Distinct Constitutive and Low-CO2-Induced CO2 Uptake

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Distinct constitutive and low-CO2-induced CO2 uptake systems in cyanobacteria: Genes involved and their phylogenetic relationship with homologous genes in other organisms

Mari Shibata*, Hiroshi Ohkawa*, Takakazu Kaneko†, Hideya Fukuzawa‡, Satoshi Tabata†, Aaron Kaplan§, and Teruo Ogawa*¶

*Bioscience Center, Nagoya University, Chikusa, Nagoya 464-8601, Japan; †Kazusa DNA Research Institute, Kisarazu, Chiba 292-0814, Japan; ‡Graduate School of Biostudies, Kyoto University, Kyoto 606-8502, Japan; and §Department of Plant Sciences, The Hebrew University of Jerusalem, 91904 Jerusalem, Israel

Edited by William L. Ogren, U.S. Department of Agriculture, Hilton Head Island, SC, and approved July 24, 2001 (received for review May 23, 2001)

Cyanobacteria possess a CO2-concentating mechanism that in- unable to grow under an air level of CO2 (7). The single ؊ volves active CO2 uptake and HCO3 transport. For CO2 uptake, we mutants ⌬ndhD3 and ⌬ndhD4, on the other hand, possess have identified two systems in the cyanobacterium Synechocystis CO2-uptake activity and can grow under low CO2 conditions sp. strain PCC 6803, one induced at low CO2 and one constitutive. (7). These results raised the possibility of multiple systems for The low CO2-induced system showed higher maximal activity and CO2 uptake. In this report, we bring evidence for the presence higher affinity for CO2 than the constitutive system. On the basis of two CO2-uptake systems, one constitutive and one induc- of speculation that separate NAD(P)H dehydrogenase complexes ible, in Synechocystis sp. PCC 6803, and further identify two were essential for each of these systems, we reasoned that inac- genes, sll1734 and slr1302 (designated cupA and cupB for their tivation of one system would allow selection of mutants defective involvement in CO2 uptake) as essential components of the in the other. Thus, mutants unable to grow at pH 7.0 in air were inducible and constitutive systems, respectively. recovered after transformation of a ⌬ndhD3 mutant with a trans- To assess the presence of homologous genes encoding con- poson-bearing library. Four of them had tags within slr1302 (des- stitutive and inducible CO2 uptake systems in other organisms, ignated cupB), a homologue of sll1734 (cupA), which is cotrans- we used databases available in web sites (ref. 8; http:͞͞www. cribed with ndhF3 and ndhD3. The ⌬cupB, ⌬ndhD4, and ⌬ndhF4 kazusa.or.jp͞cyano͞; http:͞͞www.jgi.doe.gov͞tempweb͞JGI࿝ ͞ ͞ mutants showed CO2-uptake characteristics of the low CO2- microbial html index.html). We also made use of the genome induced system observed in wild type. In contrast, mutants ⌬cupA, sequence of two cyanobacterial strains, Thermosynechococcus ⌬ndhD3, and ⌬ndhF3 showed characteristics of the constitutive elongatus and Gloeobacter violaceus PCC 7421, recently com- CO2-uptake system. Double mutants impaired in one component of pleted at Kazusa DNA Research Institute. These sequences each of the systems were unable to take up CO2 and required high enabled us to construct phylogenetic trees for genes essential to CO2 for growth. Phylogenetic analysis indicated that the ndhD3͞ CO2 uptake in Synechocystis sp. PCC 6803. We show that most ndhD4-, ndhF3͞ndhF4-, and cupA͞cupB-type genes are present of the cyanobacterial strains investigated possess sets of genes only in cyanobacteria. Most of the cyanobacterial strains studied encoding components of NDH-1 complexes involved in both the possess the ndhD3͞ndhD4-, ndhF3͞ndhF4-, and cupA͞cupB-type constitutive and the inducible CO2-uptake systems, but that genes in pairs. Thus, the two types of NAD(P)H dehydrogenase these genes apparently are missing in green algae. complexes essential for low CO2-induced and constitutive CO2- Materials and Methods uptake systems associated with the NdhD3͞NdhF3͞CupA-homo- Growth Conditions. logues and NdhD4͞NdhF4͞CupB-homologues, respectively, appear Wild-type (WT) and mutant cells of Synecho- to be present in these cyanobacterial strains but not in other cystis sp. strain PCC 6803 (hereafter Synechocystis 6803) were grown at 30°C in BG11 medium (9), buffered at pH 8.0, and organisms. ͞ bubbled with either 3% (vol vol) CO2 in air or air alone, as NAD(P)H dehydrogenase ͉ constitutive CO uptake ͉ affinity to CO ͉ described (6). Solid medium was BG11 buffered at pH 7.0, 2 2 supplemented with 1.5% agar and 5 mM sodium thiosulfate. CO -concentrating mechanism 2 Continuous illumination was provided by fluorescent lamps at 50 ␮mol photons mϪ2⅐sϪ1. n cyanobacteria, NAD(P)H dehydrogenase (NDH-1) is es- Isential for both CO2 uptake (1–3) and photosystem-1 (PSI) Construction and Isolation of Mutants. Construction of the ⌬ndhD3, cyclic electron transport (4). It has been postulated that uptake ⌬ndhD4, ⌬ndhF3, ⌬ndhF4, and ⌬cupA mutants has been of CO2 is energized by NDH-1-dependent PSI-cyclic electron described in a previous paper (6) and͞or deposited in the web transport (1). However, observations that mutants defective in site ‘‘CyanoMutants’’ (http:͞͞www.kazusa.or.jp͞cyano͞ ndhD3 display normal cyclic electron transport but are unable mutants͞). The constructs used to