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

Abstract

Cyanobacteria possess a CO₂-concentating mechanism that involves active CO₂ uptake and HCO transport. For CO₂ uptake, we have identified two systems in the cyanobacterium Synechocystis sp. strain PCC 6803, one induced at low CO₂ and one constitutive. The low CO₂-induced system showed higher maximal activity and higher affinity for CO₂ than the constitutive system. On the basis of speculation that separate NAD(P)H dehydrogenase complexes were essential for each of these systems, we reasoned that inactivation of one system would allow selection of mutants defective in the other. Thus, mutants unable to grow at pH 7.0 in air were recovered after transformation of a ΔndhD3 mutant with a transposon-bearing library. Four of them had tags within slr1302 (designated cupB), a homologue of sll1734 (cupA), which is cotranscribed with ndhF3 and ndhD3. The ΔcupB, ΔndhD4, and ΔndhF4 mutants showed CO₂-uptake characteristics of the low CO₂induced system observed in wild type. In contrast, mutants ΔcupA, ΔndhD3, and ΔndhF3 showed characteristics of the constitutive CO₂-uptake system. Double mutants impaired in one component of each of the systems were unable to take up CO₂ and required high CO₂ for growth. Phylogenetic analysis indicated that the ndhD3/ndhD4-, ndhF3/ndhF4-, and cupA/cupB-type genes are present only in cyanobacteria. Most of the cyanobacterial strains studied possess the ndhD3/ndhD4-, ndhF3/ndhF4-, and cupA/cupB-type genes in pairs. Thus, the two types of NAD(P)H dehydrogenase complexes essential for low CO₂-induced and constitutive CO₂-uptake systems associated with the NdhD3/NdhF3/CupA-homologues and NdhD4/NdhF4/CupB-homologues, respectively, appear to be present in these cyanobacterial strains but not in other organisms.

In cyanobacteria, NAD(P)H dehydrogenase (NDH-1) is essential for both CO₂ uptake (1–3) and photosystem-1 (PSI) cyclic electron transport (4). It has been postulated that uptake of CO₂ is energized by NDH-1-dependent PSI-cyclic electron transport (1). However, observations that mutants defective in ndhD3 display normal cyclic electron transport but are unable to induce high-affinity CO₂ uptake suggest the presence of multiple NDH-1 complexes (5–7). Two types of functionally distinct NDH-1 complexes were recently recognized in Synechocystis sp. strain PCC 6803 with the aid of mutants impaired in one or more subunits of NDH-1 (7). One complex, containing NdhD1 or NdhD2, plays a major role in PSI-cyclic electron flow but is not involved in CO₂ uptake (7). When the second type of NDH-1 complex is inactivated (in the double mutant ΔndhD3/ΔndhD4), nearly normal PSI-cyclic electron flow is observed, but the mutant does not take up CO₂ and is unable to grow under an air level of CO₂ (7). The single mutants ΔndhD3 and ΔndhD4, on the other hand, possess CO₂-uptake activity and can grow under low CO₂ conditions (7). These results raised the possibility of multiple systems for CO₂ uptake. In this report, we bring evidence for the presence of two CO₂-uptake systems, one constitutive and one inducible, in Synechocystis sp. PCC 6803, and further identify two genes, sll1734 and slr1302 (designated cupA and cupB for their involvement in CO₂ uptake) as essential components of the inducible and constitutive systems, respectively.

To assess the presence of homologous genes encoding constitutive and inducible CO₂ uptake systems in other organisms, we used databases available in web sites (ref. 8; http://www.kazusa.or.jp/cyano/; http://www.jgi.doe.gov/tempweb/JGI_ microbial/html/index.html). We also made use of the genome sequence of two cyanobacterial strains, Thermosynechococcus elongatus and Gloeobacter violaceus PCC 7421, recently completed at Kazusa DNA Research Institute. These sequences enabled us to construct phylogenetic trees for genes essential to CO₂ uptake in Synechocystis sp. PCC 6803. We show that most of the cyanobacterial strains investigated possess sets of genes encoding components of NDH-1 complexes involved in both the constitutive and the inducible CO₂-uptake systems, but that these genes apparently are missing in green algae.

Materials and Methods

Growth Conditions.

Wild-type (WT) and mutant cells of Synechocystis sp. strain PCC 6803 (hereafter Synechocystis 6803) were grown at 30°C in BG11 medium (9), buffered at pH 8.0, and bubbled with either 3% (vol/vol) CO₂ in air or air alone, as described (6). Solid medium was BG11 buffered at pH 7.0, supplemented with 1.5% agar and 5 mM sodium thiosulfate. Continuous illumination was provided by fluorescent lamps at 50 μmol photons m⁻²⋅s⁻¹.

