Na-Independent HCO(3) Transport and Accumulation in the Cyanobacterium Synechococcus UTEX 625

The active transport and intracellular accumulation of HCO₃⁻ by air-grown cells of the cyanobacterium Synechococcus UTEX 625 (PCC 6301) was strongly promoted by 25 millimolar Na⁺.Na⁺-dependent HCO₃⁻ accumulation also resulted in a characteristic enhancement in the rate of photosynthetic O₂ evolution and CO₂ fixation. However, when Synechococcus was grown in standing culture, high rates of HCO₃⁻ transport and photosynthesis were observed in the absence of added Na⁺. The internal HCO₃⁻ pool reached levels up to 50 millimolar, and an accumulation ratio as high as 970 was observed. Sodium enhanced HCO₃⁻ transport and accumulation in standing culture cells by about 25 to 30% compared with the five- to eightfold enhancement observed with air-grown cells. The ability of standing culture cells to utilize HCO₃⁻ from the medium in the absence of Na⁺ was lost within 16 hours after transfer to air-grown culture and was reacquired during subsequent growth in standing culture. Studies using a mass spectrometer indicated that standing culture cells were also capable of active CO₂ transport involving a high-affinity transport system which was reversibly inhibited by H₂S, as in the case for air-grown cells. The data are interpreted to indicate that Synechococcus possesses a constitutive CO₂ transport system, whereas Na⁺-dependent and Na⁺-independent HCO₃⁻ transport are inducible, depending upon the conditions of growth. Intracellular accumulation of HCO₃⁻ was always accompanied by a quenching of chlorophyll a fluorescence which was independent of CO₂ fixation. The extent of fluorescence quenching was highly dependent upon the size of the internal pool of HCO₃⁻ + CO₂. The pattern of fluorescence quenching observed in response to added HCO₃⁻ and Na⁺ in air-grown and standing culture cells was highly characteristic for Na⁺-dependent and Na⁺-independent HCO₃⁻ accumulation. It was concluded that measurements of fluorescence quenching provide an indirect means for following HCO₃⁻ transport and the dynamics of intracellular HCO₃⁻ accumulation and dissipation.     

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.     

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.     

High Affinity Transport of CO(2) in the Cyanobacterium Synechococcus UTEX 625

The active transport of CO₂ in Synechococcus UTEX 625 was measured by mass spectrometry under conditions that preclude HCO₃⁻ transport. The substrate concentration required to give one half the maximum rate for whole cell CO₂ transport was determined to be 0.4 ± 0.2 micromolar (mean ± standard deviation; n = 7) with a range between 0.2 and 0.66 micromolar. The maximum rates of CO₂ transport ranged between 400 and 735 micromoles per milligram of chlorophyll per hour with an average rate of 522 for seven experiments. This rate of transport was about three times greater than the dissolved inorganic carbon saturated rate of photosynthetic O₂ evolution observed under these conditions. The initial rate of chlorophyll a fluorescence quenching was highly correlated with the initial rate of CO₂ transport (correlation coefficient = 0.98) and could be used as an indirect method to detect CO₂ transport and calculate the substrate concentration required to give one half the maximum rate of transport. Little, if any, inhibition of CO₂ transport was caused by HCO₃⁻ or by Na⁺-dependent HCO₃⁻ transport. However, ¹²CO₂ readily interfered with ¹³CO₂ transport. CO₂ transport and Na⁺-dependent HCO₃⁻ transport are separate, independent processes and the high affinity CO₂ transporter is not only responsible for the initial transport of CO₂ into the cell but also for scavenging any CO₂ that may leak from the cell during ongoing photosynthesis.     

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.     

Simultaneous Transport of CO(2) and HCO(3) by the Cyanobacterium Synechococcus UTEX 625

A mass spectrometer was used to simultaneously follow the time course of photosynthetic O₂ evolution and CO₂ depletion of the medium by cells of the cyanobacterium Synechococcus leopoliensis UTEX 625. Analysis of the data indicated that both CO₂ and HCO₃⁻ were simultaneously and continuously transported by the cells as a source of substrate for photosynthesis. Initiation of HCO₃⁻ transport by Na⁺ addition had no effect on ongoing CO₂ transport. This result is interpreted to indicate that the CO₂ and HCO₃⁻ transport systems are separate and distinctly different transport systems. Measurement of CO₂-dependent photosynthesis indicated that CO₂ uptake involved active transport and that diffusion played only a minor role in CO₂ acquisition in cyanobacteria.     

