Dieter Sültemeyer

The involvement of NAD(P)H dehydrogenase subunits, NdhD3 and NdhF3, in high-affinity CO2 uptake in Synechococcus sp PCC7002 gives evidence for multiple NDH-1 complexes with specific roles in cyanobacteria

Random gene tagging was used to obtain new mutants of the marine cyanobacterium, Synechococcus sp. PCC7002, with defects in the CO2‐concentrating mechanism (CCM). Two of these mutants, K22 and A41, showed poor growth at limiting CO2. Isolation and sequencing of a 6.6u2003kb genomic region revealed the existence of five potential protein‐coding regions, all arranged in the same transcriptional direction. These regions code for an RbcR homologue, NdhF3 (subunit 5 of type 1 NAD(P)H dehydrogenase; NDH‐1 complex), NdhD3 (subunit 4 of NDH‐1), ORF427 and ORF133 (hypothetical proteins). Insertional mutants in ndhD3, ndhF3 and ORF427, like A41 and K22, were all incapable of inducing high‐affinity CO2 uptake and were not fully capable of inducing high‐affinity HCO3− transport. ndhD3 and ndhF3 mutants displayed P700 re‐reduction rates identical to wild‐type cells, suggesting that NdhD3 is part of a specific NDH‐1 complex that is not involved in photosynthetic cyclic electron transport. Thus, it is feasible that NdhD3, NdhF3 and ORF427 might form part of a novel NDH‐1 complex located on the cytoplasmic membrane and involved in tightly coupled energization of high‐affinity CO2 transport. The possibility of multiple, functionally distinct NDH‐1 complexes in cyanobacteria is discussed.

Isolation of ccmKLMN genes from the marine cyanobacterium, Synechococcus sp. PCC7002 (Cyanophyceae), and evidence that CcmM is essential for carboxysome assembly

A high CO2 requiring mutant of the marine cyanobacterium Synechococcus PCC7002 was generated using a random gene‐tagging procedure. This mutant demonstrated a reduced photosynthetic affinity for inorganic carbon (Ci) and accumulated high internal levels of Ci that could not be used for photosynthesis. Analysis of the mutant genomic DNA showed that the mutagenesis had disrupted a cluster of genes involved in the cyanobacterial CO2 concentrating mechanism (CCM), the so‐called ccm genes. These characteristics are consistent with a cyanobacterial mutant with defects in carboxysome assembly and/or functioning. Further genomic analyses indicated that the genes of the Synechococcus PCC7002 operon, ccmKLMN, are structurally similar to those of two closely related cyanobacteria, Synechococcus PCC7942 and Synechocystis PCC6803. The Synechococcus PCC7002 ccmM gene, which encodes a polypeptide with a predicted size of 70 kDa, was the direct target of the mutagenesis event. The CcmM protein has two distinct regions: an N‐terminal region that shows similarity to an archaeon gamma carbonic anhydrase and a C‐terminal region that contains repeated domains demonstrating sequence similarity to the small subunit of Rubisco. Physiological analysis of a ccmM‐defined mutant showed that these cells were essentially identical to the original mutant; they required high CO2 concentrations for growth, they had a low photosynthetic affinity for Ci, and they internalized Ci to high levels. Moreover, ultrastructural examination showed that both the original and the defined mutants lack carboxysomes. Thus, our results demonstrate that the ccmM gene of Synechococcus PCC7002 encodes a polypeptide that is essential for carboxysome assembly and therefore for proper functioning of the cyanobacterial CCM.

The CO2 permeability of the plasma membrane of Chlamydomonas reinhardtii: mass-spectrometric 18O-exchange measurements from 13C18O2 in suspensions of carbonic anhydrase-loaded plasma-membrane vesicles

The unicellular green alga Chlamydomonas reinhardtii possesses a CO2-concentrating mechanism. In order to measure the CO2 permeability coefficients of the plasma membranes (PMs), carbonic anhydrase (CA) loaded vesicles were isolated from C. reinhardtii grown either in air enriched with 50 mL CO2 · L−1} (high-Ci cells) or in ambient air (350 μL CO2 · L−1}; low-Ci cells). Marker-enzyme measurements indicated less than 1% contamination with thylakoid and mitochondrial membranes, and that more than 90% of the PMs from high and low-Ci cells were orientated right-side-out. The PMs appeared to be sealed as judged from the ability of vesicles to accumulate [14C]acetate along a proton gradient for at least 10 min. Carbonic anhydrase-loaded PMs from high and low-Ci cells of C. reinhardtii were used to measure the exchange of 18O between doubly labelled CO2 (13C18O2) and H2O in stirred suspensions by mass spectrometry. Analysis of the kinetics of the 18O depletion from 13C18O2 in the external medium provides a powerful tool to study CO2 diffusion across the PM to the active site of CA which catalyses 18O exchange only inside the vesicles but not in the external medium (Silverman et al., 1976, J Biol Chem 251: 4428–4435). The activity of CA within loaded PM vesicles was sufficient to speed-up the 18O loss to H2O to 45360–128800 times the uncatalysed rate, depending on the efficiency of CA-loading and PM isolation. From the 18O-depletion kinetics performed at pH 7.3 and 7.8, CO2 permeability coefficients of 0.76 and 1.49·10−3} cm·s−1}, respectively, were calculated for high Ci cells. The corresponding values for low-Ci cells were 1.21 and 1.8·10−3} cm·s−1}. The implications of the similar and rather high CO2 permeability coefficients (low CO2 resistance) in high and low-Ci cells for the COi-concentrating mechanism of C. reinhardtii are discussed.