Construction and Isolation of Mutants.

Construction of the ΔndhD3, ΔndhD4, ΔndhF3, ΔndhF4, and ΔcupA mutants has been described in a previous paper (6) and/or deposited in the web site “CyanoMutants” (http://www.kazusa.or.jp/cyano/ mutants/). The constructs used to generate the single mutants were also used to transform appropriate mutants of Synechocystis 6803 to introduce multiple mutations. The sll1732 (designated ndhF3), sll1733 (ndhD3), and sll1734 (cupA) genes are expressed as an operon, and the mutants constructed by inactivating any of these genes grew much more slowly than the WT cells under 50 ppm CO₂ (5, 6). This suggested that these genes are essential to the induced high-affinity CO₂-uptake system. If so, isolation and analysis of high CO₂-requiring mutants after inactivation of ΔndhD3 mutant also should enable us to identify the genes involved in the constitutive low-affinity CO₂-uptake system.

By using a Genomic Priming System (New England Biolabs), a transposon containing a gene that confers chloramphenicol resistance (Cm^R) was randomly inserted into the DNA in each insert of 110 cosmids, which contained DNA fragments of Synechocystis 6803 previously used for genome sequencing (8). The ΔndhD3 strain of Synechocystis 6803 was transformed with this transposon inactivation library, and Cm^R mutants unable to grow at pH 7.0 in air were isolated. Genomic DNA isolated from each mutant was digested with HhaI and, after self ligation, was used as a template for inverse PCR with primers complementary to the N- and C-terminal regions of the Cm^R cassette. The exact position of the cassette in the mutant genome was determined by sequencing the PCR product.

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 N-Tris(hydroxymethyl)methyl-2-amino-ethanesulfonic acid (TES)-KOH buffer, pH 7.0, containing 15 mM NaCl to a cell density corresponding to 4.3 μg chlorophyll ml⁻¹ and placed in a reaction vessel (10). CO₂ exchange of the cell suspension was measured at 30°C by using an open infrared gas-analysis system that records the rate of CO₂ exchange as a function of time. N₂, O₂, and CO₂ were mixed (using a standard gas generator model 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 μl) 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 μmol photons m⁻²⋅s⁻¹. The OD_{730 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 ΔndhD3/ΔndhD4 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 CO₂ uptake and possibly associated with NDH-1, we transformed mutant ΔndhD3 with a transposon-bearing library, tagging and inactivating many genes. Thirteen new mutants unable to grow under an air level of CO₂, at pH 7.0, were isolated. Table 1 summarizes the genes inactivated in these mutants and the positions and directions of the Cm^R tag. Two of these mutants had interruptions in slr1347, a homologue of icfA in Synechococcus sp. strain PCC 7942, which encodes a carboxysome-localized carbonic anhydrase essential for the growth of cells in an air level of CO₂ (11), and four mutants had interruptions in the genes encoding hypothetical proteins. NB-33 had the tag in ndhE, which is present as a single copy in Synechocystis 6803 and encodes a component essential to all types of NDH-1 complexes. Four of the mutants, NB29, 30, 31, and 32, contained 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 high-CO₂-requiring mutants bore the Cm^R tag within slr1301 or slr1303. These results suggested that the inability of the ΔcupB/ΔndhD3 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 ΔcupB/ΔndhD3 mutant. Inactivation of cupB or sll0026 (ndhF4) in the WT (not shown) or in the ΔndhD4 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 ΔndhF4/ΔndhD3, ΔcupB/ΔndhD3, and ΔndhD4/ΔndhD3 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).

Table 1.

Mutants unable to grow at pH 7.0 in air isolated after transformation of a ΔndhD3 mutant with a transposon-bearing library

Mutant names	Tagged genes	Positions interrupted*	Directions†	Gene products‡
NB-29	slr1302	305854	F	CupB
NB-30	slr1302	306595	R	CupB
NB-31	slr1302	306299	R	CupB
NB-32	slr1302	306580	F	CupB
NB-33	sll0522	3267274	F	NdhE
NB-34	slr1347	1742934	R	CA
NB-35	slr1347	1743132	F	CA
NB-38	sll0247	1518083	R	LHC
NB-39	slr0364	2354056	F	H.P.
NB-41	slr0684	429962	F	H.P.
NB-42	sll1488	3379184	F	H.P.
NB-44	slr0687	431822	F	PleD
NB-45	slr1521	1613061	R	H.P.