generate the single mutants to induce high-affinity CO2 uptake suggest the presence of PLANT BIOLOGY multiple NDH-1 complexes (5–7). Two types of functionally distinct NDH-1 complexes were recently recognized in Syn- This paper was submitted directly (Track II) to the PNAS office. ͞ echocystis sp. strain PCC 6803 with the aid of mutants impaired Abbreviations: H cells, cells grown under 3% (vol vol) CO2 in air; L cells, cells acclimated in one or more subunits of NDH-1 (7). One complex, con- to air for 18 h in the light; NDH-1, NAD(P)H dehydrogenase; WT, wild type; PSI, photosystem-1; CmR, chloramphenicol resistance. taining NdhD1 or NdhD2, plays a major role in PSI-cyclic ¶To whom reprint requests should be addressed. E-mail: [email protected] electron flow but is not involved in CO2 uptake (7). When the u.ac.jp. second type of NDH-1 complex is inactivated (in the double ⌬ ͞⌬ The publication costs of this article were defrayed in part by page charge payment. This mutant ndhD3 ndhD4), nearly normal PSI-cyclic electron article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. flow is observed, but the mutant does not take up CO2 and is §1734 solely to indicate this fact.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.191258298 PNAS ͉ September 25, 2001 ͉ vol. 98 ͉ no. 20 ͉ 11789–11794 Downloaded by guest on September 28, 2021 were also used to transform appropriate mutants of Synechocystis Table 1. Mutants unable to grow at pH 7.0 in air isolated after 6803 to introduce multiple mutations. The sll1732 (designated transformation of a ⌬ndhD3 mutant with a ndhF3), sll1733 (ndhD3), and sll1734 (cupA) genes are expressed transposon-bearing library as an operon, and the mutants constructed by inactivating any of Mutant Tagged Positions Gene these genes grew much more slowly than the WT cells under 50 names genes interrupted* Directions† products‡ ppm CO2 (5, 6). This suggested that these genes are essential to the induced high-affinity CO2-uptake system. If so, isolation and NB-29 slr1302 305854 F CupB analysis of high CO2-requiring mutants after inactivation of NB-30 slr1302 306595 R CupB ⌬ndhD3 mutant also should enable us to identify the genes NB-31 slr1302 306299 R CupB involved in the constitutive low-affinity CO2-uptake system. NB-32 slr1302 306580 F CupB By using a Genomic Priming System (New England Biolabs), NB-33 sll0522 3267274 F NdhE a transposon containing a gene that confers chloramphenicol NB-34 slr1347 1742934 R CA resistance (CmR) was randomly inserted into the DNA in each NB-35 slr1347 1743132 F CA insert of 110 cosmids, which contained DNA fragments of NB-38 sll0247 1518083 R LHC Synechocystis 6803 previously used for genome sequencing (8). NB-39 slr0364 2354056 F H.P. The ⌬ndhD3 strain of Synechocystis 6803 was transformed with NB-41 slr0684 429962 F H.P. this transposon inactivation library, and CmR mutants unable to NB-42 sll1488 3379184 F H.P. grow at pH 7.0 in air were isolated. Genomic DNA isolated from NB-44 slr0687 431822 F PleD each mutant was digested with HhaI and, after self ligation, was NB-45 slr1521 1613061 R H.P. used as a template for inverse PCR with primers complementary ͞͞ R *Numbers represent those of nucleotide sequences in the Cyanobase (http: to the N- and C-terminal regions of the Cm cassette. The exact www.kazusa.or.jp͞cyano͞). position of the cassette in the mutant genome was determined by †F, forward; R, reverse. sequencing the PCR product. ‡CA, carboxysome-localized CA; LHC, light-harvesting chlorophyll induced under iron stress conditions; H.P., hypothetical protein. ͞ CO2 Exchange Measurements. Cells grown under 3% (vol vol) CO2 in air (H cells) or acclimated to air for 18 h in the light (L cells) were harvested by centrifugation, resuspended in 25 ml of 20 mM had interruptions in slr1347, a homologue of icfA in Synecho- N-Tris(hydroxymethyl)methyl-2-amino-ethanesulfonic acid coccus sp. strain PCC 7942, which encodes a carboxysome- (TES)-KOH buffer, pH 7.0, containing 15 mM NaCl to a cell localized carbonic anhydrase essential for the growth of cells in ␮ Ϫ1 density corresponding to 4.3 g chlorophyll ml and placed in an air level of CO2 (11), and four mutants had interruptions a reaction vessel (10). CO2 exchange of the cell suspension was in the genes encoding hypothetical proteins. NB-33 had the tag measured at 30°C by using an open infrared gas-analysis system in ndhE, which is present as a single copy in Synechocystis 6803 that records the rate of CO2 exchange as a function of time. N2, and encodes a component essential to all types of NDH-1 O2, and CO2 were mixed (using a standard gas generator model complexes. Four of the mutants, NB29, 30, 31, and 32, contained SGGU-712, STECH, Tokyo) to generate N2 gas containing 20% O2 in combination with various concentrations of CO2. The mixed gas was provided to the reaction vessel at a flow rate of ͞ 1.0 liter min. The gas leaving the chamber was dried and its CO2 concentration analyzed by using an infrared CO2 analyzer (model URA-106, Shimadzu). The CO2 concentration in the medium surrounding the cells was calculated from its concentration in the gas produced by the standard gas generator, assuming that CO2 in the gas is in equilibrium with CO2 in the medium.

