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Cyanobacteria possess a CO2-concentating mechanism that involves active CO2 uptake and HCO 3 ؊ transport. For CO2 uptake, we have identified two systems in the cyanobacterium Synechocystis sp. strain PCC 6803, one induced at low CO 2 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 ⌬ndhD3 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 ⌬cupB, ⌬ndhD4, and ⌬ndhF4 mutants showed CO2-uptake characteristics of the low CO2-induced system observed in wild type. In contrast, mutants ⌬cupA, ⌬ndhD3, and ⌬ndhF3 showed characteristics of the constitutive CO2-uptake system. Double mutants impaired in one component of each of the systems were unable to take up CO 2 and required high CO2 for growth. Phylogenetic analysis indicated that the ndhD3͞ ndhD4-, ndhF3͞ndhF4-, and cupA͞cupB-type genes are present only in cyanobacteria. Most of the cyanobacterial strains studied possess the ndhD3͞ndhD4-, ndhF3͞ndhF4-, and cupA͞cupB-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 NdhD3͞NdhF3͞CupA-homologues and NdhD4͞NdhF4͞CupB-homologues, respectively, appear to be present in these cyanobacterial strains but not in other organisms.NAD(P)H dehydrogenase ͉ constitutive CO2 uptake ͉ affinity to CO2 ͉ CO2-concentrating mechanism I n cyanobacteria, NAD(P)H dehydrogenase (NDH-1) is essential for both CO 2 uptake (1-3) and photosystem-1 (PSI) cyclic electron transport (4). It has been postulated that uptake of CO 2 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 CO 2 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 f low but is not involved in CO 2 uptake (7). When the second type of NDH-1 complex is inactivated (in the double mutant ⌬ndhD3͞⌬ndhD4), nearly normal PSI-cyclic electron f low is observed, but the mutant does not take up CO 2 and is unable to grow under an air level of CO 2 (7). The single mutants ⌬ndhD3 and ⌬ndhD4, on the other hand, possess CO 2 -uptake activity and can grow under low CO 2 conditions (7). These results raised the possibility of multiple systems for CO 2 uptake. In...

The mechanism of oxygen evolution by photosystem II (PSII) has remained highly conserved during the course of evolution from ancestral cyanobacteria to green plants. A cluster of manganese, calcium, and chloride ions, whose binding environment is optimized by PSII extrinsic proteins, catalyzes this water-splitting reaction. The accepted view is that in plants and green algae, the three extrinsic proteins are PsbO, PsbP, and PsbQ, whereas in cyanobacteria, they are PsbO, PsbV, and PsbU. Our previous proteomic analysis established the presence of a PsbQ homolog in the cyanobacterium Synechocystis 6803. The current study additionally demonstrates the presence of a PsbP homolog in cyanobacterial PSII. Both psbP and psbQ inactivation mutants exhibited reduced photoautotrophic growth as well as decreased water oxidation activity under CaCl 2 -depleted conditions. Moreover, purified PSII complexes from each mutant had significantly reduced activity. In cyanobacteria, one PsbQ is present per PSII complex, whereas PsbP is significantly substoichiometric. These findings indicate that both PsbP and PsbQ proteins are regulators that are necessary for the biogenesis of optimally active PSII in Synechocystis 6803. The new picture emerging from these data is that five extrinsic PSII proteins, PsbO, PsbP, PsbQ, PsbU, and PsbV, are present in cyanobacteria, two of which, PsbU and PsbV, have been lost during the evolution of green plants.