^*Numbers represent those of nucleotide sequences in the Cyanobase (http://www.kazusa.or.jp/cyano/). 

^† F, forward; R, reverse. 

^‡ CA, carboxysome-localized CA; LHC, light-harvesting chlorophyll induced under iron stress conditions; H.P., hypothetical protein. 

Figure 1.

(A) A schematic map of the s1r1302(cupB) region and the position and direction of the Cm^R cassette tags interrupting the genes and (B) growth of WT and mutants on agar plates at pH 7.0 in air. (A) The Cm^R cassette was inserted at 259, 704, 985, and 1,000 bp downstream of the initiation codon of cupB in NB-29, 31, 30, and 32, respectively. The horizontal arrows indicate the direction of the cassettes. (B) Two microliters of the cell suspensions with the OD_{730 nm} values of 0.1 (Top), 0. 01 (Middle), and 0.001 (Bottom) were spotted on agar plates containing BG11 buffered at pH 7.0 and grown in an air level of CO₂.

Figure 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). The mixed gas containing 100 μl CO₂/liter, and 20% O₂ was provided to the reaction vessel. Light intensity was 800 μmol photons m⁻²⋅s⁻¹. 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 μg chlorophyll/ml.

Figure 3.

The rate of CO₂ uptake by H cells (closed symbols) and L cells (open symbols) of the WT (circles, Upper) and mutant strains of Synechocystis PCC 6803, ΔndhD3 (triangles) and ΔndhD4 (circles, Lower), as a function of CO₂ concentrations in the medium. The CO₂ concentration in the medium in equilibrium with the gas containing 300 μl CO₂/liter was taken as 9.2 μM at 30°C. Other conditions were as in Fig. 2.

Constitutive and Inducible CO₂-Uptake Systems Operate in Synechocystis 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 of the cell suspensions, the rate of CO₂ uptake (Fig. 2, shown by V on curve b) increased and reached a maximum in ≈20 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 ΔndhD4 mutant (Fig. 2, c and d, in which only NdhD3 is present), and the ΔcupB mutant (Fig. 2, g and h). In contrast, no appreciable difference was observed between the rates of CO₂ uptake in H and L cells of mutants ΔndhD3 (Fig. 2, e and f) and ΔcupA (Fig. 2, i and j). Confirming our previous observation (7), the double mutant ΔndhD3/ΔndhD4 was unable to take up CO₂ (V virtually zero; Fig. 2, k) after acclimation to low CO₂ conditions This mutant exhibited high rates of HCO uptake (T.O., unpublished work), confirming that the V value represents the rate of CO₂ uptake and is little affected by HCO uptake. The double mutants, ΔcupB/ΔndhD3 (l) and ΔndhF4/ΔndhD3 (Fig. 2, m) were unable to take up CO₂. The single mutants ΔcupA and ΔcupB showed CO₂-exchange characteristics similar to those of ΔnhD3 and ΔndhD4, respectively (Fig. 2, g–j). The V value for H cells of mutants ΔndhD4 and ΔcupB was considerably lower than that for WT. Taken together, the data support the suggestion that two CO₂-uptake systems operate in Synechocystis 6803. One is constitutive in H cells (impaired in mutants ΔndhD4 and ΔcupB), and the other is induced by exposing the cells to low CO₂ conditions (impaired in mutants ΔndhD3 and ΔcupA). The double mutants ΔndhD3/ΔndhD4, ΔcupB/ΔndhD3, and ΔndhF4/ΔndhD3 are apparently deprived of both the constitutive and the induced CO₂ uptake systems and thus are unable to take up CO₂.

Rates of CO₂ uptake (V) are plotted as a function of CO₂ concentrations in Fig. 3. During acclimation of high-CO₂-grown WT cells to low CO₂ conditions, the maximal rate of CO₂ uptake increased substantially (from 190 to 320 μmol/mg chlorophyll h), and the K_1/2 (CO₂) decreased from 3.3 to 1.2 μM. H cells of ΔndhD4 (Fig. 3 Lower) and ΔcupB (not shown) mutants exhibited a much lower capacity for CO₂ uptake than H cells of the WT (Fig. 3 Upper). On the other hand, when grown under low CO₂, the ΔndhD4 and ΔcupB mutants exhibited CO₂ uptake characteristics similar to WT. Unlike the case of mutants ΔndhD4 and ΔcupB, there was no significant difference between the maximal rates and the K_1/2 (CO₂) values observed in H and L cells of mutants ΔndhD3 (Fig. 3 Lower) and ΔcupA (not shown). The K_1/2 (CO₂) and maximal rates of CO₂ uptake in both H and L cells of mutants ΔndhD3 and ΔcupA were similar to those of WT H cells, but the maximal rates were significantly higher than those of H cells of ΔndhD4 and ΔcupB. The K_1/2 (CO₂) values in ΔndhD4 and ΔndhD3 mutants were 0.9 and 2.8 μM, respectively, regardless of the CO₂ concentration during growth.