Determination of Growth Characteristics. WT and mutant strains grown under 3% CO2 were collected and resuspended in fresh BG11 medium. Cell suspensions (2 ␮l) were spotted onto BG11 agar plates buffered at pH 7.0, which then were incubated under air for 5 days with continuous illumination by fluorescent lamps at 50 ␮ Ϫ2⅐ Ϫ1 mol photons m s . The OD730 nm was measured by using a recording spectrophotometer, model V-550 (Jasco, Tokyo). Results

Genes Encoding Components Involved in Two CO2-Uptake Systems. Our earlier results showed that the ⌬ndhD3͞⌬ndhD4 double mutant could not take up CO2 and required high CO2 for growth (7). The single mutants, on the other hand, could grow under low CO2 (6). These observations suggested that there are functionally distinct NDH-1 complexes and that at least one, containing either NdhD3 or NdhD4, must be functioning for CO2 uptake in Synechocystis 6803 to proceed. To clarify the role of these NDH-1 complexes and identify other components that are essential for Fig. 1. (A) A schematic map of the s1r1302(cupB) region and the position and direction of the CmR cassette tags interrupting the genes and (B) growth of WT CO2 uptake and possibly associated with NDH-1, we trans- ⌬ and mutants on agar plates at pH 7.0 in air. (A) The CmR cassette was inserted at formed mutant ndhD3 with a transposon-bearing library, tag- 259, 704, 985, and 1,000 bp downstream of the initiation codon of cupB in NB-29, ging and inactivating many genes. Thirteen new mutants unable 31, 30, and 32, respectively. The horizontal arrows indicate the direction of the to grow under an air level of CO2, at pH 7.0, were isolated. Table cassettes. (B) Two microliters of the cell suspensions with the OD730 nm values of 0.1 1 summarizes the genes inactivated in these mutants and the (Top), 0. 01 (Middle), and 0.001 (Bottom) were spotted on agar plates containing R positions and directions of the Cm tag. Two of these mutants BG11 buffered at pH 7.0 and grown in an air level of CO2.

