Identification and Characterization of the ictA/ndhL Gene Product Essential to Inorganic Carbon Transport of Synechocystis PCC6803

The ictA gene, renamed ndhL in this paper, essential to inorganic carbon transport of Synechocystis PCC6803, was expressed in Eschericia coli as a fusion protein with glutathione S-transferase. An antibody was raised against this fusion protein. Western analysis of the thylakoid membrane of wild-type (WT) Synechocystis revealed that a protein with an apparent molecular mass of 6.7 kilodaltons cross-reacted with this antibody. No immunoreactive protein was present in the thylakoid membranes of the Synechocystis mutants, RKb and M9, which have defects in the ictA/ndhL gene, or in the cytoplasmic membranes of the WT and mutant cells. Thus, the protein reacted with the antibody is the ictA gene product (IctA) and is localized in the thylakoid membrane of WT cells. IctA was absent in the thylakoid membranes of the M55 mutant, in which the ndhB gene is inactivated, and was poorly immunostained in the membranes of the mutants (M-ndhC and M-ndhK) constructed by inactivating the ndhC and ndhK genes of WT Synechocystis, respectively. The carbon dioxide uptake activity was nearly zero in M-ndhK and was about 40% of the activity of WT cells in M-ndhC. The RKb, M-ndhC, and M-ndhK mutants were unable to grow or grew very slowly under photoheterotrophic conditions. These results indicated that NADH dehydrogenase is essential to inorganic carbon transport and photoheterotrophic growth of Synechocystis and that IctA is one of the subunits of this enzyme.     

Mutants of Synechocystis PCC6803 Defective in Inorganic Carbon Transport

Eighty mutants of Synechocystis PCC6803 that require high CO₂ for growth were examined with a mass spectrometer for their ability to take up CO₂ in the light. Two of these mutants (type A) did not show any CO₂ uptake while the rest of the mutants (type B) took up CO₂ actively. Type A mutants (RKa and RKb) and one type B mutant (RK11) were partially characterized. At 3% CO₂, growth rates of the mutants and the wild type (WT) were similar. Under air levels of CO₂, growth of RKa and RKb was very slow, and RK11 did not grow at all. The photosynthetic affinities for inorganic carbon (C_i) in these three mutants were about 100 times lower than the affinity in WT. The following characteristics of type A mutants indicated that the mutants have a defect in their CO₂-transport system: (a) the activity of ¹³C¹⁸O₂ uptake in RKa and RKb in the light was less than 5% the activity in WT, and (b) each mutant had only a low level of activity of ¹⁴CO₂ uptake as measured by the method of silicone oil-filtering centrifugation. The HCO₃⁻-transport system was also impaired in these mutants. The activity of H¹⁴CO₃⁻ uptake was negligibly low in RKb and was one-third the activity of WT in RKa. On the other hand, the type B mutant, RK11, transported CO₂ and HCO₃⁻ into the intracellular C_i pool as actively as WT but was unable to utilize it for photosynthesis. Complementation analysis of type A mutants indicated that RKa and RKb have mutations in different regions of the genome. These results suggested that at least two kinds of proteins are involved in the C_i-transport system.     

Reduced Inorganic Carbon Transport in a CO(2)-Requiring Mutant of Chlamydomonas reinhardii

A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated that is deficient in inorganic carbon transport. This mutant strain, designated pmp-1-16-5K (gene locus pmp-1), was selected on the basis of a requirement of elevated CO₂ concentration for photoautrophic growth. Inorganic carbon accumulation in the mutant was considerably reduced in comparison to wild type, and the CO₂ response of photosynthesis indicated a reduced affinity for CO₂ in the mutant. At air levels of CO₂ (0.03-0.04%), O₂ inhibited photosynthesis and stimulated the synthesis of photorespiratory intermediates in the mutant but not in wild type. Neither strain was significantly affected by O₂ at saturating CO₂ concentration. Thus, the primary consequence of inorganic carbon transport deficiency in the mutant was a much lower internal CO₂ concentration compared to wild type. From these observations, we conclude that enzyme-mediated transport of inorganic carbon is an essential component of the CO₂ concentrating system in C. reinhardii photosynthesis.     