Characterisation of carbon dioxide and bicarbonate transport during steady-state photosynthesis in the marine cyanobacterium Synechococcus strain PCC7002

Net O2 evolution, gross CO2 uptake and net HCOinf3su− uptake during steady-state photosynthesis were investigated by a recently developed mass-spectrometric technique for disequilibrium flux analysis with cells of the marine cyanobacterium Synechococcus PCC7002 grown at different CO2 concentrations. Regardless of the CO2 concentration during growth, all cells had the capacity to transport both CO2 and HCOinf3su−; however, the activity of HCOinf3su− transport was more than twofold higher than CO2 transport even in cyanobacteria grown at high concentration of inorganic carbon (Ci = CO2 + HCOinf3su−). In low-Ci cells, the affinities of CO2 and HCOinf3su− transport for their substrates were about 5 (CO2 uptake) and 10 (HCOinf3su− uptake) times higher than in high-Ci cells, while air-grown cells formed an intermediate state. For the same cells, the intracellular accumulated Ci pool reached 18, 32 and 55 mM in high-Ci, air-grown and low-Ci cells, respectively, when measured at 1 mM external Ci. Photosynthetic O2 evolution, maximal CO2 and HCOinf3su− transport activities, and consequently their relative contribution to photosynthesis, were largely unaffected by the CO2 provided during growth. When the cells were adapted to freshwater medium, results similar to those for artificial seawater were obtained for all CO2 concentrations. Transport studies with high-Ci cells revealed that CO2 and HCOinf3su− uptake were equally inhibited when CO2 fixation was reduced by the addition of glycolaldehyde. In contrast, in low-Ci cells steady-state CO2 transport was preferably reduced by the same inhibitor. The inhibitor of carbonic anhydrase ethoxyzolamide inhibited both CO2 and HCOinf3su− uptake as well as O2 evolution in both cell types. In high-Ci cells, the degree of inhibition was similar for HCOinf3su− transport and O2 evolution with 50% inhibition occurring at around 1 mM ethoxyzolamide. However, the uptake of CO2 was much more sensitive to the inhibitor than HCOinf3su− transport, with an apparent I50 value of around 250 μM ethoxyzolamide for CO2 uptake. The implications of our results are discussed with respect to Ci utilisation in the marine Synechococcus strain.

Induction of intracellular carbonic anhydrases during the adaptation to low inorganic carbon concentrations in wild-type and ca-1 mutant cells of Chlamydomonas reinhardtii

Mass-spectrometric measurements of 18O exchange from 13C18O2 were used to follow changes in the intracellular carbonic anhydrase (CA) activity of cells of Chlamydomonas reinhardtii Dang, wild type and the ca-1 mutant during adaptation to air. With intact cells as well as with crude homogenates total intracellular CA activity in wild-type cells increased six to tenfold within 4 h after transferring cells from 5% CO2 (high inorganic carbon, Ci) to ambient air (air adapted). After that time the activity slowly declined to a level similar to that observed with cells which had been continuously grown in air (low-Ci grown). In the ca-1 mutant, total CA was induced to a similar extent during 4 h of adaptation; however, absolute activities were two to three times lower in ca-1 than in the wild type regardless of the CO2 supply. When crude extracts from wild-type cells were separated into soluble and insoluble fractions, each fraction contained about half of the internal CA activity. Within 4 h of adaptation, both forms of CA activity were simultaneously enhanced by nine to tenfold, reaching levels similar to those found in low-Cigrown cells. In contrast, in the ca-1 mutant the soluble CA activity was only enhanced by about eightfold while the level of insoluble CA was very low even in low-Ci cells. After isolation of intact chloroplasts from wild-type cells and further subfractionation, around 70–80% of total chloroplastic CA activity was found to be in the insoluble fraction while 17–20% remained in the soluble fraction. Both chloroplastic CA activities were inducible within the first 4 h of adaptation to air, with each of them being eight to ten times higher than in high-Ci algae. After that time their activities were similar to the corresponding CA values in low-Ci-grown cells. In contrast, plastids from high-Ci cells of the ca-1 mutant showed 40% less insoluble-CA activity compared to the wild type and this insoluble-CA activity was not increased at all by transferring algae to air. In addition, no soluble-CA activity was detected in chloroplasts from high-Ci and air-adapted ca-1 cells. These results indicate the presence of three intracellular CA activities in high-Ci air-adapted and low-Ci cells of the wild type and that two of them are associated with the chloroplasts. All three activities are completely induced within the first 4 h of adaptation to air in wild-type cells. In contrast, it was not possible to induce any of the chloroplastic CA activities in the ca-1 mutant. The possibility that the soluble chloroplastic CA represents a pyrenoid-located CA is discussed.