11790 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.191258298 Shibata et al. Downloaded by guest on September 28, 2021 Fig. 2. CO2 exchange proﬁles of the WT and various mutants of Synechocystis 6803 on switching on the light. The mutants were grown at 3% CO2 (H) or acclimated to air overnight (L). An open gas-exchange system capable of recording the rate of CO2 exchange as a function of time was used for this analysis (10). Ϫ2 Ϫ1 The mixed gas containing 100 ␮lCO2͞liter, and 20% O2 was provided to the reaction vessel. Light intensity was 800 ␮mol photons m ⅐s . Cells were suspended in 25 ml of 20 mM Tes-KOH buffer, pH 7.0, containing 15 mM NaCl, to ﬁnal concentration corresponding to 4.2 ␮g chlorophyll͞ml.

the tag at various sites within slr1302 (cupB, Fig. 1A). The putative protein encoded by cupB showed significant amino acid sequence similarity to that encoded by sll1734 (cupA), which is cotranscribed with sll1732 (ndhF3) and sll1733 (ndhD3) (6). No R high-CO2-requiring mutants bore the Cm tag within slr1301 or slr1303. These results suggested that the inability of the ⌬cupB͞⌬ndhD3 mutants to grow at pH 7.0 in air (Fig. 1B) was not because of a pleiotropic effect. In the experiments described here (Fig. 1B), we used NB-29 as the ⌬cupB͞⌬ndhD3 mutant. Inactivation of cupB or sll0026 (ndhF4) in the WT (not shown) or in the ⌬ndhD4 mutant scarcely affected their growth under an air level of CO2 (Fig. 1B). The transcript originating from sll1732–4 is hardly detectable in cells grown under high CO2 conditions but accumulates many-fold during acclimation to low CO2 (12, 13). The results of present (Fig. 1B) and earlier (7) growth experiments lend support to the hypothesis that two CO2-uptake systems occur in Synechocystis 6803. One is induced under low CO2 and involves NdhF3, NdhD3, and CupA, and the other is constitutive and involves NdhF4, NdhD4, and CupB. That the double mutants ⌬ndhF4͞⌬ndhD3, ⌬cupB͞⌬ndhD3, and ⌬ndhD4͞⌬ndhD3 could not grow at pH 7.0 in air (Fig. 1B; figure 1 in ref. 7) could, among other possibilities, stem from inactivation of both the inducible and the constitutive CO2 uptake systems. These possibilities were further examined by following the CO2 exchange characteristics of the mutants (Figs. 2 and 3).

Constitutive and Inducible CO2-Uptake Systems Operate in Synecho- cystis 6803. Fig. 2 shows the CO2 exchange profiles (measured in an open gas-exchange system; ref. 10) of cells grown under high CO2 (H) or acclimated to low CO2 (Fig. 2, L). On illumination PLANT BIOLOGY of the cell suspensions, the rate of CO2 uptake (Fig. 2, shown by V on curve b) increased and reached a maximum in Ϸ20 s. The V exhibited by L cells was much greater than that for H cells in the case of the WT (Fig. 2, a and b), the ⌬ndhD4 mutant (Fig. ⌬cupB Fig. 3. The rate of CO2 uptake by H cells (closed symbols) and L cells (open 2, c and d, in which only NdhD3 is present), and the symbols) of the WT (circles, Upper) and mutant strains of Synechocystis PCC mutant (Fig. 2, g and h). In contrast, no appreciable difference 6803, ⌬ndhD3 (triangles) and ⌬ndhD4 (circles, Lower), as a function of CO2 was observed between the rates of CO2 uptake in H and L cells concentrations in the medium. The CO concentration in the medium in ⌬ ⌬ 2 of mutants ndhD3 (Fig. 2, e and f) and cupA (Fig. 2, i and j). equilibrium with the gas containing 300 ␮lCO2͞liter was taken as 9.2 ␮Mat Confirming our previous observation (7), the double mutant 30°C. Other conditions were as in Fig. 2.

