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Background: Bacteria routinely utilize two-component signal transduction pathways to sense and alter gene expression in response to environmental cues. While cyanobacteria express numerous two-component systems, these pathways do not regulate all of the genes within many of the identified abiotic stress-induced regulons., Methods: Electron transport inhibitors combined with western analysis and measurement of chlorophyll a fluorescent yield, using pulse amplitude modulation fluorometry, were used to detect the effect of a diverse range of abiotic stresses on the redox status of the photosynthetic electron transport chain and the accumulation and degradation of the Synechocystis sp. PCC 6803 DEAD box RNA helicase, CrhR., Results: Alterations in CrhR abundance were tightly correlated with the redox poise of the electron transport chain between Q A and cytochrome b 6 f, with reduction favoring CrhR accumulation., Conclusions: The results provide evidence for an alternative, convergent sensing mechanism mediated through the redox poise of Q B /PQH 2 that senses multiple, divergent forms of abiotic stress and regulates accumulation of CrhR. The RNA helicase activity of CrhR could then function as a post-translational effector to regulate downstream gene expression., General Significance: The potential for a related system in Staphylococcus aureus and higher plant chloroplasts suggest convergent sensing mechanisms may be evolutionarily conserved and occur more widely than anticipated., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Carbonic anhydrases (CAs) are involved in CO 2 uptake and conversion, a fundamental process in photosynthetic organisms. Nevertheless, the mechanism underlying the regulation of CO 2 uptake and intracellular conversion in cyanobacteria is largely unknown. We report the characterization of a previously unrecognized thylakoid-located CA Slr0051 (EcaB) from the cyanobacterium Synechocystis sp. PCC 6803, which possesses CA activity to regulate CO 2 uptake. Inactivation of ecaB stimulated CO 2 hydration in the thylakoids, suppressed by the classical CA inhibitor acetazolamide. Absence of ecaB increased the reduced state of the photosynthetic electron transport system, lowered the rate of photosynthetic O 2 evolution at high light (HL) and pH, and decreased the cellular affinity for extracellular inorganic carbon. Furthermore, EcaB was upregulated in cells grown at limiting CO 2 concentration or HL in tandem with CupA. EcaB is mainly located in the thylakoid membranes where it interacts with CupA and CupB involved in CO 2 uptake by converting it to bicarbonate. We propose that modulation of the EcaB level and activity in response to CO 2 changes, illumination or pH reversibly regulates its conversion to HCO 3 by the two CO 2 -uptake systems (CupA, CupB), dissipating the excess HCO 3 - and alleviating photoinhibition, and thereby optimizes photosynthesis, especially under HL and alkaline conditions., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

A paper-based platform was investigated in which the selective detection of oligonucleotide targets by hybridization was accomplished via the enhancement of fluorescence emission from intrinsically labeled DNA probes that were immobilized on the surface of quantum dots (QDs). Multiple copies of a derivative of thiazole orange, an intercalating dye known to form non-emissive dimers, were conjugated to single-stranded oligonucleotide probes. Dimerization resulted in the formation of H-aggregates where excitonic interactions led to the suppression of fluorescence. The hybridization of the oligonucleotide probe with a complementary target resulted in the enhancement of fluorescence emission as the dimers dissociated and the dyes preferentially intercalated with the duplex. The detection of oligonucleotide targets using this configuration eliminated the need for labeling the target strands, and fluorescence intensity was proportional to the extent of hybridization. In addition, the dye molecules were excited using Foerster Resonance Energy Transfer (FRET) from QD donors, which resulted in improved selectivity and allowed for ratiometric detection. A solution-phase hybridization assay based on similar operational principles has been previously reported, and this new work investigated the advantages offered for this transduction scheme using paper-based solid-phase substrates. QD-probe conjugates were immobilized in sufficient density on the paper matrix to provide for multiple-donor-multiple-acceptor interactions that resulted in a 20-fold enhancement of acceptor emission compared to the solution-based assay, providing a limit of detection of 0.1 pmol. The paper-based assay provided for the reduction of the time needed for sample preparation and data acquisition, demonstrated that transduction was possible in a complex matrix (goat serum) without compromising on the performance observed in buffer solution, and that oligonucleotides generated from standard PCR amplification could be detected.

CcaA is a β-carbonic anhydrase (CA) that is a component of the carboxysomes of a subset of β-cyanobacteria. This protein, which has a characteristic C-terminal extension of unknown function, is recruited to the carboxysome via interactions with CcmM, which is itself a γ-CA homolog with enzymatic activity in many, but not all cyanobacteria. We have determined the structure of CcaA from Synechocystis sp. PCC 6803 at 1.45 Å. In contrast with the dimer-of-dimers organization of most bacterial β-CAs, or the loose dimer-of-dimers-of-dimers organization found in the plant enzymes, CcaA shows a well-packed trimer-of-dimers organization. The proximal part of the characteristic C-terminal extension is ordered by binding at a site that passes through the two-fold symmetry axis shared with an adjacent dimer; as a result, only one of a pair of converging termini can be ordered at any given time. Docking in Rosetta failed to find well-packed solutions, indicating that formation of the CcaA/CcmM complex probably requires significant backbone movements in at least one of the binding partners. Surface plasmon resonance experiments showed that CcaA forms a complex with CcmM with sub-picomolar affinity, with contributions from residues in CcmM's αA helix and CcaA's C-terminal tail. Catalytic characterization showed CcaA to be among the least active β-CAs characterized to date, with activity comparable with the γ-CA, CcmM, it either complements or replaces. Intriguingly, the C-terminal tail appears to partly inhibit activity, possibly indicating a role in minimizing the activity of unencapsulated enzyme., (© 2016 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Algal biofilms were grown to investigate the interaction effects of bulk medium CO2 concentration and photon flux density (PFD) on biomass productivities. When increasing the CO2 concentration from 0.04% to 2%, while maintaining a PFD of 100μmol/m(2)/s, biomass productivities increased from ∼0.5 to 2.0g/m(2)/d; however, the productivities plateaued when CO2 concentrations were incrementally increased above 2-12%. Statistical analysis demonstrates that there is a significant interaction between PFD and CO2 concentrations on biomass productivities. By simultaneously increasing PFD and CO2 concentrations, biomass productivities were significantly increased to 4.0 and 4.1g/m(2)/d in the experimental and modeled data, respectively. The second order model predicted increases in biomass productivities as both PFD and CO2 simultaneously increased yielding an optimum at 440μmol/m(2)/s and 7.1%; however, when conditions were extended to the highest end of their respective ranges, the conditions were detrimental to growth and productivities decreased., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Paper is a promising platform for the development of decentralized diagnostic assays owing to the low cost and ease of use of paper-based analytical devices (PADs). It can be challenging to detect on PADs very low concentrations of nucleic acid biomarkers of lengths as used in clinical assays. Herein we report the use of thermophilic helicase-dependent amplification (tHDA) in combination with a paper-based platform for fluorescence detection of probe-target hybridization. Paper substrates were patterned using wax printing. The cellulosic fibers were chemically derivatized with imidazole groups for the assembly of the transduction interface that consisted of immobilized quantum dot (QD)-probe oligonucleotide conjugates. Green-emitting QDs (gQDs) served as donors with Cy3 as the acceptor dye in a fluorescence resonance energy transfer (FRET)-based transduction method. After probe-target hybridization, a further hybridization event with a reporter sequence brought the Cy3 acceptor dye in close proximity to the surface of immobilized gQDs, triggering a FRET sensitized emission that served as an analytical signal. Ratiometric detection was evaluated using both an epifluorescence microscope and a low-cost iPad camera as detectors. Addition of the tHDA method for target amplification to produce sequences of ∼100 base length allowed for the detection of zmol quantities of nucleic acid targets using the two detection platforms. The ratiometric QD-FRET transduction method not only offered improved assay precision, but also lowered the limit of detection of the assay when compared with the non-ratiometric QD-FRET transduction method. The selectivity of the hybridization assays was demonstrated by the detection of single nucleotide polymorphism., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Algae biofilms were grown in a semicontinuous flat plate biofilm photobioreactor to study the effects of light direction and suspended algal cell populations on algal biofilm growth. It was determined that, under the growth conditions and biofilm thicknesses studied, light direction had no effect on long-term algal biofilm growth (26 days); however, light direction did affect the concentration of suspended algal cells by influencing the photon flux density in the growth medium in the photobioreactors. This suspended algal cell population affected short-term (7 days) algae cell recruitment and algal biofilm growth, but additional studies showed that enhanced suspended algal cell populations did not affect biofilm growth rates over the long term (26 days). Studying profiles of light transmittance through biofilms as they grew showed that most of the light became attenuated by the biomass after just a few days of growth (88 % after 3 days). The estimated biofilm thicknesses after these few days of growth were approximately 150 μm. The light attenuation data suggests that, although the biofilms grew to 700-900 μm, under these light intensities, only the first few hundred micrometers of the biofilm is receiving enough light to be photosynthetically active. We postulate that this photosynthetically active layer of the biofilm grows adjacent to the light source, while the rest of the biofilm is in a stationary growth phase. The results of this study have implications for algal biofilm photobioreactor design and operation.