Genetic and physiological analysis of the CO2-concentrating system of Chlamydomonas reinhardii

When grown photoautotrophically at air levels of CO₂, Chlamydomonas reinhardii possesses a system involving active transport of inorganic carbon which increases the intracellular CO₂ concentration considerably above ambient, thereby stimulating photosynthetic CO₂ assimilation. In previous investigations, two mutant strains of this unicellular green alga deficient in some component of this CO₂-concentrating system were recovered as strains requiring high levels of CO₂ to support photoautotrophic growth. One of the mutants, ca-1-12-1C, is a leaky (nonstringent) CO₂-requiring strain deficient in carbonic anhydrase (EC 4.2.1.1) activity, while the other, pmp-1-16-5K, is a stringent CO₂-requiring strain deficient in inorganic carbon transport. In the present study a double mutant (ca pmp) was constructed to investigate the physiological and biochemical interaction of the two mutations. The two mutations are unlinked and inherited in a Mendelian fashion. The double mutant was found to have a leaky CO₂-requiring phenotype, indicating that the mutation ca-1 overcomes the stringent CO₂-requirement conferred by the mutation pmp-1. Several physiological characteristics of the double mutant were very similar to the carbonic-anhydrase-deficient mutant, including high CO₂ compensation concentration, photosynthetic CO₂ response curve, and deficiency of carbonic-anhydrase activity. However, the labeling pattern of metabolites during photosynthesis in ¹⁴CO₂ was more like that of the bicarbonatetransport-deficient mutant, and accumulation of internal inorganic carbon was intermediate between that of the two original mutants. These data indicate a previously unsuspected complexity in the Chlamydomonas CO₂-concentrating system.     

Sequencing and Modification of the Gene Encoding the 42-Kilodalton Protein in the Cytoplasmic Membrane of Synechococcus PCC 7942

A 42-kilodalton cytoplasmic membrane protein is synthesized when high CO₂-grown cells of Synechococcus PCC 7942 (Anacystis nidulans R2) are exposed to low CO₂. The structural gene for this protein (cmpA) has been cloned and sequenced and shown to encode a 450 amino acid polypeptide with a molecular mass of 49 kilodalton. A deletion mutant lacking the 42-kilodalton protein was obtained by transformation of Synechococcus PCC 7942 following in vitro mutagenesis of the cloned gene. There were no significant differences between the mutant and wild-type cells in their growth rates under either low or high CO₂ conditions. The activity of inorganic carbon (C_i) transport in the mutant was as high as that in the wild-type strain. In both types of cells, CO₂ was the main species of C_i transported and the activities of CO₂ and HCO₃⁻ transport increased when high CO₂-grown cells were exposed to low CO₂. We conclude that the 42-kilodalton protein is not directly involved in the C_i-accumulating mechanism of Synechococcus PCC 7942.     

Carbon Oxysulfide Is an Inhibitor of Both CO(2) and HCO(3) Uptake in the Cyanobacterium Synechococcus PCC7942

Carbon oxysulfide (COS) was reinvestigated as an inhibitor of active inorganic carbon transport in cells of Synechococcus PCC7942 adapted to growth at low inorganic carbon. COS inhibited both CO₂ and HCO₃⁻ transport processes in a reversible (in the short term) and mixed competitive manner. The inhibition of COS was established using both silicone oil centrifugation experiments and O₂-evolution studies. The K_i for COS inhibition was 29 micromolar for CO₂ transport and 110 micromolar for HCO₃⁻ transport. These results support a model of inorganic carbon transport with a central CO₂ pump and an inducible HCO₃⁻ utilizing accessory protein which supplies CO₂ to the primary pump.     

Isolation and characterization of the ccmM gene required by the cyanobacterium Synechocystis PCC6803 for inorganic carbon utilization