Taken together, these data clearly support the hypothesis that there are two types of CO₂ uptake systems in Synechocystis 6803: a constitutive, NdhD4-dependent system and a low-CO₂-induced, NdhD3-dependent system. The inducible NdhD3-dependent system, exhibited a maximal rate of CO₂ uptake 2-fold higher, and a K_1/2 (CO₂) values 3-fold lower_, than the corresponding values for the constitutive, NdhD4-dependent system.

Phylogenetic Analysis.

The present and earlier (7) studies implicate four of the ndh genes, ndhD3, ndhD4, ndhF3, and ndhF4, as well as ΔcupA and ΔcupB, in CO₂ uptake by Synechocystis 6803. It was interesting to examine whether other photosynthetic microorganisms rely on homologous genes for CO₂ uptake. A phylogenetic tree for NdhD/NdhF (Fig. 4A) indicates that NdhD and NdhF proteins are both members of a larger family and may be related by an ancient gene duplication event. Three lineages were propagated from the ndhD line. One of them, which branched to the ndhD3- and ndhD4-types, is present only in cyanobacteria. An evolutionary relationship between cyanobacterial ndhD1/ndhD2-type and ndhD genes in chloroplast genomes is noted. On the basis of the relationship shown in Fig. 4A, the proteins designated as NdhD5 and NdhD6 according to previous papers (7, 14) should probably be designated NdhF. Prochlorococcus marinus possesses only the ndhD1-type gene, whereas Gloeobacter violaceus, a cyanobacterial strain that supposedly diverged at an early stage of evolution (15), possesses both the ndhD3- and ndhD4-type genes.

Figure 4.

Phylogenetic trees of NdhD/NdhF (A) and CupA/CupB (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.

The phylogenetic tree shows evolutionary lineages for ndhF1, ndhF3, and ndhF4 propagated from the ndhF line (Fig. 4A). Whereas the ndhF1 is related to the chloroplast ndhF, the ndhF3/ndhF4-type genes are present only in cyanobacteria. The cupA/cupB-type genes are also confined to the cyanobacteria (Fig. 4B). All of the cyanobacterial strains studied except the marine Synechococcus possess both ndhF3 and ndhF4 genes as well as cupA- and cupB-type genes. The ndhD3/ndhF3/cupA-type genes were not found in the marine Synechococcus. However, because the genome sequencing of this strain has not yet been completed, it is too early to conclude the absence of these genes. It appears that the presence of two types of NDH-1 complexes is common in cyanobacteria.

Discussion

Data presented here show that two NDH-1-dependent CO₂-uptake systems operate in Synechocystis 6803. One of them, involving NdhD3, NdhF3, and CupA, is induced during acclimation to low CO₂ conditions. The other system, involving NdhD4, NdhF4 and CupB, exhibits a 3-fold lower affinity for CO₂, and its maximal activity (approximately one-half that of the induced system) is not significantly affected by acclimation of the cells to low CO₂ (Fig. 3). The conclusion that two separate NDH-1-dependent systems are involved is based on the results shown in Figs. 1–3, in which the growth and CO₂ uptake characteristics of single and double mutants with lesions in the above components are presented. Mutants impaired in a component of the induced system, either NdhD3, NdhF3, or CupA, exhibited CO₂ uptake characteristics similar to those observed in H cells of the WT regardless of the CO₂ concentration during growth. L cells of mutants in which a component of the constitutive system was inactivated were able to take up CO₂ like L cells of the WT, but uptake was severely depressed under high CO₂. Double mutants, in which the components of the induced and the constitutive systems were inactivated, were unable to take up CO₂ and required high CO₂ for growth.

The inability of a mutant to increase CO₂ uptake after exposure to low CO₂ might stem either from a defect in the sensing/signal transduction process or from lack of an essential component of the induced CO₂ uptake system. As an example, the direct involvement of NdhD3 in CO₂ uptake indicates that ΔndhD3 mutants of Synechococcus PCC 7002 (5) and Synechocystis 6803 (6) are deprived of a component of the specific CO₂ uptake system that operates under low CO₂ conditions rather than being unable to acclimate to low CO₂.