Nonlinear optical microscopy was used to image the localization of astaxanthin accumulation in the green alga, Haematococcus pluvialis. Polarization-in, polarization-out (PIPO) second harmonic generation (SHG) and third harmonic generation (THG) microscopy was applied to study the crystalline organization of astaxanthin molecules in light-stressed H. pluvialis in vivo. Since astaxanthin readily forms H- and J-aggregates in aqueous solutions, PIPO THG studies of astaxanthin aggregates contained in red aplanospores were compared to PIPO THG of in vitro self-assembled H- and J-aggregates of astaxanthin. The PIPO THG data clearly showed an isotropic organization of astaxanthin in red aplanospores of H. pluvialis. This is in contrast to the highly anisotropic organization of astaxanthin in synthetic H- and J-aggregates, which showed to be uniaxial. Since carotenoids in vitro preferentially form H- and J-aggregates, but in vivo form a randomly organized structure, this implies that astaxanthin undergoes a different way of packing in biological organisms, which is either due to the unique physical environment of the alga or is controlled enzymatically.

Carboxysomes are proteinaceous microcompartments that encapsulate carbonic anhydrase (CA) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco); carboxysomes, therefore, catalyze reversible HCO3 (-) dehydration and the subsequent fixation of CO2. The N- and C-terminal domains of the β-carboxysome scaffold protein CcmM participate in a network of protein-protein interactions that are essential for carboxysome biogenesis, organization, and function. The N-terminal domain of CcmM in the thermophile Thermosynechococcus elongatus BP-1 is also a catalytically active, redox regulated γ-CA. To experimentally determine if CcmM from a mesophilic cyanobacterium is active, we cloned, expressed and purified recombinant, full-length CcmM from Nostoc sp. PCC 7120 as well as the N-terminal 209 amino acid γ-CA-like domain. Both recombinant proteins displayed ethoxyzolamide-sensitive CA activity in mass spectrometric assays, as did the carboxysome-enriched TP fraction. NstCcmM209 was characterized as a moderately active and efficient γ-CA with a k cat of 2.0 × 10(4) s(-1) and k cat/K m of 4.1 × 10(6) M(-1) s(-1) at 25 °C and pH 8, a pH optimum between 8 and 9.5 and a temperature optimum spanning 25-35 °C. NstCcmM209 also catalyzed the hydrolysis of the CO2 analog carbonyl sulfide. Circular dichroism and intrinsic tryptophan fluorescence analysis demonstrated that NstCcmM209 was progressively and irreversibly denatured above 50 °C. NstCcmM209 activity was inhibited by the reducing agent tris(hydroxymethyl)phosphine, an effect that was fully reversed by a molar excess of diamide, a thiol oxidizing agent, consistent with oxidative activation being a universal regulatory mechanism of CcmM orthologs. Immunogold electron microscopy and Western blot analysis of TP pellets indicated that Rubisco and CcmM co-localize and are concentrated in Nostoc sp. PCC 7120 carboxysomes.

Oxygenic photosynthesis would not be possible without photorespiration in the present day O2 -rich atmosphere. It is now generally accepted that cyanobacteria-like prokaryotes first evolved oxygenic photosynthesis, which was later conveyed via endosymbiosis into a eukaryotic host, which then gave rise to the different groups of algae and streptophytes. For photosynthetic CO2 fixation, all these organisms use RubisCO, which catalyses both the carboxylation and the oxygenation of ribulose 1,5-bisphosphate. One of the reaction products of the oxygenase reaction, 2-phosphoglycolate (2PG), represents the starting point of the photorespiratory C2 cycle, which is considered largely responsible for recapturing organic carbon via conversion to the Calvin-Benson cycle (CBC) intermediate 3-phosphoglycerate, thereby detoxifying critical intermediates. Here we discuss possible scenarios for the evolution of this process toward the well-defined 2PG metabolism in extant plants. While the origin of the C2 cycle core enzymes can be clearly dated back towards the different endosymbiotic events, the evolutionary scenario that allowed the compartmentalised high flux photorespiratory cycle is uncertain, but probably occurred early during the algal radiation. The change in atmospheric CO2 /O2 ratios promoting the acquisition of different modes for inorganic carbon concentration mechanisms, as well as the evolutionary specialisation of peroxisomes, clearly had a dramatic impact on further aspects of land plant photorespiration., (© 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.)

Reduction of flux through photorespiration has been viewed as a major way to improve crop carbon fixation and yield since the energy-consuming reactions associated with this pathway were discovered. This view has been supported by the biomasses increases observed in model species that expressed artificial bypass reactions to photorespiration. Here, we present an overview about the major current attempts to reduce photorespiratory losses in crop species and provide suggestions for future research priorities., (© 2012 German Botanical Society and The Royal Botanical Society of the Netherlands.)

An algae biofilm growth system was developed to study the growth kinetics and neutral lipid productivities of Scenedesmus obliquus and Nitzschia palea, and to determine if algal biofilms can be starved of key nutrients to increase their neutral lipid concentrations. Linear growth curves were determined for each species until nutrient starvation commenced, at which point growth ceased and/or biofilms sloughed from their substratum. Nutrient starvation did not increase neutral lipid concentrations in any of the biofilms; however, it approximately doubled their lipid concentrations when grown in suspension. Biomass productivities of 2.8 and 2.1g/m(2)/d and lipid productivities of 0.45 and 0.18 g/m(2)/d were determined for N. palea and S. obliquus, respectively. The results suggest that nutrient starvation of biofilms is not a desirable method of lipid production for algae biofilm biofuel production systems, but that lipid production rates compare favorably with conventional terrestrial biofuel sources., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Inactivation of the DEAD box RNA helicase, crhR, has dramatic effects on the physiology and morphology of the photosynthetic cyanobacterium, Synechocystis sp. PCC 6803. These effects are observed at both normal growth temperature (30°C) and under cold stress (20°C), indicating that CrhR performs crucial function(s) at all temperatures. A major physiological effect is the rapid cessation of photosynthesis upon temperature downshift from 30 to 20°C. This defect does not originate from an inability to transport or accumulate inorganic carbon or a deficiency in photosynthetic capacity as the mutant has sufficient electron transport and enzymatic capacity to sustain photosynthesis at 30°C and inorganic carbon (Ci) accumulation at 20°C. Oxygen consumption in the presence of methyl viologen indicated that while electron transport capacity is sufficient to accumulate Ci, the mutant does not possess sufficient activity to sustain carbon fixation at maximal rates. These defects are correlated with severely impaired cell growth and decreased viability, cell size and DNA content at low temperature. The ΔcrhR mutant also progressively accumulates structural abnormalities at low temperature that cannot be attributed solely to reactive oxygen species (ROS)-induced photooxidative damage, suggesting that they are manifestations of pre-existing defects that are amplified over time. The data indicate that the observed physiological and morphological effects are intimately related to crhR mutation, implying that the lack of CrhR RNA unwinding/annealing activity results in the inability to execute one or more vital steps in photosynthesis that are required at all temperatures but are crucial at low temperature.

We studied the interactions of the CO(2)-concentrating mechanism and variable light in the filamentous cyanobacterium Leptolyngbya sp. CPCC 696 acclimated to low light (15 μmol m(-2) s(-1) PPFD) and low inorganic carbon (50 μM Ci). Mass spectrometric and polarographic analysis revealed that mediated CO(2) uptake along with both active Na(+)-independent and Na(+)-dependent HCO(3)(-) transport, likely through Na(+)/HCO(3)(-) symport, were employed to concentrate Ci internally. Combined transport of CO(2) and HCO(3)(-) required about 30 kJ mol(-1) of energy from photosynthetic electron transport to support an intracellular Ci accumulation 550-fold greater than the external Ci. Initially, Leptolyngbya rapidly induced oxygen evolution and Ci transport to reach 40-50% of maximum values by 50 μmol m(-2) s(-1) PPFD. Thereafter, photosynthesis and Ci transport increased gradually to saturation around 1,800 μmol m(-2) s(-1) PPFD. Leptolyngbya showed a low intrinsic susceptibility to photoinhibition of oxygen evolution up to PPFD of 3,000 μmol m(-2) s(-1). Intracellular Ci accumulation showed a lag under low light but then peaked at about 500 μmol photons m(-2) s(-1) and remained high thereafter. Ci influx was accompanied by a simultaneous, light-dependent, outward flux of CO(2) and by internal CO(2)/HCO(3)(-) cycling. The high-affinity and high-capacity CCM of Leptolyngbya responded dynamically to fluctuating PPFD and used excitation energy in excess of the needs of CO(2) fixation by increasing Ci transport, accumulation and Ci cycling. This capacity may allow Leptolyngbya to tolerate periodic exposure to excess high light by consuming electron equivalents and keeping PSII open.