A high CO₂-requiring mutant of Synechocystis PCC6803 (G3) capable of C_i transport but unable to utilize the intracellular C_i pool for photosynthesis was constructed. A DNA clone of 6.1 kbp that transforms the G3 mutant to the wild-type phenotype was isolated from a Synechocystis PCC6803 genomic library. Complementation test with subclones allocated the mutation site within a DNA fragment of 674 bp nucleotides. Sequencing analysis of the mutation region elucidated an open reading frame encoding a 534 amino-acid protein with a significant sequence homology to the protein coded by the ccmN gene of Synechococcus PCC7942. The ccmM-like gene product of Synechocystis PCC6803 contains four internal repeats with a week similarity to the rbcS gene product. An open reading frame homologous to the ccmN gene of Synechococcus PCC7942 was found downstream to the ccmM-like gene. As opposed to the Synechococcus PCC7942 ccmM and ccmN genes located 2 kbp upstream to, and oriented in the same direction as, the rbc operon, the ccm-like genes in Synechocystis PCC6803 are not located within 22 kbp upstream to the rbcL gene of the Rubisco operon. Thus, despite the resemblance in clustering of the ccmM and ccmN genes in both cyanobacterial species, the difference in their genomic location relative to the rbc genes demonstrates variability in structural organization of the genes involved in inorganic carbon acquisition.Abbreviations CCM CO₂-concentrating mechanism - C_i inorganic carbon - HCR high CO₂-requiring - kbp kilobase pair - ORF open reading frame - Rubisco ribulose 1,5-bisphosphate carboxylase-oxygenase gene - SSC sodium chloride and sodium citrate - WT wild-type     

Chlorophyll a Fluorescence Yield as a Monitor of Both Active CO(2) and HCO(3) Transport by the Cyanobacterium Synechococcus UTEX 625

Simultaneous measurements have been made of inorganic carbon accumulation (by mass spectrometry) and chlorophyll a fluorescence yield of the cyanobacterium Synechococcus UTEX 625. The accumulation of inorganic carbon by the cells was accompanied by a substantial quenching of chlorophyll a fluorescence. The quenching occurred even when CO₂ fixation was inhibited by iodoacetamide and whether the accumulation of inorganic carbon resulted from either active CO₂ or HCO₃⁻ transport. Measurement of chlorophyll a fluorescence yield of cyanobacteria may prove to be a rapid and convenient means of screening for mutants of inorganic carbon accumulation.     

A Model for HCO(3) Accumulation and Photosynthesis in the Cyanobacterium Synechococcus sp: Theoretical Predictions and Experimental Observations

A simple model based on HCO₃⁻ transport has been developed to relate photosynthesis and inorganic carbon fluxes for the marine cyanobacterium, Synechococcus sp. Nägeli (strain RRIMP N1). Predicted relationships between inorganic carbon transport, CO₂ fixation, internal carbonic anhydrase activity, and leakage of CO₂ out of the cell, allow comparisons to be made with experimentally obtained data. Measurements of inorganic carbon fluxes and internal inorganic carbon pool sizes in these cells were made by monitoring time-courses of CO₂ changes (using a mass spectrometer) during light/dark transients. At just saturating CO₂ conditions, total inorganic carbon transport did not exceed net CO₂ fixation by more than 30%. This indicates CO₂ leakage similar to that estimated for C₄ plants.

For this leakage rate, the model predicts the cell would need a conductance to CO₂ of around 10⁻⁵ centimeters per second. This is similar to estimates made for the same cells using inorganic carbon pool sizes and CO₂ efflux measurements. The model predicts that carbonic anhydrase is necessary internally to allow a sufficiently fast rate of CO₂ production to prevent a large accumulation of HCO₃⁻. Intact cells show light stimulated carbonic anhydrase activity when assayed using ¹⁸O-labeled CO₂ techniques. This is also supported by low but detectable levels of carbonic anhydrase activity in cell extracts, sufficient to meet the requirements of the model.

     

Effect of photon flux density on inorganic carbon accumulation and net CO2 exchange in a high-CO2-requiring mutant of Chlamydomonas reinhardtii