Shibata et al. PNAS ͉ September 25, 2001 ͉ vol. 98 ͉ no. 20 ͉ 11791 Downloaded by guest on September 28, 2021 Fig. 4. Phylogenetic trees of NdhD͞NdhF (A) and CupA͞CupB (B). Multiple sequence alignments were performed by using CLUSTAL (26). Ana, Anabaena sp. PCC 7120; Glo, Gloeobacter violaceus PCC 7421; MSy, Marine Synechococcus sp. WH8502; Nos, Nostoc punctiforme; Pro, Prochlorococcus marinus MED4; Syn, Synechocystis sp. PCC 6803; Tsy, Thermosynechococcus elongatus; Cmy, Chlamydomonas reinhardtii; Ara, Arabidopsis thaliana; Mar, Marchantia polymorpha; Tob, Nicotiana tabacum; Zea, Zea mays. D and F after the organism names in A indicate ndhD and ndhF. These genes were denoted as ndhD1 (slr0331 in Synechocystis 6803), ndhD2 (slr1291), ndhD3 (sll1733), ndhD4 (sll0027), ndhD5 (slr2007), ndhD6 (slr2009), ndhF1 (sll0844), ndhF3 (sll1732), and ndhF4 (sll0026) (7, 14). A and B after the organism names in B indicate CupA (sll1734 in Synechocystis 6803) and CupB (slr1302), respectively. C and M in parentheses indicate the genes in chloroplast and mitochondrial genomes, respectively.

⌬ ͞⌬ ⌬ ⌬ ndhD3 ndhD4 was unable to take up CO2 (V virtually zero; ndhD4 and cupB, there was no significant difference between Fig. 2, k) after acclimation to low CO2 conditions This mutant the maximal rates and the K1/2 (CO2) values observed in H and Ϫ ⌬ ⌬ exhibited high rates of HCO3 uptake (T.O., unpublished work), L cells of mutants ndhD3 (Fig. 3 Lower) and cupA (not confirming that the V value represents the rate of CO2 uptake shown). The K1/2 (CO2) and maximal rates of CO2 uptake in both Ϫ ⌬ ⌬ and is little affected by HCO3 uptake. The double mutants, H and L cells of mutants ndhD3 and cupA were similar to ⌬cupB͞⌬ndhD3 (l) and ⌬ndhF4͞⌬ndhD3 (Fig. 2, m) were those of WT H cells, but the maximal rates were significantly ⌬ ⌬ ⌬ ⌬ unable to take up CO2. The single mutants cupA and cupB higher than those of H cells of ndhD4 and cupB. The K1/2 ⌬ ⌬ ⌬ showed CO2-exchange characteristics similar to those of nhD3 (CO2) values in ndhD4 and ndhD3 mutants were 0.9 and 2.8 ⌬ ␮ and ndhD4, respectively (Fig. 2, g–j). The V value for H cells M, respectively, regardless of the CO2 concentration during of mutants ⌬ndhD4 and ⌬cupB was considerably lower than that growth. for WT. Taken together, the data support the suggestion that two Taken together, these data clearly support the hypothesis that CO2-uptake systems operate in Synechocystis 6803. One is con- there are two types of CO2 uptake systems in Synechocystis 6803: a ⌬ ⌬ stitutive in H cells (impaired in mutants ndhD4 and cupB), constitutive, NdhD4-dependent system and a low-CO2-induced, and the other is induced by exposing the cells to low CO2 NdhD3-dependent system. The inducible NdhD3-dependent sys- ⌬ ⌬ conditions (impaired in mutants ndhD3 and cupA). The tem, exhibited a maximal rate of CO2 uptake 2-fold higher, and a ⌬ ͞⌬ ⌬ ͞⌬ double mutants ndhD3 ndhD4, cupB ndhD3, and K1/2 (CO2) values 3-fold lower, than the corresponding values for ⌬ndhF4͞⌬ndhD3 are apparently deprived of both the constitu- the constitutive, NdhD4-dependent system. tive and the induced CO2 uptake systems and thus are unable to take up CO2. Phylogenetic Analysis. The present and earlier (7) studies impli- Rates of CO2 uptake (V) are plotted as a function of CO2 cate four of the ndh genes, ndhD3, ndhD4, ndhF3, and ndhF4,as ⌬ ⌬ concentrations in Fig. 3. During acclimation of high-CO2-grown well as cupA and cupB,inCO2 uptake by Synechocystis 6803. WT cells to low CO2 conditions, the maximal rate of CO2 uptake It was interesting to examine whether other photosynthetic ␮ ͞ increased substantially (from 190 to 320 mol mg chlorophyll microorganisms rely on homologous genes for CO2 uptake. A ␮ ͞ h), and the K1/2 (CO2) decreased from 3.3 to 1.2 M. H cells of phylogenetic tree for NdhD NdhF (Fig. 4A) indicates that ⌬ndhD4 (Fig. 3 Lower) and ⌬cupB (not shown) mutants exhib- NdhD and NdhF proteins are both members of a larger family ited a much lower capacity for CO2 uptake than H cells of the and may be related by an ancient gene duplication event. Three WT (Fig. 3 Upper). On the other hand, when grown under low lineages were propagated from the ndhD line. One of them, ⌬ ⌬ CO2, the ndhD4 and cupB mutants exhibited CO2 uptake which branched to the ndhD3- and ndhD4-types, is present only characteristics similar to WT. Unlike the case of mutants in cyanobacteria. An evolutionary relationship between cya-