Cyanobacteria (as well as many chemoautotrophs) actively pump inorganic carbon (in the form of HCO(3)(-)) into the cytosol in order to enhance the overall efficiency of carbon fixation. The success of this approach is dependent upon the presence of carboxysomes-large, polyhedral, cytosolic bodies which sequester ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) and carbonic anhydrase. Carboxysomes seem to function by allowing ready passage of HCO(3)(-) into the body, but hindering the escape of evolved CO(2), promoting the accumulation of CO(2) in the vicinity of RubisCO and, consequently, efficient carbon fixation. This selectivity is mediated by a thin shell of protein, which envelops the carboxysome's enzymatic core and uses narrow pores to control the passage of small molecules. In this review, we summarize recent advances in understanding the organization and functioning of these intriguing, and ecologically very important molecular machines.

Cyanobacterial RuBisCO is sequestered in large, icosahedral, protein-bounded microcompartments called carboxysomes. Bicarbonate is pumped into the cytosol, diffuses into the carboxysome through small pores in its shell, and is then converted to CO(2) by carbonic anhydrase (CA) prior to fixation. Paradoxically, many beta-cyanobacteria, including Thermosynechococcus elongatus BP-1, lack the conventional carboxysomal beta-CA, ccaA. The N-terminal domain of the carboxysomal protein CcmM is homologous to gamma-CA from Methanosarcina thermophila (Cam) but recombinant CcmM derived from ccaA-containing cyanobacteria show no CA activity. We demonstrate here that either full length CcmM from T. elongatus, or a construct truncated after 209 residues (CcmM209), is active as a CA-the first catalytically active bacterial gamma-CA reported. The 2.0 A structure of CcmM209 reveals a trimeric, left-handed beta-helix structure that closely resembles Cam, except that residues 198-207 form a third alpha-helix stabilized by an essential Cys194-Cys200 disulfide bond. Deleting residues 194-209 (CcmM193) results in an inactive protein whose 1.1 A structure shows disordering of the N- and C-termini, and reorganization of the trimeric interface and active site. Under reducing conditions, CcmM209 is similarly partially disordered and inactive as a CA. CcmM protein in fresh E. coli cell extracts is inactive, implying that the cellular reducing machinery can reduce and inactivate CcmM, while diamide, a thiol oxidizing agent, activates the enzyme. Thus, like membrane-bound eukaryotic cellular compartments, the beta-carboxysome appears to be able to maintain an oxidizing interior by precluding the entry of thioredoxin and other endogenous reducing agents.

Emerging as well as the most frequently used optical microscopy techniques are reviewed and image contrast generation methods in a microscope are presented, focusing on the nonlinear contrasts such as harmonic generation and multiphoton excitation fluorescence. Nonlinear microscopy presents numerous advantages over linear microscopy techniques including improved deep tissue imaging, optical sectioning, and imaging of live unstained samples. Nonetheless, with the exception of multiphoton excitation fluorescence, nonlinear microscopy is in its infancy, lacking protocols, users and applications; hence, this review focuses on the potential of nonlinear microscopy for studying photosynthetic organisms. Examples of nonlinear microscopic imaging are presented including isolated light-harvesting antenna complexes from higher plants, starch granules, chloroplasts, unicellular alga Chlamydomonas reinhardtii, and cyanobacteria Leptolyngbya sp. and Anabaena sp. While focusing on nonlinear microscopy techniques, second and third harmonic generation and multiphoton excitation fluorescence microscopy, other emerging nonlinear imaging modalities are described and several linear optical microscopy techniques are reviewed in order to clearly describe their capabilities and to highlight the advantages of nonlinear microscopy., (© Springer Science+Business Media B.V. 2009)

Carboxysomes are proteinaceous biochemical compartments that constitute the enzymatic "back end" of the cyanobacterial CO2-concentrating mechanism. These protein-bound organelles catalyze HCO3- dehydration and photosynthetic CO2 fixation. In Synechocystis sp. strain PCC6803 these reactions involve the beta-class carbonic anhydrase (CA), CcaA, and Form 1B ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The surrounding shell is thought to be composed of proteins encoded by the ccmKLMN operon, although little is known about how structural and catalytic proteins integrate to form a functional carboxysome. Using biochemical activity assays and molecular approaches we have identified a catalytic, multiprotein HCO3- dehydration complex (BDC) associated with the protein shell of Synechocystis carboxysomes. The complex was minimally composed of a CcmM73 trimer, CcaA dimer, and CcmN. Larger native complexes also contained RbcL, RbcS, and two or three immunologically identified smaller forms of CcmM (62, 52, and 36 kDa). Yeast two-hybrid analyses indicated that the BDC was associated with the carboxysome shell through CcmM73-specific protein interactions with CcmK and CcmL. Protein interactions between CcmM73 and CcaA, CcmM73 and CcmN, or CcmM73 and itself required the N-terminal gamma-CA-like domain of CcmM73. The specificity of the CcmM73-CcaA interaction provided both a mechanism to integrate CcaA into the fabric of the carboxysome shell and a means to recruit this enzyme to the BDC during carboxysome biogenesis. Functionally, CcaA was the catalytic core of the BDC. CcmM73 bound H14CO3- but was unable to catalyze HCO3- dehydration, suggesting that it may potentially regulate BDC activity.

The cyanobacteria Synechococcus elongatus strain PCC7942 and Synechococcus sp. strain UTEX625 decomposed exogenously supplied cyanate (NCO-) to CO2 and NH3 through the action of a cytosolic cyanase which required HCO3- as a second substrate. The ability to metabolize NCO- relied on three essential elements: proteins encoded by the cynABDS operon, the biophysical activity of the CO2-concentrating mechanism (CCM), and light. Inactivation of cynS, encoding cyanase, and cynA yielded mutants unable to decompose cyanate. Furthermore, loss of CynA, the periplasmic binding protein of a multicomponent ABC-type transporter, resulted in loss of active cyanate transport. Competition experiments revealed that native transport systems for CO2, HCO3-, NO3-, NO2-, Cl-, PO4(2-), and SO4(2-) did not contribute to the cellular flux of NCO- and that CynABD did not contribute to the flux of these nutrients, implicating CynABD as a novel primary active NCO- transporter. In the S. elongatus strain PCC7942 DeltachpX DeltachpY mutant that is defective in the full expression of the CCM, mass spectrometry revealed that the cellular rate of cyanate decomposition depended upon the size of the internal inorganic carbon (Ci) (HCO3- + CO2) pool. Unlike wild-type cells, the rate of NCO- decomposition by the DeltachpX DeltachpY mutant was severely depressed at low external Ci concentrations, indicating that the CCM was essential in providing HCO3- for cyanase under typical growth conditions. Light was required to activate and/or energize the active transport of both NCO- and Ci. Putative cynABDS operons were identified in the genomes of diverse Proteobacteria, suggesting that CynABDS-mediated cyanate metabolism is not restricted to cyanobacteria.

Exposure to blue light has previously been shown to induce the reversible quenching of fluorescence in cyanobacteria, indicative of a photoprotective mechanism responsible for the down regulation of photosynthesis. We have investigated the molecular mechanism behind fluorescence quenching by characterizing changes in excitation energy transfer through the phycobilin pigments of the phycobilisome to chlorophyll with steady-state and time-resolved fluorescence excitation and emission spectroscopy. Quenching was investigated in both a photosystem II-less mutant, and DCMU-poisoned wild-type Synechocystis sp. PCC 6803. The action spectra for blue-light-induced quenching was identical in both cell types and was dominated by a band in the blue region, peaking at 480 nm. Fluorescence quenching and its dark recovery was inhibited by the protein cross-linking agent glutaraldehyde, which could maintain cells in either the quenched or the unquenched state. We found that high phosphate concentrations that inhibit phycobilisome mobility and the regulation of energy transfer by the light-state transition did not affect blue-light-induced fluorescence quenching. Both room temperature and 77 K fluorescence emission spectra revealed that fluorescence quenching was associated with phycobilin emission. Quenching was characterized by a decrease in the emission of allophycocyanin and long wavelength phycobilisome terminal emitters relative to that of phycocyanin. A global analysis of the room-temperature fluorescence decay kinetics revealed that phycocyanin and photosystem I decay components were unaffected by quenching, whereas the decay components originating from allophycocyanin and phycobilisome terminal emitters were altered. Our data support a regulatory mechanism involving a protein conformational change and/or change in protein-protein interaction which quenches excitation energy at the core of the phycobilisome.