The effect of photon flux density on inorganic carbon accumulation and photosynthetic CO₂ assimilation was determined by CO₂ exchange studies at three, limiting CO₂ concentrations with a ca-1 mutant of Chlamydomonas reinhardiii. This mutant accumulates a large internal inorganic carbon pool in the light which apparently is unavailable for photosynthetic assimilation. Although steady-state photosynthetic CO₂ assimilation did not respond to the varying photon flux densities because of CO₂ limitation, components of inorganic-carbon accumulation were not clearly light saturated even at 1100 mol photons m^-2 s^-1, indicating a substantial energy requirement for inorganic carbon transport and accumulation. Steady-state photosynthetic CO₂ assimilation responded to external CO₂ concentrations but not to changing internal inorganic carbon concentrations, confirming that diffusion of CO₂ into the cells supplies most of the CO₂ for photosynthetic assimilation and that the internal inorganic carbon pool is essentially unavailable for photosynthetic assimilation. The estimated concentration of the internal inorganic carbon pool was found to be relatively insensitive to the external CO₂ concentration over the small range tested, as would be expected if the concentration of this pool is limited by the internal to external inorganic carbon gradient. An attempt to use this CO₂ exchange method to determine whether inorganic carbon accumulation and photosynthetic CO₂ assimilation compete for energy at low photon flux densities proved inconclusive.     

Active Transport of CO(2) by the Cyanobacterium Synechococcus UTEX 625 : Measurement by Mass Spectrometry

Mass spectrometry has been used to confirm the presence of an active transport system for CO₂ in Synechococcus UTEX 625. Cells were incubated at pH 8.0 in 100 micromolar KHCO₃ in the absence of Na⁺ (to prevent HCO₃⁻ transport). Upon illumination the cells rapidly removed almost all the free CO₂ from the medium. Addition of carbonic anhydrase revealed that the CO₂ depletion resulted from a selective uptake of CO₂, rather than a total uptake of all inorganic carbon species. CO₂ transport stopped rapidly (<3 seconds) when the light was turned off. Iodoacetamide (3.3 millimolar) completely inhibited CO₂ fixation but had little effect on CO₂ transport. In iodoacetamide poisoned cells, transport of CO₂ occurred against a concentration gradient of about 18,000 to 1. Transport of CO₂ was completely inhibited by 10 micromolar diethylstilbestrol, a membrane-bound ATPase inhibitor. Studies with DCMU and PSI light indicated that CO₂ transport was driven by ATP produced by cyclic or pseudocyclic photophosphorylation. Low concentrations of Na⁺ (<100 microequivalents per liter), but not of K⁺, stimulated CO₂ transport as much as 2.4-fold. Unlike Na⁺-dependent HCO₃⁻ transport, the transport of CO₂ was not inhibited by high concentrations (30 milliequivalents per liter) of Li⁺. During illumination, the CO₂ concentration in the medium remained far below its equilibrium value for periods up to 15 minutes. This could only happen if CO₂ transport was continuously occurring at a rapid rate, since the continuing dehydration of HCO₃⁻ to CO₂ would rapidly raise the CO₂ concentration to its equilibrium value if transport ceased. Measurement of the rate of dissolved inorganic carbon accumulation under these conditions indicated that at least part of the continuing CO₂ transport was balanced by HCO₃⁻ efflux.     

Inorganic Carbon Uptake during Photosynthesis : II. Uptake by Isolated Asparagus Mesophyll Cells during Isotopic Disequilibrium

The species of inorganic carbon (CO₂ or HCO₃⁻) taken up a source of substrate for photosynthetic fixation by isolated Asparagus sprengeri mesophyll cells is investigated. Discrimination between CO₂ or HCO₃⁻ transport, during steady state photosynthesis, is achieved by monitoring the changes (by ¹⁴C fixation) which occur in the specific activity of the intracellular pool of inorganic carbon when the inorganic carbon present in the suspending medium is in a state of isotopic disequilibrium. Quantitative comparisons between theoretical (CO₂ or HCO₃⁻ transport) and experimental time-courses of ¹⁴C incorporation, over the pH range of 5.2 to 7.5, indicate that the specific activity of extracellular CO₂, rather than HCO₃⁻, is the appropriate predictor of the intracellular specific activity. It is concluded, therefore, that CO₂ is the major source of exogenous inorganic carbon taken up by Asparagus cells. However, at high pH (8.5), a component of net DIC uptake may be attributable to HCO₃⁻ transport, as the incorporation of ¹⁴C during isotopic disequilibrium exceeds the maximum possible incorporation predicted on the basis of CO₂ uptake alone. The contribution of HCO₃⁻ to net inorganic carbon uptake (pH 8.5) is variable, ranging from 5 to 16%, but is independent of the extracellular HCO₃⁻ concentration. The evidence for direct HCO₃⁻ transport is subject to alternative explanations and must, therefore, be regarded as equivocal. Nonlinear regression analysis of the rate of ¹⁴C incorporation as a function of time indicates the presence of a small extracellular resistance to the diffusion of CO₂, which is partially alleviated by a high extracellular concentration of HCO₃⁻.     