Photorespiration and Internal CO(2) Accumulation in Chara corallina as Inferred from the Influence of DIC and O(2) on Photosynthesis

An O₂ electrode system with a specially designed chamber for `whorl' cell complexes of Chara corallina was used to study the combined effects of inorganic carbon and O₂ concentrations on photosynthetic O₂ evolution. At pH = 5.5 and 20% O₂, cells grown in HCO₃⁻ medium (low CO₂, pH ≥ 9.0) exhibited a higher affinity for external CO₂ (K_½(CO₂) = 40 ± 6 micromolar) than the cells grown for at least 24 hours in high-CO₂ medium (pH = 6.5), (K_½(CO₂) = 94 ± 16 micromolar). With O₂ ≤ 2% in contrast, both types of cells showed a high apparent affinity (K_½(CO₂) = 50 − 52 micromolar). A Warburg effect was detectable only in the low affinity cells previously cultivated in high-CO₂ medium (pH = 6.5). The high-pH, HCO₃⁻-grown cells, when exposed to low pH (5.5) conditions, exhibited a response indicating an ability to fix CO₂ which exceeded the CO₂ externally supplied, and the reverse situation has been observed in high-CO₂-grown cells. At pH 8.2, the apparent photosynthetic affinity for external HCO₃⁻ (K_½[HCO₃⁻]) was 0.6 ± 0.2 millimolar, at 20% O₂. But under low O₂ concentrations (≤2%), surprisingly, an inhibition of net O₂ evolution was elicited, which was maximal at low HCO₃⁻ concentrations. These results indicate that: (a) photorespiration occurs in this alga and can be revealed by cultivation in high-CO₂ medium, (b) Chara cells are able to accumulate CO₂ internally by means of a process apparently independent of the plasmalemma HCO₃⁻ transport system, (c) molecular oxygen appears to be required for photosynthetic utilization of exogenous HCO₃⁻: pseudocyclic electron flow, sustained by O₂ photoreduction, may produce the additional ATP needed for the HCO₃⁻ transport.     

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⁻.     

Carbon Oxysulfide Inhibition of the CO(2)-Concentrating Process of Unicellular Green Algae

Carbonyl sulfide (COS), a substrate for carbonic anhydrase, inhibited alkalization of the medium, O₂ evolution, dissolved inorganic carbon accumulation, and photosynthetic CO₂ fixation at pH 7 or higher by five species of unicellular green algae that had been air-adapted for forming a CO₂-concentrating process. This COS inhibition can be attributed to inhibition of external HCO₃⁻ conversion to CO₂ and OH⁻ by the carbonic anhydrase component of an active CO₂ pump. At a low pH of 5 to 6, COS stimulated O₂ evolution during photosynthesis by algae with low CO₂ in the media without alkalization of the media. This is attributed to some COS hydrolysis by carbonic anhydrase to CO₂. Although COS had less effect on HCO₃⁻ accumulation at pH 9 by a HCO₃⁻ pump in Scenedesmus, COS reduced O₂ evolution probably by inhibiting internal carbonic anhydrases. Because COS is hydrolyzed to CO₂ and H₂S, its inhibition of the CO₂ pump activity and photosynthesis is not accurate, when measured by O₂ evolution, by NaH¹⁴CO₃ accumulation, or by ¹⁴CO₂ fixation.     

Nature of the Inorganic Carbon Species Actively Taken Up by the Cyanobacterium Anabaena variabilis

The nature of the inorganic carbon (C_i) species actively taken up by cyanobacteria CO₂ or HCO₃⁻ has been investigated. The kinetics of CO₂ uptake, as well as that of HCO₃⁻ uptake, indicated the involvement of a saturable process. The apparent affinity of the uptake mechanism for CO₂ was higher than that for HCO₃⁻. Though the calculated V_max was the same in both cases, the maximum rate of uptake actually observed was higher when HCO₃⁻ was supplied. C_i uptake was far more sensitive to the carbonic anhydrase inhibitor ethoxyzolamide when CO₂ was the species supplied. Observations of photosynthetic rate as a function of intracellular C_i level (following supply of CO₂ or HCO₃⁻ for 5 seconds) led to the inference that HCO₃⁻ is the species which arrives at the inner membrane surface, regardless of the species supplied. When the two species were supplied simultaneously, mutual inhibition of uptake was observed.