A significant portion of the total carbon fixed in the biosphere is attributed to the autotrophic metabolism of prokaryotes. In cyanobacteria and many chemolithoautotrophic bacteria, CO(2) fixation is catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO), most if not all of which is packaged in protein microcompartments called carboxysomes. These structures play an integral role in a cellular CO(2)-concentrating mechanism and are essential components for autotrophic growth. Here we report that the carboxysomal shell protein, CsoS3, from Halothiobacillus neapolitanus is a novel carbonic anhydrase (epsilon-class CA) that has an evolutionary lineage distinct from those previously recognized in animals, plants, and other prokaryotes. Functional CAs encoded by csoS3 homologues were also identified in the cyanobacteria Prochlorococcus sp. and Synechococcus sp., which dominate the oligotrophic oceans and are major contributors to primary productivity. The location of the carboxysomal CA in the shell suggests that it could supply the active sites of RuBisCO in the carboxysome with the high concentrations of CO(2) necessary for optimal RuBisCO activity and efficient carbon fixation in these prokaryotes, which are important contributors to the global carbon cycle.

The CO(2)-concentrating mechanism (CCM) of the marine eustigmatophycean microalga Nannochloropsis gaditana consists of an active HCO(3)(-) transport system and an internal carbonic anhydrase to facilitate accumulation and conversion of HCO(3)(-) to CO(2) for photosynthetic fixation. Aqueous inlet mass spectrometry revealed that a portion of the CO(2) generated within the cells leaked to the medium, resulting in a significant rise in the extracellular CO(2) concentration to a level above its chemical equilibrium that was diagnostic for active HCO(3)(-) transport. The transient rise in extracellular CO(2) occurred in the light and the dark and was resolved from concurrent respiratory CO(2) efflux using H(13)CO(3)(-) stable isotope techniques. H(13)CO(3)(-) pump-(13)CO(2) leak activity of the CCM was unaffected by 10 microM 3(3,4-dichlorophenyl)-1,1-dimethylurea, an inhibitor of chloroplast linear electron transport, although photosynthetic O(2) evolution was reduced by 90%. However, low concentrations of cyanide, azide, and rotenone along with anoxia significantly reduced or abolished (13)CO(2) efflux in the dark and light. These results indicate that H(13)CO(3)(-) transport was supported by mitochondrial energy production in contrast to other algae and cyanobacteria in which it is supported by photosynthetic electron transport. This is the first report of a direct role for mitochondria in the energization and functioning of the CCM in a photosynthetic organism.

Sequence analysis of the carboxysomal carbonic anhydrase (CcaA) from Synechocystis PCC6803, Synechococcus PCC7942 and Nostoc ATCC29133, indicated high sequence identity to the β class of plant and bacterial carbonic anhydrases (CA), and conservation of the active site region. However, the cyanobacterial enzyme has a C-terminal extension of about 75 amino acids (aa) not found in the plant enzymes, and largely absent from other bacterial enzymes. Using recombinant DNA technology, genes encoding C-terminal truncation products of up to 127 aa were overexpressed in E. coli, and partially purified lysates were analysed for CA-mediated exchange of 18 O between 13 C 18 O2and H2 16 O. Recombinant CcaA proteins with up to 60 aa removed (CcaAΔ60) were catalytically competent, but beyond this there was an abrupt loss of activity. CcaAΔ0, along with CcaAΔ40 and CcaAΔ60, also catalysed the hydrolysis of carbon oxysulfide (COS; an isoelectronic structural analogue of CO2), but CcaAΔ63 and CcaAΔ127 did not, indicating that truncations greater than 62 aa resulted in a general loss of catalytic competency. Analysis of protein-protein interaction using the yeast two-hybrid system revealed that CcaA did not interact with the large or small Rubisco subunits (RbcL and RbcS, respectively) of Synechocystis, but there was strong CcaA-CcaA interaction. This protein interaction also ceased with C-terminal truncations in CcaA greater than 60 aa. The correlation between loss of CcaA-CcaA interaction and CcaA catalytic activity suggests that the proximal portion of the C-terminal extension is required for oligomerization, and that this oligomerization is essential for catalysis by the cyanobacterial enzyme. Thus, the C-terminal extension may play an important role in the function of CA within cyanobacterial carboxysomes, which is not required by the higher plant enzymes.

The eustigmatophyceans are primitive unicellular algae that represent the most basal group of ochrophytes. They are believed to be obligate photoautotrophs, occurring mainly in freshwater and soil but with some marine representatives. The freshwater eustigmatophytes Monodus subterraneus and Vischeria stellata, and the marine eustigmatophyte Nannochloropsis gaditana, have been studied by mass spectrometry with respect to their characteristics for inorganic carbon (Ci) uptake. A CO2 concentrating mechanism was found in all three, but an external carbonic anhydrase (CA) was not detected. The acquisition of Ci from the external medium was based on the active transport of HCO3 - , CO2, or both. In particular, N. gaditana was able to use HCO3 - exclusively as an exogenous carbon source for photosynthesis, with this HCO3 - being subsequently converted to CO2 by an intracellular CA for photosynthetic fixation. A unique characteristic of this species was its capacity to transport HCO3 - during prolonged periods of time in the dark. In contrast, M. subterraneus utilized CO2 alone through an active transport process, whereas V. stellataexhibited the capacity to transport both HCO3 - and CO2. The uptake of CO2 also continued in the dark in V. stellatacells. Regardless of the Ci species taken up, transport was abolished by anoxia and by inhibitors of mitochondrial respiration. These results indicate that that the supply of Ci for photosynthetic CO2 fixation is partly dependent upon mitochondrial activity in these primitive eukaryotes.

CO2 fluxes in the marine microalga Nannochloris atomus were studied by mass spectrometry using inhibitors and artificial acceptors of photosynthetic electron transport to investigate the energy source for CO2 uptake. This algal species is capable of taking up CO2 from the external medium by active transport but lacks active HCO(3)(-) transport and extracellular carbonic anhydrase. The capacity of cells to take up CO2 was a function of photosynthetic photon flux density. Dark respiration rates were also dependent upon the light intensity during the preceding illumination period, indicating the presence of light-enhanced dark respiration. Addition of 3-(3',4'-dichlorophenyl)-1,1-dimethylurea to illuminated cell suspensions that had been allowed to concentrate inorganic carbon internally during photosynthesis caused a rapid burst of CO2, demonstrating that active CO2 transport had been abolished. A similar response was obtained when cell suspensions were treated with 2,5-dibromo-6-isopropyl-3methyl-1,4-benzoqinone or hydroxylamine. When methyl viologen was used to drain electrons from ferredoxin, cells were still able to take up CO2 from the external medium, although C-fixation decreased with time. These results demonstrate that active CO2 transport in N. atomus is supported by photosynthetic linear electron transport.

We have examined the effect of inorganic and organic carbon nutrition on the physiological expression of HCO3- transport and the CO2-concentrating mechanism (CCM) in the nutritionally versatile cyanobacterium Chlorogloeopsis sp. ATCC 27193. Cells grown under photoautotrophic conditions in the presence of limiting or replete levels of inorganic carbon (Ci), or grown under mixotrophic (light) or chemoheterotrophic (dark) conditions in the presence of sucrose retained both active CO2 and Na(+)-independent HCO3- transport activity. However, two distinct effects on the kinetic properties of HCO3- transport were observed, which segregated on the basis of phototrophic and chemoheterotrophic growth in the dark. In the former, the apparent substrate affinity of the HCO3- transport system (K0.5) varied (12-fold) in response to the growth Ci or mixotrophy while the maximum rate of HCO3- transport was approximately constant. In the latter case, the K0.5 value was unchanged from the starting value (35 microM) of Ci-limited photoautotrophic cells used to initiate the dark-grown cultures, but transport capacity declined 3-fold. Modulation of the K0.5 (HCO3- transport) value required light. Cellular carboxysome content was unaffected by growth under any of the regimes employed and these structures were the predominant location of ribulose-1,5-bisphosphate carboxylase/oxygenase, as indicated by immunogold electron microscopy. Mixotrophic and chemoheterotrophic growth resulted in a diminished ability to concentrate Ci internally and a reduction in Ci accumulation ratios at low external Ci concentrations. The relationship between photosynthetic carbon fixation and the internal Ci pool varied by 2-fold, with high-Ci-grown cells being the most efficient and mixotrophically grown cells the least, indicating that there was limited capacity to modulate this relationship in response to changes in carbon nutrition. Within broad limits this relationship appeared to be a fixed trait of the strain and an important factor in determining growth rate.