The Role of External Carbonic Anhydrase in Inorganic Carbon Acquisition by Chlamydomonas reinhardii at Alkaline pH

The role of external carbonic anhydrase in inorganic carbon acquisition and photosynthesis by Chlamydomonas reinhardii at alkaline pH (8.0) was studied. Acetazolamide (50 micromolar) completely inhibited external carbonic anhydrase (CA) activity as determined from isotopic disequilibrium experiments. Under these conditions, photosynthetic rates at low dissolved inorganic carbon (DIC) were far greater than could be maintained by CO₂ supplied from the spontaneous dehydration of HCO₃⁻ thereby showing that C. reinhardii has the ability to utilize exogenous HCO₃⁻. Acetazolamide increased the concentration of DIC required to half-saturate photosynthesis from 38 to 80 micromolar, while it did not affect the maximum photosynthetic rate. External CA activity was also removed from the cell-wall-less mutant (CW-15) by washing. This had no effect on the photosynthetic kinetics of the algae while the addition of acetazolamide to washed cells (CW-15) increased the K_½^DIC from 38 to 80 micromolar. Acetazolamide also caused a buildup of the inorganic carbon pool upon NaHCO₃ addition, indicating that this compound partially inhibited internal CA activity. The effects of acetazolamide on the photosynthetic kinetics of C. reinhardii are likely due to the inhibition of internal rather than a consequence of the inhibition of external CA. Further analysis of the isotopic disequilibrium experiments at saturating concentration of DIC provided evidence consistent with active CO₂ transport by C. reinhardii. The observation that C. reinhardii has the ability to take up both CO₂ and bicarbonate throws into question the role of external CA in the accumulation of DIC in this alga.     

Carbonic Anhydrase-Deficient Mutant of Chlamydomonas reinhardii Requires Elevated Carbon Dioxide Concentration for Photoautotrophic Growth

A mendelian mutant of the unicellular green alga Chlamydomonas reinhardii has been isolated which is deficient in carbonic anhydrase (EC 4.2.1.1) activity. This mutant strain, designated ca-1-12-1C (gene locus ca-1), was selected on the basis of a high CO₂ requirement for photoautotrophic growth. Photosynthesis by the mutant at atmospheric CO₂ concentration was very much reduced compared to wild type and, unlike wild type, was strongly inhibited by O₂. In contrast to a CO₂ compensation concentration of near zero in wild type at all O₂ concentrations examined, the mutant exhibited a high, O₂-stimulated CO₂ compensation concentration. Evidence of photorespiratory activity in the mutant but not in wild type was obtained from the analysis of photosynthetic products in the presence of ¹⁴CO₂. At air levels of CO₂ and O₂, the mutant synthesized large amounts of glycolate, while little glycolate was synthesized by wild type under identical conditions. Both mutant and wild type strains formed only small amounts of glycolate at saturating CO₂ concentration. At ambient CO₂, wild type accumulated inorganic carbon to a concentration several-fold higher than that in the suspension medium. The mutant cells accumulated inorganic carbon internally to a concentration 6-fold greater than found in wild type, yet photosynthesis was CO₂ limited. The mutant phenotype was mimicked by wild type cells treated with ethoxyzolamide, an inhibitor of carbonic anhydrase activity. These observations indicate a requirement for carbonic anhydrase-catalyzed dehydration of bicarbonate in maintaining high internal CO₂ concentrations and high photosynthesis rates. Thus, in wild type cells, carbonic anhydrase rapidly converts the bicarbonate taken up to CO₂, creating a high internal CO₂ concentration which stimulates photosynthesis and suppresses photorespiration. In mutant cells, bicarbonate is taken up rapidly but, because of a carbonic anhydrase deficiency, is not dehydrated at a rate sufficiently rapid to maintain a high internal CO₂ concentration.     