11792 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.191258298 Shibata et al. Downloaded by guest on September 28, 2021 nobacterial ndhD1͞ndhD2-type and ndhD genes in chloroplast to plastoquinone pool) appears to be small, because these genomes is noted. On the basis of the relationship shown in Fig. processes were scarcely impaired in the ⌬ndhD3͞⌬ndhD4 mu- 4A, the proteins designated as NdhD5 and NdhD6 according to tants (7), even though CO2 uptake was depressed. In contrast, previous papers (7, 14) should probably be designated NdhF. NdhD1͞NdhD2 types of NDH-1 are essential for cyclic PSI Prochlorococcus marinus possesses only the ndhD1-type gene, electron transport, but their absence hardly affected CO2 uptake whereas Gloeobacter violaceus, a cyanobacterial strain that sup- (7). It was earlier suggested that cyclic PSI electron transport is posedly diverged at an early stage of evolution (15), possesses essential for CO2 uptake. To reconcile the data from mutants both the ndhD3- and ndhD4-type genes. with this suggestion, we proposed (16) that multiple PSI types or The phylogenetic tree shows evolutionary lineages for ndhF1, alternative routes of cyclic electron transport (23) operate in ndhF3, and ndhF4 propagated from the ndhF line (Fig. 4A). Synechocystis 6803. The present conclusion that NdhD3 and Whereas the ndhF1 is related to the chloroplast ndhF, the NdhD4 actually belong to two functionally distinct NDH-I ndhF3͞ndhF4-type genes are present only in cyanobacteria. The complexes, induced and constitutive, respectively, supports the cupA͞cupB-type genes are also confined to the cyanobacteria suggestion of multiple routes for electron transport. (Fig. 4B). All of the cyanobacterial strains studied except the The mechanism for CO2 uptake and the role of the NDH-1 marine Synechococcus possess both ndhF3 and ndhF4 genes as complexes in this process are not fully understood. A recent model ͞ ͞ (18) proposed that CO uptake by cyanobacteria and its intracel- well as cupA- and cupB-type genes. The ndhD3 ndhF3 cupA- 2 Ϫ type genes were not found in the marine Synechococcus. How- lular conversion to HCO3 is energized by photosynthetic electron ever, because the genome sequencing of this strain has not yet transport via the formation of alkaline domains on the stromal face of the thylakoid membrane. The formation of these putative been completed, it is too early to conclude the absence of these Ϫ genes. It appears that the presence of two types of NDH-1 domains would enable the vectoral conversion of CO2 to HCO3 , complexes is common in cyanobacteria. which accumulates in the cytoplasm (17, 18). The carbonic anhydrase-like moiety essential for accelerating the conversion of CO Ϫ 2 Discussion to HCO3 has not been identified, and the roles of the components of the induced and constitutive CO uptake systems in the route of Data presented here show that two NDH-1-dependent CO2- 2 uptake systems operate in Synechocystis 6803. One of them, involv- electron transport have not yet been clarified. The presence of two ing NdhD3, NdhF3, and CupA, is induced during acclimation to low typesofCO2 uptake systems suggests the involvement of two proteins (or protein complexes) that bind CO2 followed by hydra- CO2 conditions. The other system, involving NdhD4, NdhF4 and Ϫ tion and release of HCO to the cytoplasm. Hydrophobicity CupB, exhibits a 3-fold lower affinity for CO2, and its maximal 3 activity (approximately one-half that of the induced system) is not analyses (not shown) indicated that NdhD3, NdhD4, NdhF3, and NdhF4 are most likely embedded in the membrane, whereas CupA significantly affected by acclimation of the cells to low CO2 (Fig. 3). The conclusion that two separate NDH-1-dependent systems are and CupB are not. Because CupA and CupB are functionally associated with NDH-1 complexes involved in CO uptake, they involved is based on the results shown in Figs. 1–3, in which the 2 Ϫ may have a role in the conversion of CO2 to HCO3 . However, growth and CO2 uptake characteristics of single and double mutants with lesions in the above components are presented. Mutants