A fully-segregated mutant (ccaA::kanR) defective in the ccaA gene, encoding a carboxysome-associated beta-carbonic anhydrase (CA), was generated in the cyanobacterium Synechocystis sp. PCC6803 by insertional mutagenesis. Immunoblot analysis indicated that the CcaA polypeptide was absent from the carboxysome-enriched fraction obtained from ccaA::kanR, but was present in wild-type (WT) cells. The carboxysome-enriched fraction isolated from WT cells catalyzed 18O exchange between 13C18O2 and H2O, indicative of CA activity, while ccaA::kanR carboxysomes did not. Transmission and immunogold electron microscopy revealed that carboxysomes of WT and ccaA::kanR were of similar size, shape and cellular distribution, and contained most of the cellular complement of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The ccaA::kanR cells were substantially smaller than WT and were unable to grow autotrophically at air levels of CO2. However, cell division occurred at near-WT rates when ccaA::kanR was supplied with 5% CO2 (v/v) in air. The apparent photosynthetic affinity of the mutant for inorganic carbon (Ci) was 500-fold lower than that of WT cells although intracellular Ci accumulation was comparable to WT measurements. Mass spectrometric analysis revealed that the CA-like activity associated with the active CO2 transport system was retained by ccaA::kanR cells and was inhibited by H2S, indicating that CO2 transport was distinct from the CcaA-mediated dehydration of intracellular HCO3-. The data suggest that the ccaA mutant was unable to efficiently utilize the internal Ci pool for carbon fixation and that the high-CO2-requiring phenotype of ccaA::kanR was due primarily to an inability to generate enough CO2 in the carboxysomes to sustain normal rates of photosynthesis.

Inorganic carbon (Ci) uptake and efflux has been investigated in the marine microalga Nannochloropsis gaditana Lubian by monitoring CO2 fluxes in cell suspensions using mass spectrometry. Addition of H13CO3- to cell suspensions in the dark caused a transient increase in the CO2 concentration in the medium far in excess of the equilibrium CO2 concentration. The magnitude of this release was dependent on the length of time the cells had been kept in the dark. Once equilibrium between the Ci species had been achieved, a CO2 efflux was observed after saturating light intensity was applied to the cells. External carbonic anhydrase (CA) was not detected nor does this species demonstrate a capacity to take up CO2 by active transport. Photosynthetic O2 evolution and the release CO2 in the dark depend on HCO3- uptake since both were inhibited by the anion exchange inhibitor, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). The bicarbonate uptake mechanism requires light but can also continue for short periods in the dark. Ethoxyzolamide, a CA inhibitor, markedly inhibited CO2 efflux in the dark, indicating that CO2 efflux was dependent upon the intracellular dehydration of HCO3-. These results indicate that Nannochloropsis possesses a bicarbonate uptake system which causes the accumulation of high intracellular Ci levels and an internal CA which maintains the equilibrium between CO2 and HCO3- and thus causes a subsequent release of CO2 to the external medium.

Iron limitation led to a large increase in extracellular ferricyanide (Fe[III]) reductase activity in cells of the green alga Chlamydomonas reinhardtii Dangeard. Mass-spectrometric measurement of gas exchange indicated that ferricyanide reduction in the dark resulted in a stimulation of respiratory CO2 production without affecting the rate of respiratory O2 consumption, consistent with the previously postulated activation of the oxidative pentose phosphate pathway in support of Fe(III) reduction by iron-limited Chlamydomonas cells (X. Xue et al., 1998, J. Phycol. 34: 939-944). At saturating irradiance, the rate of ferricyanide reduction was stimulated almost 3-fold, and this stimulation was inhibited by 3-(3',4'-dichlorophenyl)-1,1-dimethylurea. Ferricyanide reduction during photosynthesis resulted in approximately a 50% inhibition of photosynthetic CO2 fixation at saturating irradiance, and almost 100% inhibition of CO2 fixation at sub-saturating irradiance. Photosynthesis by iron-sufficient cells was not affected by ferricyanide addition. Addition of 250 microM ferricyanide to iron-limited cells in which photosynthesis was inhibited (either by the presence of glycolaldehyde, or by maintaining the cells at the CO2 compensation point) resulted in a stimulation in the rate of gross photosynthetic O2 evolution. Chlorophyll a fluorescence measurements indicated a large increase in non-photochemical quenching during ferricyanide reduction in the light; the increase in nonphotochemical quenching was abolished by the addition of nigericin. These results suggest that reduction of extracellular ferricyanide (mediated at the plasma membrane) interacts with both photosynthesis and respiration, and that both of these processes contribute NADPH in the light.

A 3.3 kb HindIII restriction-digest DNA fragment was isolated from a Synechocystis sp. strain PCC6803 subgenomic plasmid library which strongly hybridized to a 349 bp fragment of the icfA (ccaA) gene from Synechococcus sp. strain PCC7942. DNA sequence analysis of the fragment revealed three open reading frames (ORFs), two of which potentially coded for pantothenate synthetase (ORF275) and cytidylate kinase (ORF230). The third, ORF274, was 825 bp in length, encoding a deduced polypeptide of 274 aa (Mr, 30747) that bears 55% sequence identity to the Synechococcus icfA (ccaA) translation product, a beta-type carbonic anhydrase (CA). A 932 bp EcoRI fragment containing ORF274 was subcloned into an expression vector and the construct was transformed into Escherichia coli for overexpression. Electrometric assays for CA activity revealed that whole cell extracts containing the recombinant protein significantly enhanced the rate of conversion of CO2 to HCO3- and that 98% of this catalytic activity was inhibited by ethoxyzolamide, a well-characterized CA inhibitor. Antisera derived against the overexpressed protein recognized a 30.7 kDa protein that was predominantly associated with the isolated carboxysome fraction from Synechocystis. These results provide molecular and physiological evidence for the identification of a ccaA homologue in Synechocystis PCC6803 that encodes a carboxysomal beta-type CA.

Previous work has shown that the maximum fluorescence yield from PS 2 of Synechococcus PCC 7942 occurs when the cells are at the CO2 compensation point. The addition of inorganic carbon (Ci), as CO2 or HCO3 (-), causes a lowering of the fluorescence yield due to both photochemical (qp) and non-photochemical (qN) quenching. In this paper, we characterize the qN that is induced by Ci addition to cells grown at high light intensities (500 μmol photons m(-2) s(-1)). The Ci-induced qN was considerably greater in these cells than in cells grown at low light intensities (50 μmol photons m(-2) s(-1)), when assayed at a white light (WL) intensity of 250 μmol photons m(-2) s(-1). In high-light grown cells we measured qN values as high as 70%, while in low-light grown cells the qN was about 16%. The qN was relieved when cells regained the CO2 compensation point, when cells were illuminated by supplemental far-red light (FRL) absorbed mainly by PS 1, or when cells were illuminated with increased WL intensities. These characteristics indicate that the qN was not a form of energy quenching (qE). Supplemental FRL illumination caused significant enhancement of photosynthetic O2 evolution that could be correlated with the changes in qp and qN. The increases in qp induced by Ci addition represent increases in the effective quantum yield of PS 2 due to increased levels of oxidized QA. The increase in qN induced by Ci represents a decrease in PS 2 activity related to decreases in the potential quantum yield. The lack of diagnostic changes in the 77 K fluorescence emission spectrum argue against qN being related to classical state transitions, in which the decrease in potential quantum yield of PS 2 is due either to a decrease in absorption cross-section or by increased 'spill-over' of excitation energy to PS 1. Both the Ci-induced qp (t 0.5<0.5 s) and qN (t 0.5≃1.6 s) were rapidly relieved by the addition of DCMU. The two time constants give further support for two separate quenching mechanisms. We have thus characterized a novel form of qN in cyanobacteria, not related to state transitions or energy quenching, which is induced by the addition of Ci to cells at the CO2-compensation point.