Internal Inorganic Carbon Pool of Chlamydomonas reinhardtii: EVIDENCE FOR A CARBON DIOXIDE-CONCENTRATING MECHANISM

The external inorganic carbon pool (CO₂ + HCO₃⁻) was measured in both high and low CO₂-grown cells of Chlamydomonas reinhardtii, using a silicone oil layer centrifugal filtering technique. The average internal pH values were measured for each cell type using [¹⁴C]dimethyloxazolidinedione, and the internal inorganic carbon pools were recalculated on a free CO₂ basis. These measurements indicated that low CO₂-grown cells were able to concentrate CO₂ up to 40-fold in relation to the external medium. Low and high CO₂-grown cells differed in their photosynthetic affinity for external CO₂. These differences could be most readily explained as being due to the relative CO₂-concentrating capacity of each cell type. This physiological adaptation appeared to be based on changes in the abilities of the cells actively to accumulate inorganic carbon using an energy-dependent transport system.     

Photosynthesis and Inorganic Carbon Usage by the Marine Cyanobacterium, Synechococcus sp

The marine cyanobacterium, Synechococcus sp. Nägeli (strain RRIMP N1) changes its affinity for external inorganic carbon used in photosynthesis, depending on the concentration of CO₂ provided during growth. The high affinity for CO₂ + HCO₃⁻ of air-grown cells (K_½ < 80 nanomoles [pH 8.2]) would seem to be the result of the presence of an inducible mechanism which concentrates inorganic carbon (and thus CO₂) within the cells. Silicone-oil centrifugation experiments indicate that the inorganic carbon concentration inside suitably induced cells may be in excess of 1,000-fold greater than that in the surrounding medium, and that this accumulation is dependent upon light energy. The quantum requirements for O₂ evolution appear to be some 2-fold greater for low CO₂-grown cells, compared with high CO₂-grown cells. This presumably is due to the diversion of greater amounts of light energy into inorganic carbon transport in these cells.

A number of experimental approaches to the question of whether CO₂ or HCO₃⁻ is primarily utilized by the inorganic carbon transport system in these cells show that in fact both species are capable of acting as substrate. CO₂, however, is more readily taken up when provided at an equivalent concentration to HCO₃⁻. This discovery suggests that the mechanistic basis for the inorganic carbon concentrating system may not be a simple HCO₃⁻ pump as has been suggested. It is clear, however, that during steady-state photosynthesis in seawater equilibrated with air, HCO₃⁻ uptake into the cell is the primary source of internal inorganic carbon.

     

Fluvial transport of carbon along the river-to-ocean continuum and its potential impacts on a brackish water food web in the Iwaki River watershed, northern Japan

Riverine transport of dissolved inorganic carbon (DIC) from land to the ocean is an important carbon flux that influences the carbon budget at the watershed scale. However, the dynamics of DIC in an entire river network has remained unknown, especially in mountainous Japanese watersheds. We examined the effects of watershed land use and geology on the transports of inorganic carbon as well as weathered silica (Si) and calcium (Ca) in the Iwaki River system where agricultural and residential areas have developed in the middle and lower parts of the watershed. The concentration and stable carbon isotope ratios (δ¹³C) of DIC showed the longitudinal increase of ¹³C-depleted inorganic carbon along the river. As a result, most streams and rivers were supersaturated in dissolved CO₂ that will eventually be emitted to the atmosphere. The possible origin of ¹³C-depleted carbon is CO₂ derived from the decomposition of organic matter in agricultural and urban landscapes, as well as from in-stream respiration. In addition, agricultural and urban areas, respectively, exported the large amount of dissolved Si and Ca to the rivers, suggesting that CO₂ increased by respiration accelerates the chemical weathering of silicate and carbonate materials in soils, river sediments, and/or urban infrastructure. Furthermore, riverine bicarbonate flux is likely to enter shell carbonates of Corbicula japonica, an aragonitic bivalve, in the downstream brackish lake (Lake Jusan). These results revealed that the flux of DIC from the human-dominated watersheds is a key to understanding the carbon dynamics and food-web structure along the land-to-river-to-ocean continuum.     