On the basis of these and other results, a model is proposed postulating that a carboic anhydrase-like subunit of the C_i transport apparatus binds CO₂ and releases HCO₃⁻ at or near a membrane porter. The latter transports HCO₃⁻ ions to the cell interior.

     

Uptake of HCO3? and CO2 in Cells and Chloroplasts from the Microalgae Chlamydomonas reinhardtii and Dunaliella tertiolecta

Mass-spectrometric disequilibrium analysis was applied to investigate CO₂ uptake and HCO₃⁻ transport in cells and chloroplasts of the microalgae Dunaliella tertiolecta and Chlamydomonas reinhardtii, which were grown in air enriched with 5% (v/v) CO₂ (high-Ci cells) or in ambient air (low-Ci cells). High- and low-Ci cells of both species had the capacity to transport CO₂ and HCO₃⁻, with maximum rates being largely unaffected by the growth conditions. In high- and low-Ci cells of D. tertiolecta, HCO₃⁻ was the dominant inorganic C species taken up, whereas HCO₃⁻ and CO₂ were used at similar rates by C. reinhardtii. The apparent affinities of HCO₃⁻ transport and CO₂ uptake increased 3- to 9-fold in both species upon acclimation to air. Photosynthetically active chloroplasts isolated from both species were able to transport CO₂ and HCO₃⁻. For chloroplasts from C. reinhardtii, the concentrations of HCO₃⁻ and CO₂ required for half-maximal activity declined from 446 to 33 μm and 6.8 to 0.6 μm, respectively, after acclimation of the parent cells to air; the corresponding values for chloroplasts from D. tertiolecta decreased from 203 to 58 μm and 5.8 to 0.5 μm, respectively. These results indicate the presence of inducible high-affinity HCO₃⁻ and CO₂ transporters at the chloroplast envelope membrane.     

Characterization of the na-requirement in cyanobacterial photosynthesis

The Na⁺ requirement for photosynthesis and its relationship to dissolved inorganic carbon (DIC) concentration and Li⁺ concentration was examined in air-grown cells of the cyanobacterium Synechococcus leopoliensis UTEX 625 at pH 8. Analysis of the rate of photosynthesis (O₂ evolution) as a function of Na⁺ concentration, at fixed DIC concentration, revealed two distinct regions to the response curve, for which half-saturation values for Na⁺ (K_½[Na⁺]) were calculated. The value of both the low and the high K_½(Na⁺) was dependent upon extracellular DIC concentration. The low K_½(Na⁺) decreased from 1000 micromolar at 5 micromolar DIC to 200 micromolar at 140 micromolar DIC whereas over the same DIC concentration range the high K_½(Na⁺) decreased from 10 millimolar to 1 millimolar. The most significant increases in photosynthesis occurred in the 1 to 20 millimolar range. A fraction of total photosynthesis, however, was independent of added Na⁺ and this fraction increased with increased DIC concentration. A number of factors were identified as contributing to the complexity of interaction between Na⁺ and DIC concentration in the photosynthesis of Synechococcus. First, as revealed by transport studies and mass spectrometry, both CO₂ and HCO₃⁻ transport contributed to the intracellular supply of DIC and hence to photosynthesis. Second, both the CO₂ and HCO₃⁻ transport systems required Na⁺, directly or indirectly, for full activity. However, micromolar levels of Na⁺ were required for CO₂ transport while millimolar levels were required for HCO₃⁻ transport. These levels corresponded to those found for the low and high K_½(Na⁺) for photosynthesis. Third, the contribution of each transport system to intracellular DIC was dependent on extracellular DIC concentration, where the contribution from CO₂ transport increased with increased DIC concentration relative to HCO₃⁻ transport. This change was reflected in a decrease in the Na⁺ concentration required for maximum photosynthesis, in accord with the lower Na⁺-requirement for CO₂ transport. Lithium competitively inhibited Na⁺-stimulated photosynthesis by blocking the cells' ability to form an intracellular DIC pool through Na⁺-dependent HCO₃⁻ transport. Lithium had little effect on CO₂ transport and only a small effect on the size of the pool it generated. Thus, CO₂ transport did not require a functional HCO₃⁻ transport system for full activity. Based on these observations and the differential requirement for Na⁺ in the CO₂ and HCO₃⁻ transport system, it was proposed that CO₂ and HCO₃⁻ were transported across the membrane by different transport systems.     