The effects of ethoxyzolamide (EZ), a carbonic anhydrase inhibitor, on the active CO2 and Na+-independent and Na+-dependent HCO3- transport systems of the unicellular cyanobacterium Synechococcus sp. UTEX 625 were examined. Measurements of transport and accumulation using radiochemical, fluorometric, and mass spectrometric assays indicated that active CO2 transport and active Na+-independent HCO3- transport were inhibited by EZ. However, Na+-independent HCO3- transport was about 1 order of magnitude more sensitive to EZ inhibition than was CO2 transport (50% inhibition = 12 [mu]M versus 80 [mu]M). The data suggest that both the active CO2 (G.D. Price, M.R. Badger [1989] Plant Physiol 89: 37-43) and the Na+ -independent HCO3 - transport systems possessed carbonic anhydrase-like activity as part of their mechanism of action. In contrast, Na+-dependent HCO3- transport was only partially (50% inhibition = 230 [mu]M) and noncompetitively inhibited by EZ. The collective evidence suggested that EZ inhibition of Na+ -dependent HCO3- transport was an indirect consequence of the action of EZ on the CO2 transport system, rather than a direct effect on HCO3- transport. A model is presented in which the core of the inorganic carbon translocating system is formed by Na+-dependent HCO3- transport and the CO2 transport system. It is argued that the Na+-independent HCO3 - utilizing system was not directly involved in translocation, but converted HCO3- to CO2 for use in CO2 transport.

The effect of monensin, an ionophore that mediates Na+/H+ exchange, on the activity of the inorganic carbon transport systems of the cyanobacterium Synechococcus UTEX 625 was investigated using transport assays based on the measurement of chlorophyll a fluorescence emission or 14C uptake. In Synechococcus cells grown in standing culture at about 20 [mu]M CO2 + HCO3-, 50 [mu]M monensin transiently inhibited active CO2 and Na+-independent HCO3- transport, intracellular CO2 and HCO3- accumulation, and photosynthesis in the presence but not in the absence of 25 mM Na+. These activities returned to near-normal levels within 15 min. Transient inhibition was attributed to monensin-mediated intracellular alkalinization, whereas recovery may have been facilitated by cellular mechanisms involved in pH homeostasis or by monensin-mediated H+ uptake with concomitant K+ efflux. In air-grown cells grown at 200 [mu]M CO2 + HCO3- and standing culture cells, Na+-dependent HCO3- transport, intracellular HCO3- accumulation, and photosynthesis were also inhibited by monensin, but there was little recovery in activity over time. However, normal photosynthetic activity could be restored to air-grown cells by the addition of carbonic anhydrase, which increased the rate of CO2 supply to the cells. This observation indicated that of all the processes required to support photosynthesis only Na+-dependent HCO3- transport was significantly inhibited by monensin. Monensin-mediated dissipation of the Na+ chemical gradient between the medium and the cells largely accounted for the decline in the HCO3- accumulation ratio from 751 to 55. The two HCO3- transport systems were further distinguished in that Na+-dependent HCO3- transport was inhibited by Li+, whereas Na+-independent HCO3- transport was not. It is suggested that Na+-dependent HCO3- transport involves an Na+/HCO3- symport mechanism that is energized by the Na+ electrochemical potential.

In the cyanobacterium Synechococcus UTEX 625, the yield of chlorophyll a fluorescence decreased in response to the transport-mediated accumulation of intracellular inorganic carbon (CO2 + HCO3- + CO32- = dissolved inorganic carbon [DIC]) and subsequently increased to a near-maximum level following photosynthetic depletion of the DIC pool. When DIC accumulation was mediated by the active Na+-dependent HCO3- transport system, the initial rate of fluorescence quenching was found to be highly correlated with the initial rate of H14CO3- transport (r = 0.96), and the extent of fluorescence quenching was correlated with the size of the internal DIC pool (r = 0.99). Na+-dependent HCO3- transport-mediated accumulation of DIC caused fluorescence quenching in either the presence or absence of the CO2 fixation inhibitor glycolaldehyde, indicating that quenching was not due simply to NADP+ reduction. The concentration of Na+ required to attain one-half the maximum rate of H14CO3- transport, at 20 [mu]M external HCO3-, declined from 9 to 1 mM as the external pH increased from 8 to 9.6. A similar pH dependency was observed when fluorescence quenching was used to determine the kinetic constants for HCO3- transport. In cells capable of Na+-dependent HCO3- transport, both the initial rate and extent of fluorescence quenching increased with increasing external HCO3-, saturating at about 150 [mu]M. In contrast Na+-independent HCO3- transport-mediated fluorescence quenching saturated at an HCO3- concentration of about 10 [mu]M. It was concluded that measurement of chlorophyll a fluorescence emission provided a convenient, but indirect, means of following Na+-dependent HCO3- transport and accumulation in Synechococcus.

The active transport and intracellular accumulation of HCO(3) (-) by air-grown cells of the cyanobacterium Synechococcus UTEX 625 (PCC 6301) was strongly promoted by 25 millimolar Na(+).Na(+)-dependent HCO(3) (-) accumulation also resulted in a characteristic enhancement in the rate of photosynthetic O(2) evolution and CO(2) fixation. However, when Synechococcus was grown in standing culture, high rates of HCO(3) (-) transport and photosynthesis were observed in the absence of added Na(+). The internal HCO(3) (-) pool reached levels up to 50 millimolar, and an accumulation ratio as high as 970 was observed. Sodium enhanced HCO(3) (-) 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 HCO(3) (-) 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 CO(2) transport involving a high-affinity transport system which was reversibly inhibited by H(2)S, as in the case for air-grown cells. The data are interpreted to indicate that Synechococcus possesses a constitutive CO(2) transport system, whereas Na(+)-dependent and Na(+)-independent HCO(3) (-) transport are inducible, depending upon the conditions of growth. Intracellular accumulation of HCO(3) (-) was always accompanied by a quenching of chlorophyll a fluorescence which was independent of CO(2) fixation. The extent of fluorescence quenching was highly dependent upon the size of the internal pool of HCO(3) (-) + CO(2). The pattern of fluorescence quenching observed in response to added HCO(3) (-) and Na(+) in air-grown and standing culture cells was highly characteristic for Na(+)-dependent and Na(+)-independent HCO(3) (-) accumulation. It was concluded that measurements of fluorescence quenching provide an indirect means for following HCO(3) (-) transport and the dynamics of intracellular HCO(3) (-) accumulation and dissipation.

The active transport of CO(2) in Synechococcus UTEX 625 was measured by mass spectrometry under conditions that preclude HCO(3) (-) transport. The substrate concentration required to give one half the maximum rate for whole cell CO(2) 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 CO(2) 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 O(2) evolution observed under these conditions. The initial rate of chlorophyll a fluorescence quenching was highly correlated with the initial rate of CO(2) transport (correlation coefficient = 0.98) and could be used as an indirect method to detect CO(2) transport and calculate the substrate concentration required to give one half the maximum rate of transport. Little, if any, inhibition of CO(2) transport was caused by HCO(3) (-) or by Na(+)-dependent HCO(3) (-) transport. However, (12)CO(2) readily interfered with (13)CO(2) transport. CO(2) transport and Na(+)-dependent HCO(3) (-) transport are separate, independent processes and the high affinity CO(2) transporter is not only responsible for the initial transport of CO(2) into the cell but also for scavenging any CO(2) that may leak from the cell during ongoing photosynthesis.

Chlorophyll a fluorescence of Synechococcus UTEX 625 was quenched during the transport of inorganic carbon, even when CO(2) 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 O(2) photoreduction. Hence, O(2) could serve as an electron acceptor to allow oxidation of Q(a) even in the absence of CO(2) fixation.

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 CO(2) fixation was inhibited by iodoacetamide and whether the accumulation of inorganic carbon resulted from either active CO(2) or HCO(3) (-) 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.