Inhibition of CO₂ uptake by an aquaporin blocker suggested that CO₂ enters the cells passively (16) rather than by active transport (17). Intracellular conversion of the entering CO₂ to HCO, the inorganic carbon species that accumulates in the cells, is mediated by a carbonic anhydrase-like activity (18–21). The direct energy source for the energy-requiring energy HCO formation and release to the cytoplasm is photosynthetically generated ΔμH⁺ rather than ATP hydrolysis (16). The observation that NDH-1 components essential for CO₂ uptake (ref. 6 and Figs. 1–3) are located on the thylakoid (22) provides strong evidence that conversion of CO₂ to bicarbonate at the thylakoid provides the driving force for inward diffusion of CO₂ across the cytoplasmic membrane.

Participation of NdhD3/NdhD4-type NDH-1 complexes in the respiratory and cyclic PSI electron transfer (from NADPH to plastoquinone pool) appears to be small, because these processes were scarcely impaired in the ΔndhD3/ΔndhD4 mutants (7), even though CO₂ uptake was depressed. In contrast, NdhD1/NdhD2 types of NDH-1 are essential for cyclic PSI electron transport, but their absence hardly affected CO₂ uptake (7). It was earlier suggested that cyclic PSI electron transport is essential for CO₂ uptake. To reconcile the data from mutants with this suggestion, we proposed (16) that multiple PSI types or alternative routes of cyclic electron transport (23) operate in Synechocystis 6803. The present conclusion that NdhD3 and NdhD4 actually belong to two functionally distinct NDH-I complexes, induced and constitutive, respectively, supports the suggestion of multiple routes for electron transport.

The mechanism for CO₂ uptake and the role of the NDH-1 complexes in this process are not fully understood. A recent model (18) proposed that CO₂ uptake by cyanobacteria and its intracellular conversion to HCO is energized by photosynthetic electron transport via the formation of alkaline domains on the stromal face of the thylakoid membrane. The formation of these putative domains would enable the vectoral conversion of CO₂ to HCO, which accumulates in the cytoplasm (17, 18). The carbonic anhydrase-like moiety essential for accelerating the conversion of CO₂ to HCO has not been identified, and the roles of the components of the induced and constitutive CO₂ uptake systems in the route of electron transport have not yet been clarified. The presence of two types of CO₂ uptake systems suggests the involvement of two proteins (or protein complexes) that bind CO₂ followed by hydration and release of HCO to the cytoplasm. Hydrophobicity analyses (not shown) indicated that NdhD3, NdhD4, NdhF3, and NdhF4 are most likely embedded in the membrane, whereas CupA and CupB are not. Because CupA and CupB are functionally associated with NDH-1 complexes involved in CO₂ uptake, they may have a role in the conversion of CO₂ to HCO. However, neither CupA nor CupB shows significant homology to any of the presently known carbonic anhydrases.

Recent studies (24) indicated significant oxidation of NADPH in cyanobacteria even when CO₂ fixation is completely inhibited, under which conditions massive Ci cycling in the form of CO₂ uptake and HCO release still persists (16). These data raised the possibility that the reduction of NADP⁺ to NADPH may serve as the direct source of OH⁻ for the conversion of CO₂ to HCO. This possibility is being examined.

All of the cyanobacterial strains studied except the marine Synechochoccus possess genes encoding components associated with the two types of NDH-1 complexes involved in the induced and constitutive CO₂ uptake systems (Fig. 4), suggesting that their presence is common in cyanobacteria. Gloeobacter possesses two copies of ndhD4- and cupB-type genes, suggesting that this strain has two constitutive CO₂ uptake systems. Although Prochlorococcus did not have any genes related to the CO₂ uptake systems, we are unable to conclude the absence of such genes in Prochlorococcus at present because the genome sequencing of this strain has not been completed. Green algae also possess mechanisms to concentrate CO₂ (25). However, the absence of genes homologous to ndhD3(F3), ndhD4(F4), and cupA(B) in algae indicates that different mechanism(s) may be involved in CO₂ uptake by algae.

Abbreviations

H cells

cells grown under 3% (vol/vol) CO₂ in air

L cells

cells acclimated to air for 18 h in the light

NDH-1

NAD(P)H dehydrogenase

WT

wild type

PSI

photosystem-1

Cm^R

chloramphenicol resistance