Selective and Reversible Inhibition of Active CO(2) Transport by Hydrogen Sulfide in a Cyanobacterium

The active transport of CO₂ in the cyanobacterium Synechococcus UTEX 625 was inhibited by H₂S. Treatment of the cells with up to 150 micromolar H₂S + HS⁻ at pH 8.0 had little effect on Na⁺-dependent HCO₃⁻ transport or photosynthetic O₂ evolution, but CO₂ transport was inhibited by more than 90%. CO₂ transport was restored when H₂S was removed by flushing with N₂. At constant total H₂S + HS⁻ concentrations, inhibition of CO₂ transport increased as the ratio of H₂S to HS⁻ increased, suggesting a direct role for H₂S in the inhibitory process. Hydrogen sulfide does not appear to serve as a substrate for transport. In the presence of H₂S and Na⁺ -dependent HCO₃⁻ transport, the extracellular CO₂ concentration rose considerably above its equilibrium level, but was maintained far below its equilibrium level in the absence of H₂S. The inhibition of CO₂ transport, therefore, revealed an ongoing leakage from the cells of CO₂ which was derived from the intracellular dehydration of HCO₃⁻ which itself had been recently transported into the cells. Normally, leaked CO₂ is efficiently transported back into the cell by the CO₂ transport system, thus maintaining the extracellular CO₂ concentration near zero. It is suggested that CO₂ transport not only serves as a primary means of inorganic carbon acquisition for photosynthesis but also serves as a means of recovering CO₂ lost from the cell. A schematic model describing the relationship between the CO₂ and HCO₃⁻ transport systems is presented.     

Ethoxyzolamide Inhibition of CO(2) Uptake in the Cyanobacterium Synechococcus PCC7942 without Apparent Inhibition of Internal Carbonic Anhydrase Activity

In high inorganic carbon grown (1% CO₂ [volume/volume]) cells of the cyanobacterium Synechococcus PCC7942, the carbonic anhydrase (CA) inhibitor, ethoxyzolamide (EZ), was found to inhibit the rate of CO₂ uptake and to reduce the final internal inorganic carbon (C_i) pool size reached. The relationship between CO₂ fixation rate and internal C_i concentration in high C_i grown cells was little affected by EZ. This suggests that in intact cells internal CA activity was unaffected by EZ. High C_i grown cells readily took up CO₂ but had little or no capacity for HCO₃⁻ uptake. These cells appear to possess a CO₂ utilizing C_i pump that has a CA-like function associated with the transport step such that HCO₃⁻ is the species delivered to the cell interior. This CA-like step may be the site of inhibition by EZ. Low C_i grown cells possess both CO₂ uptake and HCO₃⁻ uptake activities and EZ inhibited both activities to a similar degree, suggesting that a common step in CO₂ and HCO₃⁻ uptake (such as the C_i pump) may have been affected. The inhibitor had no apparent effect on internal CO₂/HCO₃⁻ equilibria (internal CA function) in low C_i grown cells.     

Energy Sources for HCO3? and CO2 Transport in Air-Grown Cells of Synechococcus UTEX 625

Light-dependent inorganic C (C_i) transport and accumulation in air-grown cells of Synechococcus UTEX 625 were examined with a mass spectrometer in the presence of inhibitors or artificial electron acceptors of photosynthesis in an attempt to drive CO₂ or HCO₃⁻ uptake separately by the cyclic or linear electron transport chains. In the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea, the cells were able to accumulate an intracellular C_i pool of 20 mm, even though CO₂ fixation was completely inhibited, indicating that cyclic electron flow was involved in the C_i-concentrating mechanism. When 200 μm N,N-dimethyl-p-nitrosoaniline was used to drain electrons from ferredoxin, a similar C_i accumulation was observed, suggesting that linear electron flow could support the transport of C_i. When carbonic anhydrase was not present, initial CO₂ uptake was greatly reduced and the extracellular [CO₂] eventually increased to a level higher than equilibrium, strongly suggesting that CO₂ transport was inhibited and that C_i accumulation was the result of active HCO₃⁻ transport. With 3-(3,4-dichlorophenyl)-1,1-dimethylurea-treated cells, C_i transport and accumulation were inhibited by inhibitors of CO₂ transport, such as COS and Na₂S, whereas Li⁺, an HCO₃⁻-transport inhibitor, had little effect. In the presence of N,N-dimethyl-p-nitrosoaniline, C_i transport and accumulation were not inhibited by COS and Na₂S but were inhibited by Li⁺. These results suggest that CO₂ transport is supported by cyclic electron transport and that HCO₃⁻ transport is supported by linear electron transport.     