The cyanobacterium Synechocystis sp. strain PCC 6803 possesses two CO 2 uptake systems and two HCO 3 ؊ transporters. We transformed a mutant impaired in CO 2 uptake and in cmpA-D encoding a HCO 3 ؊ transporter with a transposon inactivation library, and we recovered mutants unable to take up HCO 3 ؊ and grow in low CO 2 at pH 9.0. They are all tagged within slr1512 (designated sbtA). We show that SbtA-mediated transport is induced by low CO 2 , requires Na ؉ , and plays the major role in HCO 3 ؊ uptake in Synechocystis. Inactivation of slr1509 (homologous to ntpJ encoding a Na ؉ /K ؉ -translocating protein) abolished the ability of cells to grow at [Na ؉ ] higher than 100 mM and severely depressed the activity of the SbtA-mediated HCO 3 ؊ transport. We propose that the SbtA-mediated HCO 3 ؊ transport is driven by ⌬Na ؉ across the plasma membrane, which is disrupted by inactivating ntpJ. Phylogenetic analyses indicated that two types of sbtA exist in various cyanobacterial strains, all of which possess ntpJ. The sbtA gene is the first one identified as essential to Na ؉ -dependent HCO 3 ؊ transport in photosynthetic organisms and may play a crucial role in carbon acquisition when CO 2 supply is limited, or in Prochlorococcus strains that do not possess CO 2 uptake systems or Cmp-dependent HCO 3 ؊ transport.Growth of many photosynthetic microorganisms depends on the activity of a CO 2 -concentrating mechanism (CCM), 1 which raises the [CO 2 ] in close proximity to ribulose-1,5-bisphosphate carboxylase/oxygenase and thereby enables efficient CO 2 fixation despite the low affinity of the enzyme for CO 2 (1, 2). In the cyanobacterium Synechocystis sp. strain PCC 6803 (hereafter Synechocystis 6803), the CCM involves active CO 2 uptake and HCO 3 Ϫ transport. We have recently identified two systems for CO 2 uptake in Synechocystis 6803, one constitutive and the other inducible by low CO 2 (3). As deduced from phylogenetic analysis of proteins encoded by the genes involved, these CO 2 uptake systems are present in various cyanobacteria with the exception of Prochlorococcus marinus (3). The inducible system that depends on NdhD3/ NdhF3/CupA shows higher maximal activity and higher affinity for CO 2 than the constitutive, NdhD4/NdhF4/CupB-dependent system. Inactivation of two different genes, one encoding a component of the constitutive system and the other a constituent of the inducible system, abolished CO 2 uptake activity. The double mutants were unable to grow at pH 7.0 under air level of CO 2 (3, 4). In contrast, because the mutants possessed HCO 3 Ϫ transport capability, they could grow like the wild type (WT) at pH 9.0 in air.An ABC-type HCO 3 Ϫ transporter encoded by cmpABCD has been identified in Synechococcus sp. strain PCC 7942 (thereafter Synechococcus 7942) (5). Inactivation of cmp genes in Synechocystis 6803, however, had little effect on the HCO 3 Ϫ transport activity. This indicated that another HCO 3 Ϫ transporter, as yet unidentified, plays a central role in HCO 3 Ϫ uptake. Sodium ions are essential for ...

The ndhD gene encodes a membrane protein component of NAD(P)H dehydrogenase. The genome of Synechocystis sp. PCC6803 contains 6 ndhD genes. Three mutants were constructed by disrupting highly homologous ndhD genes in pairs. Only the ⌬ndhD1/⌬ndhD2 (⌬ndhD1/D2) mutant was unable to grow under photoheterotrophic conditions and exhibited low respiration rate, although the mutant grew normally under photoautotrophic conditions in air. The ⌬ndhD3/⌬ndhD4 (⌬ndhD3/D4) mutant grew very slowly in air and did not take up CO 2 . The results demonstrated the presence of two types of functionally distinct NAD(P)H dehydrogenases in Synechocystis PCC6803 cells. The⌬ndhD5/ ⌬ndhD6 (⌬ndhD5/D6) mutant grew like the wild-type strain. Under far-red light (>710 nm), the level of P700 ؉ was high in ⌬ndhD1/D2 and M55 (ndhB-less mutant) at low intensities. The capacity of Q A (tightly bound plastoquinone) reduction by plastoquinone pool, as measured by the fluorescence increase in darkness upon addition of KCN, was much less in ⌬ndhD1/D2 and M55 than in ⌬ndhD3/D4 and ⌬ndhD5/D6. We conclude that electrons from NADPH are transferred to the plastoquinone pool mainly by the NdhD1⅐NdhD2 type of NAD(P)H dehydrogenases.The Type I NAD(P)H dehydrogenase complex (NDH-1) 1 in cyanobacteria is involved in both the respiratory and photosynthetic electron transport chains (1). The whole genome sequence data base for Synechocystis sp. PCC6803 has shown the presence of genes for 12 subunits of NDH-1 with the large, hydrophobic