Photosynthesis and apparent affinity for dissolved inorganic carbon by cells and chloroplasts of Chlamydomonas reinhardtii grown at high and low CO2 concentrations.

Chloroplasts with high rates of photosynthetic O2 evolution (up to 120 μmol O2· (mg Chl)-1·h-1 compared with 130 μmol O2· (mg Chl)-1·h-1 of whole cells) were isolated from Chlamydomonas reinhardtii cells grown in high and low CO2 concentrations using autolysine-digitonin treatment. At 25° C and pH=7.8, no O2 uptake could be observed in the dark by high- and low-CO2 adapted chloroplasts. Light saturation of photosynthetic net oxygen evolution was reached at 800 μmol photons·m-2·s-1 for high- and low-CO2 adapted chloroplasts, a value which was almost identical to that observed for whole cells. Dissolved inorganic carbon (DIC) saturation of photosynthesis was reached between 200–300 μM for low-CO2 adapted chloroplasts, whereas high-CO2 adapted chloroplasts were not saturated even at 700 μM DIC. The concentrations of DIC required to reach half-saturated rates of net O2 evolution (Km(DIC)) was 31.1 and 156 μM DIC for low- and high-CO2 adapted chloroplasts, respectively. These results demonstrate that the CO2 concentration provided during growth influenced the photosynthetic characteristics at the whole cell as well as at the chloroplast level.

Characterisation of inorganic carbon fluxes, carbonic anhydrase(s) and ribulose-1,5-biphosphate carboxylase-oxygenase in the green unicellular alga Coccomyxa

Processes involved in the uptake and fixation of dissolved inorganic carbon (DIC) were characterised for Coccomyxa, the green algal primary photobiont of the lichen Peltigera aphthosa and compared with the freeliving alga Chlamydomonas reinhardtii Dangeard (WT cc 125+). A mass-spectrometer disequilibrium technique was used to quantify fluxes of both HCOinf3sup−and CO2 in the two algae, while activities of carbonic anhydrases (CAs) were examined in intact cells by measuring 18O exchange from doubly labelled CO2 (13C18O18O) to water and by using CA inhibitors. The CO2-fixation kinetics of intact Coccomyxa cells were also compared with the carboxylation efficiency of its isolated and purified primary carboxylating enzyme, ribulose-1,5-bisphosphate carboxylaseoxygenase (Rubisco). The two algae were found to be significantly different in their modes of acquiring CO2 for photosynthesis. Chlamydomonas was able to actively transport both HCOinf3sup−and CO2 from the external medium, while Coccomyxa clearly favoured CO2 as a substrate. Both algae were found to possess external as well as internal CAs, but the relative amounts of these as well as their overall significance for the functioning of photosynthesis differed. In Coccomyxa, the internal CA activity was significantly higher than in Chlamydomonas and also predominated over the external activity. In Chlamydomonas, both transport and fixation of DIC were severely inhibited by ethoxyzolamide, an inhibitor of external and internal CAs as well as the DIC-transporting system, while this inhibitor only caused a minor inhibition of photosynthesis in Coccomyxa. These results thus give strong support for earlier indirect observations of the absence of a CO2concentrating mechanism in Coccomyxa. In addition, Coccomyxa was found to possess a Rubisco with a higher carboxylation efficiency than Chlamydomonas, having a Km (CO2) of 12 +3 μM CO2 and a CO2/O2 specificity factor (Sc/o) of 83 +2, and it may hence be concluded that the absence of the CO2-concentrating mechanism is positively correlated with a more efficient Rubisco in this alga.