Salicylhydroxamic Acid (SHAM) Inhibition of the Dissolved Inorganic Carbon Concentrating Process in Unicellular Green Algae

Rates of photosynthetic O₂ evolution, for measuring K_0.5(CO₂ + HCO₃⁻) at pH 7, upon addition of 50 micromolar HCO₃⁻ to air-adapted Chlamydomonas, Dunaliella, or Scenedesmus cells, were inhibited up to 90% by the addition of 1.5 to 4.0 millimolar salicylhydroxamic acid (SHAM) to the aqueous medium. The apparent K₁(SHAM) for Chlamydomonas cells was about 2.5 millimolar, but due to low solubility in water effective concentrations would be lower. Salicylhydroxamic acid did not inhibit oxygen evolution or accumulation of bicarbonate by Scenedesmus cells between pH 8 to 11 or by isolated intact chloroplasts from Dunaliella. Thus, salicylhydroxamic acid appears to inhibit CO₂ uptake, whereas previous results indicate that vanadate inhibits bicarbonate uptake. These conclusions were confirmed by three test procedures with three air-adapted algae at pH 7. Salicylhydroxamic acid inhibited the cellular accumulation of dissolved inorganic carbon, the rate of photosynthetic O₂ evolution dependent on low levels of dissolved inorganic carbon (50 micromolar Na-HCO₃), and the rate of ¹⁴CO₂ fixation with 100 micromolar [¹⁴C] HCO₃⁻. Salicylhydroxamic acid inhibition of O₂ evolution and ¹⁴CO₂-fixation was reversed by higher levels of NaHCO₃. Thus, salicylhydroxamic acid inhibition was apparently not affecting steps of photosynthesis other than CO₂ accumulation. Although salicylhydroxamic acid is an inhibitor of alternative respiration in algae, it is not known whether the two processes are related.     

Fast Induction of High-Affinity HCO3− Transport in Cyanobacteria

The induction of a high-affinity state of the CO₂-concentration mechanism was investigated in two cyanobacterial species, Synechococcus sp. strain PCC7002 and Synechococcus sp. strain PCC7942. Cells grown at high CO₂ concentrations were resuspended in low-CO₂ buffer and illuminated in the presence of carbonic anhydrase for 4 to 10 min until the inorganic C compensation point was reached. Thereafter, more than 95% of a high-affinity CO₂-concentration mechanism was induced in both species. Mass-spectrometric analysis of CO₂ and HCO₃⁻ fluxes indicated that only the affinity of HCO₃⁻ transport increased during the fast-induction period, whereas maximum transport activities were not affected. The kinetic characteristics of CO₂ uptake remained unchanged. Fast induction of high-affinity HCO₃⁻ transport was not inhibited by chloramphenicol, cantharidin, or okadaic acid. In contrast, fast induction of high-affinity HCO₃⁻ transport did not occur in the presence of K252a, staurosporine, or genistein, which are known inhibitors of protein kinases. These results show that induction of high-affinity HCO₃⁻ transport can occur within minutes of exposure to low-inorganic-C conditions and that fast induction may involve posttranslational phosphorylation of existing proteins rather than de novo synthesis of new protein components.     

Utilization of Inorganic Carbon by Ulva lactuca

Thalli discs of the marine macroalga Ulva lactuca were given inorganic carbon in the form of HCO₃⁻, and the progression of photosynthetic O₂ evolution was followed and compared with predicted O₂ evolution as based on calculated external formation of CO₂ (extracellular carbonic anhydrase was not present in this species) and its carboxylation (according to the K_m(CO₂) of ribulose-1,5-bisphosphate carboxylase/oxygenase), at two different pHs, assuming a photosynthetic quotient of 1. The K_m(inorganic carbon) was some 2.5 times lower at pH 5.6 than at the natural seawater pH of 8.2, whereas V_max was similar under the two conditions, indicating that the unnaturally low pH per se had no adverse effect on U. lactuca's photosynthetic performance. These results, therefore, could be evaluated with regard to differential CO₂ and HCO₃⁻ utilization. The photosynthetic performance observed at the lower pH largely followed that predicted, with a slight discrepancy probably reflecting a minor diffusion barrier to CO₂ uptake. At pH 8.2, however, dehydration rates were too slow to supply CO₂ for the measured photosynthetic response. Given the absence of external carbonic anhydrase activity, this finding supports the view that HCO₃⁻ transport provides higher than external concentrations of CO₂ at the ribulose-1,5-bisphosphate carboxylase/oxygenase site. Uptake of HCO₃⁻ by U. lactuca was further indicated by the effects of potential inhibitors at pH 8.2. The alleged band 3 membrane anion exchange protein inhibitor 4,4′-diisothiocyanostilbene-2,2′disulphonate reduced photosynthetic rates only when HCO₃⁻ (but not CO₂) could be the extracellular inorganic carbon form taken up. A similar, but less drastic, HCO₃⁻-competitive inhibition of photosynthesis was obtained with Kl and KNO₃. It is suggested that, under ambient conditions, HCO₃⁻ is transported into cells at defined sites either via facilitated diffusion or active uptake, and that such transport is the basis for elevated internal [CO₂] at the site of ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation.     