Mass spectrometry has been used to confirm the presence of an active transport system for CO(2) in Synechococcus UTEX 625. Cells were incubated at pH 8.0 in 100 micromolar KHCO(3) in the absence of Na(+) (to prevent HCO(3) (-) transport). Upon illumination the cells rapidly removed almost all the free CO(2) from the medium. Addition of carbonic anhydrase revealed that the CO(2) depletion resulted from a selective uptake of CO(2), rather than a total uptake of all inorganic carbon species. CO(2) transport stopped rapidly (<3 seconds) when the light was turned off. Iodoacetamide (3.3 millimolar) completely inhibited CO(2) fixation but had little effect on CO(2) transport. In iodoacetamide poisoned cells, transport of CO(2) occurred against a concentration gradient of about 18,000 to 1. Transport of CO(2) was completely inhibited by 10 micromolar diethylstilbestrol, a membrane-bound ATPase inhibitor. Studies with DCMU and PSI light indicated that CO(2) transport was driven by ATP produced by cyclic or pseudocyclic photophosphorylation. Low concentrations of Na(+) (<100 microequivalents per liter), but not of K(+), stimulated CO(2) transport as much as 2.4-fold. Unlike Na(+)-dependent HCO(3) (-) transport, the transport of CO(2) was not inhibited by high concentrations (30 milliequivalents per liter) of Li(+). During illumination, the CO(2) concentration in the medium remained far below its equilibrium value for periods up to 15 minutes. This could only happen if CO(2) transport was continuously occurring at a rapid rate, since the continuing dehydration of HCO(3) (-) to CO(2) would rapidly raise the CO(2) 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 CO(2) transport was balanced by HCO(3) (-) efflux.

The species of inorganic carbon (CO(2) or HCO(3) (-)) taken up a source of substrate for photosynthetic fixation by isolated Asparagus sprengeri mesophyll cells is investigated. Discrimination between CO(2) or HCO(3) (-) transport, during steady state photosynthesis, is achieved by monitoring the changes (by (14)C 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 (CO(2) or HCO(3) (-) transport) and experimental time-courses of (14)C incorporation, over the pH range of 5.2 to 7.5, indicate that the specific activity of extracellular CO(2), rather than HCO(3) (-), is the appropriate predictor of the intracellular specific activity. It is concluded, therefore, that CO(2) 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 HCO(3) (-) transport, as the incorporation of (14)C during isotopic disequilibrium exceeds the maximum possible incorporation predicted on the basis of CO(2) uptake alone. The contribution of HCO(3) (-) to net inorganic carbon uptake (pH 8.5) is variable, ranging from 5 to 16%, but is independent of the extracellular HCO(3) (-) concentration. The evidence for direct HCO(3) (-) transport is subject to alternative explanations and must, therefore, be regarded as equivocal. Nonlinear regression analysis of the rate of (14)C incorporation as a function of time indicates the presence of a small extracellular resistance to the diffusion of CO(2), which is partially alleviated by a high extracellular concentration of HCO(3) (-).

The possibility of HCO(3) (-) transport into isolated leaf mesophyll cells of Asparagus sprengeri Regel has been investigated. Measurement of the inorganic carbon pool in these cells over an external pH range 6.2 to 8.0, using the silicone-fluid filtration technique, indicated that the pool was larger than predicted by passive (14)CO(2) distribution, suggesting that HCO(3) (-) as well as CO(2) crosses the plasmalemma. Intracellular pH values, calculated from the distribution of (14)CO(2) between the cells and the medium, were found to be higher (except at pH 8.0) than those previously determined by 5,5-dimethyl[2-(14)C]oxazolidine-2,4-dione distribution. It is suggested that the inorganic carbon accumulated above predicted concentrations may be bound to proteins and membranes and thus may not represent inorganic carbon actively accumulated by the cells, inasmuch as in a closed system at constant CO(2) concentration, the photosynthetic rates at pH 7.0 and 8.0 were 5 to 8 times lower than the maximum rate which could be supported by CO(2) arising from the spontaneous dehydration of HCO(3) (-). Furthermore, CO(2) compensation points of the cells in liquid media at 21% O(2) at pH 7.0 and 8.0, and the K((1/2)) CO(2) (CO(2) concentration supporting the half maximal rate of O(2) evolution) at 2% O(2) at pH 7.0 and 8.0 are not consistent with HCO(3) (-) transport. These results indicate that the principal inorganic carbon species crossing the plasmalemma in these cells is CO(2).

Carbon oxysulfide (carbonyl sulfide, COS) is a close structural analog of CO(2). Although hydrolysis of COS (to CO(2) and H(2)S) 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 CO(2) transport system of Synechococcus UTEX 625. A light-dependent uptake of COS was observed that was inhibited by CO(2) 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 H(2)S was observed and COS removal from the medium was inhibited by ethoxyzolamide. Bovine erythrocyte carbonic anhydrase catalysed the stoichiometric hydrolysis of COS to H(2)S. The active transport of CO(2) was inhibited by COS in an apparently competitive manner. When Na(+)-dependent HCO(3) (-) transport was allowed in the presence of COS, the extracellular [CO(2)] rose considerably above the equilibrium level. This CO(2) appearing in the medium was derived from the dehydration of transported HCO(3) (-) and was leaked from the cells. In the presence of COS the return to the cells of this leaked CO(2) was inhibited. These results showed that the Na(+)-dependent HCO(3) (-) transport was not inhibited by COS, whereas active CO(2) transport was inhibited. When COS was removed by gassing with N(2), a normal pattern of CO(2) uptake was observed. The silicone fluid centrifugation method showed that COS (100 micromolar) had little effect upon the initial rate of HCO(3) (-) transport or CO(2) fixation. The steady state rate of CO(2) fixation was, however, inhibited about 50% in the presence of COS. This inhibition can be at least partially explained by the significant leakage of CO(2) from the cells that occurred when CO(2) uptake was inhibited by COS. Neither CS(2) nor N(2)O acted like COS. It is concluded that COS is an effective and selective inhibitor of active CO(2) transport.

Equations have been developed which quantitatively predict the theoretical time-course of photosynthetic (14)C incorporation when CO(2) or HCO(3) (-) serves as the sole source of exogenous inorganic carbon taken up for fixation by cells during steady state photosynthesis. Comparison between the shape of theoretical (CO(2) or HCO(3) (-)) and experimentally derived time-courses of (14)C incorporation permits the identification of the major species of inorganic carbon which crosses the plasmalemma of photosynthetic cells and facilitates the detection of any combined contribution of CO(2) and HCO(3) (-) transport to the supply of intracellular inorganic carbon. The ability to discriminate between CO(2) or HCO(3) (-) uptake relies upon monitoring changes in the intracellular specific activity (by (14)C fixation) which occur when the inorganic carbon, present in the suspending medium, is in a state of isotopic disequilibrium (JT Lehman 1978 J Phycol 14: 33-42). The presence of intracellular carbonic anhydrase or some other catalyst of the CO(2)-HCO(3) (-) interconversion reaction is required for quantitatively accurate predictions. Analysis of equations describing the rate of (14)C incorporation provides two methods by which any contribution of HCO(3) (-) ions to net photosynthetic carbon uptake can be estimated.

The intracellular pH of isolated, photosynthetically active mesophyll cells of Asparagus sprengeri Regel has been determined, in the light and dark, by the distribution of the weak acid 5,5-dimethyl-[2-(14)C]oxazolidine-2,4-dione ([(14)C]DMO) between the cells and the liquid medium. [(14)C]DMO was taken up rapidly, reaching equilibrium in 7-10 min of incubation, but was not metabolized by the cells, and intracellular binding of the compound was minimal. The intracellular pH, measured at saturating light fluence and 1.5 mM sodium bicarbonate, was found to remain relatively constant at 6.95-7.21 over the external pH range of 5.5-7.2. Illumination of the cells increased the intracellular pH compared to dark controls. The pH of the cytoplasm, excluding and including the chloroplasts ("cytoplasmic" and "bulk cytoplasmic", respectively) was calculated from the experimentally derived intracellular [(14)C]DMO concentration and estimates of the vacuolar, chloroplastic and cytoplasmic volumes. The calculated cytoplasmic pH was similar in the light and dark, being 7.75 and 7.74, respectively, while the calculated pH of bulk cytoplasm was 7.85 in the light and 7.49 in the dark. Theoretical analysis indicated that intracellular pH is a good indicator of changes in the bulk cytoplasmic pH but insensitive to changes in vacuolar pH. The external pH optimum for photosynthesis (O2 evolution) of isolated Asparagus cells was pH 7.2. At pH 8.0 photosynthesis was inhibited by 30% and at pH 5.25 by 45%. Inhibition at alkaline pH may be the result of a decrease in the pH gradient between the cells and the medium, causing CO2 limitation in the cell. At acid pH, decrease in internal pH caused by substantial accumulation of inorganic carbon may account for the loss in photosynthetic activity.

A mass spectrometer was used to simultaneously follow the time course of photosynthetic O(2) evolution and CO(2) depletion of the medium by cells of the cyanobacterium Synechococcus leopoliensis UTEX 625. Analysis of the data indicated that both CO(2) and HCO(3) (-) were simultaneously and continuously transported by the cells as a source of substrate for photosynthesis. Initiation of HCO(3) (-) transport by Na(+) addition had no effect on ongoing CO(2) transport. This result is interpreted to indicate that the CO(2) and HCO(3) (-) transport systems are separate and distinctly different transport systems. Measurement of CO(2)-dependent photosynthesis indicated that CO(2) uptake involved active transport and that diffusion played only a minor role in CO(2) acquisition in cyanobacteria.