Effect of Carbonic Anhydrase Inhibitors on Inorganic Carbon Accumulation by Chlamydomonas reinhardtii

Membrane-permeable and impermeable inhibitors of carbonic anhydrase have been used to assess the roles of extracellular and intracellular carbonic anhydrase on the inorganic carbon concentrating system in Chlamydomonas reinhardtii. Acetazolamide, ethoxzolamide, and a membrane-impermeable, dextran-bound sulfonamide were potent inhibitors of extracellular carbonic anhydrase measured with intact cells. At pH 5.1, where CO₂ is the predominant species of inorganic carbon, both acetazolamide and the dextran-bound sulfonamide had no effect on the concentration of CO₂ required for the half-maximal rate of photosynthetic O₂ evolution (K_0.5[CO₂]) or inorganic carbon accumulation. However, a more permeable inhibitor, ethoxzolamide, inhibited CO₂ fixation but increased the accumulation of inorganic carbon as compared with untreated cells. At pH 8, the K_0.5(CO₂) was increased from 0.6 micromolar to about 2 to 3 micromolar with both acetazolamide and the dextran-bound sulfonamide, but to a higher value of 60 micromolar with ethoxzolamide. These results are consistent with the hypothesis that CO₂ is the species of inorganic carbon which crosses the plasmalemma and that extracellular carbonic anhydrase is required to replenish CO₂ from HCO₃⁻ at high pH. These data also implicate a role for intracellular carbonic anhydrase in the inorganic carbon accumulating system, and indicate that both acetazolamide and the dextran-bound sulfonamide inhibit only the extracellular enzyme. It is suggested that HCO₃⁻ transport for internal accumulation might occur at the level of the chloroplast envelope.     

Active Transport of Inorganic Carbon Increases the Rate of O(2) Photoreduction by the Cyanobacterium Synechococcus UTEX 625

Chlorophyll a fluorescence of Synechococcus UTEX 625 was quenched during the transport of inorganic carbon, even when CO₂ fixation was inhibited by iodoacetamide. Measurements with a pulse modulation fluorometer showed that at least 75% of the quenching was due to oxidation of Q_a, the primary acceptor of photosystem II. Mass spectrometry revealed that transport of inorganic carbon increased the rate of O₂ photoreduction. Hence, O₂ could serve as an electron acceptor to allow oxidation of Q_a even in the absence of CO₂ fixation.     

Uptake of inorganic carbon by isolated chloroplasts from air-adapted dunaliella

Neither Dunaliella cells grown with 5% CO₂ nor their isolated chloroplasts had a CO₂ concentrating mechanism. These cells primarily utilized CO₂ from the medium because the K_(0.5) (HCO₃⁻) increase from 57 micromolar at pH 7.0 to 1489 micromolar at pH 8.5, where as the K_(0.5) CO₂ was about 12 micromolar over the pH range. After air adaptation for 24 hours in light, a CO₂ concentrating mechanism was present that decreased the K_0.5 (CO₂) to about 0.5 micromolar and K_0.5 (HCO₃⁻) to 11 micromolar at pH 8. These K_0.5 values suggest that air-adapted cells preferentially concentrated CO₂ but could also use HCO₃⁻ from the medium. Chloroplasts isolated from air-adapted cells had a K_(0.5) for total inorganic carbon of less than 10 micromolar compared to 130 micromolar for chloroplasts from cells grown on high CO₂. Chloroplasts from air-adapted cells, but not CO₂-grown cells, concentrate inorganic carbon internally to 1 millimolar in 60 seconds from 240 micromolar in the medium. Maximum uptake rates occurred after preillumination of 45 seconds to 3 minutes. The CO₂ concentrating mechanism by chloroplasts from air-adapted cells was light dependent and inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or flurocarbonyl-cyamidephenylhydrazone (FCCP). Phenazine-methosulfate at 10 micromolar to provide cyclic phosphorylation partially reversed the inhibition by DCMU but not by FCCP. One to 0.1 millimolar vanadate, an inhibitor of plasma membrane ATPase, inhibited inorganic carbon accumulation by isolated chloroplasts. Vanadate had no effect on CO₂ concentration by whole cells, as it did not readily cross the cell plasmalemma. Addition of external ATP to the isolated chloroplast only slightly stimulated inorganic carbon uptake and did not reverse vanadate inhibition by more than 25%. These results are consistent with a CO₂ concentrating mechanism in Dunaliella cells which consists in part of an inorganic carbon transporter at the chloroplast envelope that is energized by ATP from photosynthetic electron transport.