Growth and Photosynthesis of the Cyanobacterium Synechococcus leopoliensis in HCO(3)-Limited Chemostats

Synechococcus leopoliensis was grown in HCO₃⁻-limited chemostats. Growth at 50% the maximum rate occurred when the inorganic carbon concentration was 10 to 15 micromolar (or 5.6 to 8.4 nanomolar CO₂). The O₂ to CO₂ ratios during growth were as high as 192,000 to 1. At growth rates below 80% the maximum rate, essentially all the supplied inorganic carbon was converted to organic carbon, and the cells were carbon limited. Carbon-limited cells used HCO₃⁻ rather than CO₂ for growth. They also exhibited a very high photosynthetic affinity for inorganic carbon in short-term experiments. Cells growing at greater than 80% maximum growth rate, in the presence of high dissolved inorganic carbon, were termed carbon sufficient. These cells had photosynthetic affinities that were about 1000-fold lower than HCO₃⁻-limited cells and also had a reduced capacity for HCO₃⁻ transport. HCO₃⁻-limited cells are reminiscent of the air-grown cells of batch culture studies while the carbon sufficient cells are reminiscent of high-CO₂ grown cells. However, the low affinity cells of the present study were growing at CO₂ concentrations less than air saturation. This suggests that supranormal levels of CO₂ not required to induce the physiological changes usually ascribed to high CO₂ cells.     

Use of Carbon Oxysulfide, a Structural Analog of CO(2), to Study Active CO(2) Transport in the Cyanobacterium Synechococcus UTEX 625

Carbon oxysulfide (carbonyl sulfide, COS) is a close structural analog of CO₂. Although hydrolysis of COS (to CO₂ and H₂S) does occur at alkaline pH (>9), at pH 8.0 the rate of hydrolysis is slow enough to allow investigation of COS as a possible substrate and inhibitor of the active CO₂ transport system of Synechococcus UTEX 625. A light-dependent uptake of COS was observed that was inhibited by CO₂ and the ATPase inhibitor diethylstilbestrol. The COS taken up by the cells could not be recovered when the lights were turned off or when acid was added. It was concluded that most of the COS taken up was hydrolyzed by intracellular carbonic anhydrase. The production of H₂S was observed and COS removal from the medium was inhibited by ethoxyzolamide. Bovine erythrocyte carbonic anhydrase catalysed the stoichiometric hydrolysis of COS to H₂S. The active transport of CO₂ was inhibited by COS in an apparently competitive manner. When Na⁺-dependent HCO₃⁻ transport was allowed in the presence of COS, the extracellular [CO₂] rose considerably above the equilibrium level. This CO₂ appearing in the medium was derived from the dehydration of transported HCO₃⁻ and was leaked from the cells. In the presence of COS the return to the cells of this leaked CO₂ was inhibited. These results showed that the Na⁺-dependent HCO₃⁻ transport was not inhibited by COS, whereas active CO₂ transport was inhibited. When COS was removed by gassing with N₂, a normal pattern of CO₂ uptake was observed. The silicone fluid centrifugation method showed that COS (100 micromolar) had little effect upon the initial rate of HCO₃⁻ transport or CO₂ fixation. The steady state rate of CO₂ fixation was, however, inhibited about 50% in the presence of COS. This inhibition can be at least partially explained by the significant leakage of CO₂ from the cells that occurred when CO₂ uptake was inhibited by COS. Neither CS₂ nor N₂O acted like COS. It is concluded that COS is an effective and selective inhibitor of active CO₂ transport.