 88, 4275-4279.5. Codd, G. A. & Marsden, W. J. N. (1984) Biol. Rev. 59, 389-

422.6. Price, G. D. & Badger, M. R. (1989) Plant Physiol. 89, 37-43.7. Abe, T., Tsuzuki, M. & Miyachi, S. (1987) Plant Cell Physiol.

28, 867-874.

Proc. Natl. Acad. Sci. USA 89 (1992)

Dow

nloa

ded

by g

uest

on

Oct

ober

5, 2

020

Biochemistry: Fukuzawa et al.

8. Price, G. D. & Badger, M. R. (1989) Plant Physiol. 91, 505-513.

9. Reinhold, L., Zviman, M. & Kaplan, A. (1989) Plant Physiol.Biochem. 27, 945-954.

10. Friedberg, D., Kaplan, A., Ariel, R., Kessel, M. & Seijffers, J.(1989) J. Bacteriol. 171, 6069-6076.

11. Ogawa, T. (1991) Plant Physiol. 96, 280-284.12. Price, G. D. & Badger, M. R. (1991) Can J. Bot. 69, %3-973.13. Suzuki, E., Fukuzawa, H. & Miyachi, S. (1991) Mol. Gen.

Genet. 226, 401-408.14. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M. &

Stanier, R. Y. (1979) J. Gen. Microbiol. 111, 1-61.15. Vieira, J. & Messing, J. (1982) Gene 19, 259-268.16. Barany, F. (1985) Gene 37, 111-123.17. Golden, S. S., Brazilian, J. & Haselkorn, R. (1987) Methods

Enzymol. 153, 215-231.18. Hein, J. (1990) Methods Enzymol. 183, 626-645.19. Suzuki, E., Tsuzuki, M. & Miyachi, S. (1987) Plant Cell

Physiol. 28, 1377-1388.20. Roeske, C. A. & Ogren W. L. (1990) NucleicAcids Res. 18,3413.21. Fawcett, T. W., Browse, J. A., Volokita, M. & Bartlett, S. G.

(1990) J. Biol. Chem. 265, 5414-5417.

Proc. Nati. Acad. Sci. USA 89 (1992) 4441

22. Sung, Y.-C. & Fuchs, J. A. (1988) J. Biol. Chem. 263, 14769-14775.

23. Sung, Y.-C. & Fuchs, J. A. (1989) J. Bacteriol. 171, 4674-4678.24. Badger, M. R. & Price, G. D. (1989) Plant Physiol. 89, 51-60.25. Price, G. D. & Badger, M. R. (1989) Plant Physiol. 91, 514-

525.26. Burnell, J. N. & Hatch, M. D. (1988) Plant Physiol. 86, 1252-

1256.27. Hatch, M. D. & Burnell, J. N. (1990) Plant Physiol. 93, 825-

828.28. Tashian, R. E., Hewett-Emmett, D. & Venta, P. J. (1991)

Carbonic Anhydrase: From Biochemistry and Genetics toPhysiology and Clinical Medicine (VCH, Whinheim, F.R.G.),pp. 151-161.

29. Fukuzawa, H., Fujiwara, S., Tachiki, A. & Miyachi, S. (1990)Nucleic Acids Res. 18, 6441-6442.

30. Fukuzawa, H., Fujiwara, S., Yamamoto, Y., Dionisio-Sese,M. L. & Miyachi, S. (1990) Proc. Natl. Acad. Sci. USA 87,4383-4387.

31. Fujiwara, S., Fukuzawa, H., Tachiki, A. & Miyachi, S. (1990)Proc. Natl. Acad. Sci. USA 87, 9779-9783.

Dow

nloa

ded

by g

uest

on

Oct

ober

5, 2

020