Evidence for the contribution of pseudocyclic photophosphorylation to the energy requirement of the mechanism for concentrating inorganic carbon in Chlamydomonas

Mass-spectrometric measurements of 16O2 and 18O2 were made to compare the rates of light-dependent O2 evolution and uptake by Chlamydomonas reinhardtii Dang. grown in air (0.035% CO2; low-Ci cells) or CO2-enriched air (5% CO2; high-Ci cells) at pH 5.5 and 8.0. While at pH 5.5, no differences were observed in the isotopic O2-gas exchange of high- and low-Ci cells, at pH 8.0 the rates of true O2 evolution and uptake were considerably higher in low-Ci than in high-Ci cells. The enhanced rates of O2 uptake and evolution by low-Ci cells were completely inducible within 6 h after transferring high-Ci cells to ambient air. At pH 8.0, O2 uptake in the light was inhibited by 2 μM 3-(3,4-dichlorophenyl)-1,1 dimethylurea in both types of alga, but this effect was more pronounced in low-Ci than in high-Ci cells.When the cells were grown at pH 5.5 the activities of the superoxide-radical-degrading enzymes, superoxide dismutase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase, were similar regardless of the CO2 concentration provided during growth. At pH 8.0, however, the activities of these enzymes were 4 to 20 times higher in low-Ci than in high-Ci cells. When high-Ci cells were allowed to acclimate to ambient air for 6 h at pH 8.0, the activities of superoxide dismutase, ascorbate peroxidase and monodehydroascorbate dehydrogenase increased to more than 50% of the level observed with low-Ci cells. These results are consistent with an enhanced operation of O2 photoreduction which could provide energy to the inorganic-carbon-concentrating mechanism via pseudo-cyclic photophosphorylation.

Intracellular carbonic anhydrase activities in Dunaliella tertiolecta (Butcher) and Chlamydomonas reinhardtii (Dangeard) in relation to inorganic carbon concentration during growth: further evidence for the existence of two distinct carbonic anhydrases associated with the chloroplasts

Using mass-spectrometric measurements of 18O exchange from 13C18O2 intracellular carbonic anhydrase (CA) activity was investigated in the unicellular green algae Dunaliella tertiolecta and Chlamydomonas reinhardtii which were either grown on air enriched with 5% CO2 (high-Ci cells) or on air (low-Ci cells). In D. tertiolecta high- and low-Ci cells had detectable levels of internal CA activity when measured under in-vivo conditions and this activity could be split up into three distinct forms. One CA was not associated with the chloroplasts, while two isozymes were found to be located within the plastids. The activities of all intracellular CAs were always about twofold higher in low than in high-Ci cells of D. tertiolecta and the chloroplastic enzymes were completely induced within 4 h of adaptation to air. One of the chloroplastic CAs was found to be soluble the other was insoluble. In addition to the physical differences, MgSO4 in vitro caused a more than twofold stimulation of the soluble activity while the insoluble form of CA remained rather unaffected. In C. reinhardtii, MgSO4 increased the soluble CA activity by 346% and the concentration of MgSO4 required for half-maximum stimulation was between 10 and 15 mM. Again, the insoluble CA activity was not affected by MgSO4. Furthermore, the soluble isoenzyme was considerably more sensitive to ethoxyzolamide, a potent inhibitor of CA, than the insoluble enzyme. The concentration of inhibitor causing 50% inhibition of soluble CA activity was 110 and 85 μM ethoxyzolamide for D. tertiolecta and C. reinhardtii, respectively. From these data we conclude that the two chloroplast-associated CAs are distinct enzymes.

A new chloroplast envelope carbonic anhydrase activity is induced during acclimation to low inorganic carbon concentrations in Chlamydomonas reinhardtii

Abstract. Using mass-spectrometric measurements of 18O exchange from 13C18O2 we determined the activity of carbonic anhydrase (CA; EC 4.2.1.1) in chloroplast envelope membranes isolated from Chlamydomonas reinhardtii cw-15. Our results show an enrichment of CA activity in these fractions relative to the activity in the crude chloroplast. The envelope CA activity increased about 8-fold during the acclimation to low-CO2 conditions and was completely induced within the first 4xa0h after the transfer to air levels of CO2. The CA activity was not dissociated from envelope membranes after salt treatment. In addition, no cross-reactivity with other CA isoenzymes of Chlamydomonas was observed in our chloroplast envelope membranes. All these observations indicated that the protein responsible for this activity was a new CA isoenzyme, which was an integral component of the chloroplast envelopes from Chlamydomonas. The catalytic properties of the envelope CA activity were completely different from those of the thylakoid isoenzyme, showing a high requirement for Mg2+ and a high sensitivity to ethoxyzolamide. Analysis of the integral envelope proteins showed that there were no detectable differences between high- and low-inorganic carbon (Ci) cells, suggesting that the new CA activity was constitutively expressed in both high- and low-Ci cells. Two different high-Ci-requiring mutants of C. reinhardtii, cia-3 and pmp-1, had a reduced envelope CA activity. We propose that this activity could play a role in the uptake of inorganic carbon at the chloroplast envelope membranes.