NdhB, NdhD, and NdhF subunits being core membrane components (2). The data base also reveals that ndhD and ndhF are present as gene families with six and three members, respectively (note that NdhF4 has homology to NdhD5 and has been designated as NdhD6 (3)), although most ndh genes are present as single copies. This suggests that several types of NDH-1 exist in cyanobacteria, each with different NdhD and/or NdhF subunits, and with each potential complex having differing functions (4 -6). In fact, of the five ndhD-less mutants, ⌬ndhD3 is the only mutant that displays the phenotype of slow growth at limiting CO 2 (i.e. 50 ppm CO 2 ) and reduced affinity for CO 2 uptake, whereas the other ndhDless mutants (⌬ndhD1, ⌬ndhD2, ⌬ndhD4, and ⌬ndhD5) do not show such phenotype (6).It has been demonstrated that NDH-1 is essential for inorganic carbon (CO 2 and HCO 3 Ϫ ; designated C i ) transport in cyanobacteria (3-11), and it was assumed that ATP produced by NDH-1-dependent cyclic electron flow is essential to energize the C i transport (7, 12). However, a recent observation indicated that mutations in ndh genes lead to inhibition of CO 2 uptake rather than HCO 3 Ϫ uptake (6). This suggested that CO 2 uptake is energized differently from HCO 3 Ϫ uptake. The presence of an ATP-dependent HCO 3 Ϫ transporter in Synechococcus PCC7942 has been recently demonstrated (13). In an attempt to see if there are functionally distinct NDH-1 complexes, we constructed double mutants of Synechocystis sp. PCC6803 by disrupting highly homologous ndhD genes in p...

Phosphoenolpyruvate carboxylase (PEPC) is a key enzyme of primary metabolism in bacteria, algae, and vascular plants, and is believed to be cytosolic. Here we show that rice (Oryza sativa L.) has a plant-type PEPC, Osppc4, that is targeted to the chloroplast. Osppc4 was expressed in all organs tested and showed high expression in the leaves. Its expression in the leaves was confined to mesophyll cells, and Osppc4 accounted for approximately one-third of total PEPC protein in the leaf blade. Recombinant Osppc4 was active in the PEPC reaction, showing V max comparable to cytosolic isozymes. Knockdown of Osppc4 expression by the RNAi technique resulted in stunting at the vegetative stage, which was much more marked when rice plants were grown with ammonium than with nitrate as the nitrogen source. Comparison of leaf metabolomes of ammonium-grown plants suggested that the knockdown suppressed ammonium assimilation and subsequent amino acid synthesis by reducing levels of organic acids, which are carbon skeleton donors for these processes. We also identified the chloroplastic PEPC gene in other Oryza species, all of which are adapted to waterlogged soil where the major nitrogen source is ammonium. This suggests that, in addition to glycolysis, the genus Oryza has a unique route to provide organic acids for ammonium assimilation that involves a chloroplastic PEPC, and that this route is crucial for growth with ammonium. This work provides evidence for diversity of primary ammonium assimilation in the leaves of vascular plants.amino acid synthesis | glycolysis | nitrogen assimilation | organic acid synthesis | Oryza

Photosystem II (PSII), the enzyme responsible for photosynthetic oxygen evolution, is a rapidly turned over membrane protein complex. However, the factors that regulate biogenesis of PSII are poorly defined. Previous proteomic analysis of the PSII preparations from the cyanobacterium Synechocystis sp PCC 6803 detected a novel protein, Psb29 (Sll1414), homologs of which are found in all cyanobacteria and vascular plants with sequenced genomes. Deletion of psb29 in Synechocystis 6803 results in slower growth rates under high light intensities, increased light sensitivity, and lower PSII efficiency, without affecting the PSII core electron transfer activities. A T-DNA insertion line in the PSB29 gene in Arabidopsis thaliana displays a phenotype similar to that of the Synechocystis mutant. This plant mutant grows slowly and exhibits variegated leaves, and its PSII activity is light sensitive. Low temperature fluorescence emission spectroscopy of both cyanobacterial and plant mutants shows an increase in the proportion of uncoupled proximal antennae in PSII as a function of increasing growth light intensities. The similar phenotypes observed in both plant and cyanobacterial mutants demonstrate that the function of Psb29 has been conserved throughout the evolution of oxygenic photosynthetic organisms and suggest a role for the Psb29 protein in the biogenesis of PSII.