At low levels of dissolved inorganic carbon (DIC) and alkaline pH the rate of photosynthesis by air-grown cells of Synechococcus leopoliensis (UTEX 625) was enhanced 7- to 10-fold by 20 millimolar Na(+). The rate of photosynthesis greatly exceeded the CO(2) supply rate and indicated that HCO(3) (-) was taken up by a Na(+)-dependent mechanism. In contrast, photosynthesis by Synechococcus grown in standing culture proceeded rapidly in the absence of Na(+) and exceeded the CO(2) supply rate by 8 to 45 times. The apparent photosynthetic affinity (K((1/2))) for DIC was high (6-40 micromolar) and was not markedly affected by Na(+) concentration, whereas with air-grown cells K((1/2)) (DIC) decreased by more than an order of magnitude in the presence of Na(+). Lithium, which inhibited Na(+)-dependent HCO(3) (-) uptake in air-grown cells, had little effect on Na(+)-independent HCO(3) (-) uptake by standing culture cells. A component of total HCO(3) (-) uptake in standing culture cells was also Na(+)-dependent with a K((1/2)) (Na(+)) of 4.8 millimolar and was inhibited by lithium. Analysis of (14)C-fixation during isotopic disequilibrium indicated that standing culture cells also possessed a Na(+)-independent CO(2) transport system. The conversion from Na(+)-independent to Na(+)-dependent HCO(3) (-) uptake was readily accomplished by transferring cells grown in standing to growth in cultures bubbled with air. These results demonstrated that the conditions experienced during growth influenced the mode by which Ssynechococcus acquired HCO(3) (-) for subsequent photosynthetic fixation.

The active transport of CO(2) in the cyanobacterium Synechococcus UTEX 625 was inhibited by H(2)S. Treatment of the cells with up to 150 micromolar H(2)S + HS(-) at pH 8.0 had little effect on Na(+)-dependent HCO(3) (-) transport or photosynthetic O(2) evolution, but CO(2) transport was inhibited by more than 90%. CO(2) transport was restored when H(2)S was removed by flushing with N(2). At constant total H(2)S + HS(-) concentrations, inhibition of CO(2) transport increased as the ratio of H(2)S to HS(-) increased, suggesting a direct role for H(2)S in the inhibitory process. Hydrogen sulfide does not appear to serve as a substrate for transport. In the presence of H(2)S and Na(+) -dependent HCO(3) (-) transport, the extracellular CO(2) concentration rose considerably above its equilibrium level, but was maintained far below its equilibrium level in the absence of H(2)S. The inhibition of CO(2) transport, therefore, revealed an ongoing leakage from the cells of CO(2) which was derived from the intracellular dehydration of HCO(3) (-) which itself had been recently transported into the cells. Normally, leaked CO(2) is efficiently transported back into the cell by the CO(2) transport system, thus maintaining the extracellular CO(2) concentration near zero. It is suggested that CO(2) transport not only serves as a primary means of inorganic carbon acquisition for photosynthesis but also serves as a means of recovering CO(2) lost from the cell. A schematic model describing the relationship between the CO(2) and HCO(3) (-) transport systems is presented.

Mass spectrometric measurements of dissolved free (13)CO(2) were used to monitor CO(2) uptake by air grown (low CO(2)) cells and protoplasts from the green alga Chlamydomonas reinhardtii. In the presence of 50 micromolar dissolved inorganic carbon and light, protoplasts which had been washed free of external carbonic anhydrase reduced the (13)CO(2) concentration in the medium to close to zero. Similar results were obtained with low CO(2) cells treated with 50 micromolar acetazolamide. Addition of carbonic anhydrase to protoplasts after the period of rapid CO(2) uptake revealed that the removal of CO(2) from the medium in the light was due to selective and active CO(2) transport rather than uptake of total dissolved inorganic carbon. In the light, low CO(2) cells and protoplasts incubated with carbonic anhydrase took up CO(2) at an apparently low rate which reflected the uptake of total dissolved inorganic carbon. No net CO(2) uptake occurred in the dark. Measurement of chlorophyll a fluorescence yield with low CO(2) cells and washed protoplasts showed that variable fluorescence was mainly influenced by energy quenching which was reciprocally related to photosynthetic activity with its highest value at the CO(2) compensation point. During the linear uptake of CO(2), low CO(2) cells and protoplasts incubated with carbonic anhydrase showed similar rates of net O(2) evolution (102 and 108 micromoles per milligram of chlorophyll per hour, respectively). The rate of net O(2) evolution (83 micromoles per milligram of chlorophyll per hour) with washed protoplasts was 20 to 30% lower during the period of rapid CO(2) uptake and decreased to a still lower value of 46 micromoles per milligram of chlorophyll per hour when most of the free CO(2) had been removed from the medium. The addition of carbonic anhydrase at this point resulted in more than a doubling of the rate of O(2) evolution. These results show low CO(2) cells of Chlamydomonas are able to transport both CO(2) and HCO(3) (-) but CO(2) is preferentially removed from the medium. The external carbonic anhydrase is important in the supply to the cells of free CO(2) from the dehydration of HCO(3) (-).

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 (O(2) 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((1/2))[Na(+)]) were calculated. The value of both the low and the high K((1/2))(Na(+)) was dependent upon extracellular DIC concentration. The low K((1/2))(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((1/2))(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 CO(2) and HCO(3) (-) transport contributed to the intracellular supply of DIC and hence to photosynthesis. Second, both the CO(2) and HCO(3) (-) transport systems required Na(+), directly or indirectly, for full activity. However, micromolar levels of Na(+) were required for CO(2) transport while millimolar levels were required for HCO(3) (-) transport. These levels corresponded to those found for the low and high K((1/2))(Na(+)) for photosynthesis. Third, the contribution of each transport system to intracellular DIC was dependent on extracellular DIC concentration, where the contribution from CO(2) transport increased with increased DIC concentration relative to HCO(3) (-) transport. This change was reflected in a decrease in the Na(+) concentration required for maximum photosynthesis, in accord with the lower Na(+)-requirement for CO(2) transport. Lithium competitively inhibited Na(+)-stimulated photosynthesis by blocking the cells' ability to form an intracellular DIC pool through Na(+)-dependent HCO(3) (-) transport. Lithium had little effect on CO(2) transport and only a small effect on the size of the pool it generated. Thus, CO(2) transport did not require a functional HCO(3) (-) transport system for full activity. Based on these observations and the differential requirement for Na(+) in the CO(2) and HCO(3) (-) transport system, it was proposed that CO(2) and HCO(3) (-) were transported across the membrane by different transport systems.

The effect of pH, O(2) concentration, and temperature on the CO(2) compensation point (capital GHE, Cyrillic[CO(2)]) of isolated Asparagus sprengeri Regel mesophyll cells has been determined in a closed, aqueous environment by a sensitive gas-chromatographic technique. Measured values range between 10 and 100 microliters per liter CO(2) depending upon experimental conditions. The capital GHE, Cyrillic(CO(2)) increases with increasing temperature. The rate of increase is dependent upon the O(2) concentration and is more rapid at high (250-300 micromolar), than at low (30-60 micromolar), O(2) concentrations. The differential effect of temperature on capital GHE, Cyrillic(CO(2)) is more pronounced at pH 6.2 than at pH 8.0, but this pH-dependence is not attributable to a direct, differential effect of pH on the relative rates of photosynthesis and photorespiration, as the O(2)-sensitive component of capital GHE, Cyrillic(CO(2)) remains constant over this range. The capital GHE, Cyrillic(CO(2)) of Asparagus cells at 25 degrees C decreases by 50 microliters per liter when the pH is raised from 6.2 to 8.0, regardless of the prevailing O(2) concentration. It is suggested that the pH-dependence of capital GHE, Cyrillic(CO(2)) is related to the ability of the cell to take up CO(2) from the aqueous environment. The correlation between high HCO(3) (-) concentrations and low capital GHE, Cyrillic(CO(2)) at alkaline pH indicates that extracellular HCO(3) (-) facilitates the uptake of CO(2), possibly by increasing the flux of inorganic carbon from the bulk medium to the cell surface. The strong O(2)- and temperature-dependence of capital GHE, Cyrillic(CO(2)) indicates that isolated Asparagus mesophyll cells lack an efficient means for concentrating intracellular CO(2) to a level sufficient to reduce or suppress photorespiration.