neither CupA nor CupB shows significant homology to any of the impaired in a component of the induced system, either NdhD3, presently known carbonic anhydrases. Recent studies (24) indicated significant oxidation of NADPH NdhF3, or CupA, exhibited CO uptake characteristics similar to 2 in cyanobacteria even when CO fixation is completely inhibited, those observed in H cells of the WT regardless of the CO 2 2 under which conditions massive Ci cycling in the form of CO concentration during growth. L cells of mutants in which a com- Ϫ 2 uptake and HCO3 release still persists (16). These data raised ponent of the constitutive system was inactivated were able to take ϩ the possibility that the reduction of NADP to NADPH may up CO like L cells of the WT, but uptake was severely depressed Ϫ 2 serve as the direct source of OH for the conversion of CO to under high CO . Double mutants, in which the components of the Ϫ 2 2 HCO . This possibility is being examined. induced and the constitutive systems were inactivated, were unable 3 All of the cyanobacterial strains studied except the marine to take up CO2 and required high CO2 for growth. Synechochoccus possess genes encoding components associated The inability of a mutant to increase CO2 uptake after with the two types of NDH-1 complexes involved in the induced exposure to low CO2 might stem either from a defect in the ͞ and constitutive CO2 uptake systems (Fig. 4), suggesting that sensing signal transduction process or from lack of an essential their presence is common in cyanobacteria. Gloeobacter pos- component of the induced CO2 uptake system. As an example, sesses two copies of ndhD4- and cupB-type genes, suggesting that the direct involvement of NdhD3 in CO2 uptake indicates that this strain has two constitutive CO2 uptake systems. Although ⌬ndhD3 mutants of Synechococcus PCC 7002 (5) and Synecho- Prochlorococcus did not have any genes related to the CO2 cystis 6803 (6) are deprived of a component of the specific CO2 uptake systems, we are unable to conclude the absence of such uptake system that operates under low CO2 conditions rather genes in Prochlorococcus at present because the genome se- than being unable to acclimate to low CO2. quencing of this strain has not been completed. Green algae also Inhibition of CO uptake by an aquaporin blocker suggested that 2 possess mechanisms to concentrate CO2 (25). However, the CO enters the cells passively (16) rather than by active transport 2 Ϫ absence of genes homologous to ndhD3(F3), ndhD4(F4), and (17). Intracellular conversion of the entering CO2 to HCO3 ,the cupA(B) in algae indicates that different mechanism(s) may be inorganic carbon species that accumulates in the cells, is mediated involved in CO2 uptake by algae. by a carbonic anhydrase-like activity (18–21). The direct energy Ϫ source for the energy-requiring energy HCO formation and 3 We thank Ms. Natsu Hagino, Ms. Akiko Tachibana, and Mr. Yasunori PLANT BIOLOGY ϩ release to the cytoplasm is photosynthetically generated ⌬␮H Matsui for technical assistance. This study was supported by a Grant- rather than ATP hydrolysis (16). The observation that NDH-1 in-Aid for Scientific Research (B) (2)(12440228) and a grant from the ͞ components essential for CO2 uptake (ref. 6 and Figs. 1–3) are Human Frontier Science Program (RG0051 1997 M) to T.O., a Grant- located on the thylakoid (22) provides strong evidence that in-Aid from for Scientific Research (No. 12660300) to H.F., a grant for conversion of CO to bicarbonate at the thylakoid provides the ‘‘Research for the Future’’ Program (JSPS-RFTF97R16001) to T.O. and 2 H.F., and grants from the Israeli Ministry of Science and Technology driving force for inward diffusion of CO2 across the cytoplasmic (MOST), the U.S.A.–Israel Binational Science Foundation, and Pro- membrane. gram MARS2, a cooperation between MOST and the German Minis- Participation of NdhD3͞NdhD4-type NDH-1 complexes in terium fu¨r Bildung, Wissenschaft, Forschung, und Technologie (BMBF) the respiratory and cyclic PSI electron transfer (from NADPH to A.K.

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