Six mutants (B1 to B6) that grew poorly in air on BG11 agar plates buffered at pH 8.0 were rescued after mutations were introduced into ndhB of wild-type (WT) Synechocystis sp. strain PCC 6803. In these mutants and a mutant (M55) lacking ndhB, CO 2 uptake was much more strongly inhibited than HCO 3 ؊ uptake, i.e., the activities of CO 2 and HCO 3 ؊ uptake in B1 were 9 and 85% of those in the WT, respectively. Most of the mutants grew very slowly or did not grow at all at pH 6.5 or 7.0 in air, and their ability to grow under these conditions was correlated with CO 2 uptake capacity. Detailed studies of B1 and M55 indicated that the mutants grew as fast as the WT in liquid at pH 8.0 under air, although they grew poorly on agar plates. The contribution of CO 2 uptake appears to be larger on solid medium. Five mutants were constructed by inactivating each of the five ndhD genes in Synechocystis sp. strain PCC 6803. The mutant lacking ndhD3 grew much more slowly than the WT at pH 6.5 under 50 ppm CO 2 , although other ndhD mutants grew like the WT under these conditions and showed low affinity for CO 2 uptake. These results indicated the presence of multiple NAD(P)H dehydrogenase type I complexes with specific roles.In cyanobacteria the type I NAD(P)H dehydrogenase complex (NDH-1) is a proton-pumping complex that has been shown to be involved in both the respiratory and photosynthetic electron transport chains (25). NDH-1 acts as a plastoquinone oxidoreductase with NADH or NADPH as a substrate. There is evidence that NDH-1 is located in the cytoplasmic membrane as well as the thylakoid membrane, but evidence for location in the cytoplasmic membrane has not been consistent. NDH-1 is composed of 12 recognized subunits, with the large, hydrophobic NdhB, NdhD, and NdhF subunits being core membrane components. The availability of a whole-genome database for Synechocystis sp. strain PCC 6803 has shown that most ndh genes are present as single copies; however, ndhD and ndhF are present as multiple copies with five and four members, respectively (note that NdhF4 has homology with NdhD5 and could be counted as an NdhD homolog). There is a marked degree of protein sequence divergence within the NdhD and NdhF families, and this has led to suggestions that several NDH-1 complexes may exist in cyanobacteria, each with different NdhD and/or NdhF subunits and with each potential complex having differing functions (21, 24). One main role for NDH-1 in the thylakoid membrane is to participate in cyclic electron flow around photosystem I and to pump protons into the lumen, thereby contributing to ⌬pH-driven ATP generation at the expense of NADPH (11-13). However, other roles for NDH-1 are possible.It has been demonstrated that NDH-1 is essential for inorganic carbon (C i ) transport in cyanobacteria (4,5,10,(16)(17)(18)21). Inactivation of ndhB or ndhL in Synechocystis sp. strain PCC 6803 greatly reduced the activities of CO 2 and HCO 3 Ϫ uptake, and in the past it has been assumed that the NDH-1-dependent cyclic electron transport sup...