Identification of Conjugated Dienes of Fatty Acids in Vischeria sp. IPPAS C-70 under Oxidative Stress.

The microalgae Vischeria sp. IPPAS C-70 produces eicosapentaenoic acid. Several stresses cause the formation of fatty acid peaks that resemble hexadecadienoic acids. We used the integrated technique including TLC, HPLC, and GC-MS to search and determine these fatty acids. Double bond positioning in these fatty acids indicated that they were conjugated dienes and allenes. We identified and described natural nine isomers of C16 polyunsaturated fatty acids, including common methylene-interrupted dienes (Δ6,9-16:2, Δ7,10-16:2, Δ9,12-16:2), and unusual conjugated dienes (Δ6,8-, Δ7,9-, Δ8,10-, Δ9,11-, and Δ10,12-16:2), as well as allenic diene (Δ9,10-16:2). We hypothesize that the formation of conjugated dienes and allenes among fatty acids is the result of oxidative stress caused by H2O2. Hydrogen peroxide also caused an increase in saturated at the expense of unsaturated fatty acids, suggesting inhibition either fatty acid desaturases activities or the corresponding gene expression.

Putative extracellular α-class carbonic anhydrase, EcaA, of Synechococcus elongatus PCC 7942 is an active enzyme: a sequel to an old story.

Carbonic anhydrase (CA) EcaA of Synechococcus elongatus PCC 7942 was previously characterized as a putative extracellular α-class CA, however, its activity was never verified. Here we show that EcaA possesses specific CA activity, which is inhibited by ethoxyzolamide. An active EcaA was expressed in heterologous bacterial system, which supports the formation of disulfide bonds, as a full-length protein (EcaA+L) and as a mature protein that lacks a leader peptide (EcaA-L). EcaA-L exhibited higher specific activity compared to EcaA+L. The recombinant EcaA, expressed in a bacterial system that does not support optimal disulfide bond formation, exhibited extremely low activity. This activity, however, could be enhanced by the thiol-oxidizing agent, diamide; while a disulfide bond-reducing agent, dithiothreitol, further inactivated the enzyme. Intact E. coli cells that overexpress EcaA+L possess a small amount of processed protein, EcaA-L, whereas the bulk of the full-length protein resides in the cytosol. This may indicate poor recognition of the EcaA leader peptide by protein export systems. S. elongatus possessed a relatively low level of ecaA mRNA, which varied insignificantly in response to changes in CO2 supply. However, the presence of protein in the cells is not obvious. This points to the physiological insignificance of EcaA in S. elongatus, at least under the applied experimental conditions.

New insights in cyanobacterial cold stress responses: Genes, sensors, and molecular triggers.

BACKGROUND: Cold stress strongly induces the expression of ~100 genes in cyanobacteria. Some of these genes are necessary to protect cellular functions by adjustment of membranes, as well as transcriptional and translational machineries. About a half of cold-induced genes are not functionally characterized. A part of cold-induced genes is under control of a two-component regulatory system, consisting of histidine kinase Hik33 and response regulator Rre26. The mechanism(s) that control another part of cold-inducible genes are still unknown. SCOPE OF REVIEW: The aim of this review is to summarise the latest findings in cyanobacterial cold-stress responses including transcriptomics, cold sensing, and molecular triggers. MAJOR CONCLUSIONS: A feedback loop between the membrane fluidity and transcription of genes for fatty acid desaturases operates via the transmembrane red-light-activated cold sensor Hik33, which perceives cold-induced membrane rigidification as a change in its thickness. The cold-induced kinase activity of Hik33 is facilitated by interaction with a small protein, Ssl3451 - the third contributor to a canonical two-component regulatory system, which may explain the ability of some cyanobacterial histidine kinases to interact with different response regulators under different stress conditions. Other regulatory systems that control cold-stress responses operate via Ser/Thr protein kinase, SpkE, and via temperature-dependent changes in DNA supercoiling. Transcriptomic analysis shows that universal triggers of stress responses are reactive oxygen species and changes in redox status of plastoquinone pool. GENERAL SIGNIFICANCE: Deeper understanding of molecular mechanisms of temperature sensing and regulation of cold-stress responses in photosynthetic cells provide a background for generation of cold-resistant crops.

Synechocystis mutants defective in manganese uptake regulatory system, ManSR, are hypersensitive to strong light.

High affinity transport of manganese ions (Mn2+) in cyanobacteria is carried by an ABC-type transporter, encoded by the mntCAB operon, which is derepressed by the deficiency of Mn2+. Transcription of this operon is negatively regulated by the two-component system consisting of a sensory histidine kinase ManS and DNA-binding response regulator ManR. In this study, we examined two Synechocystis mutants, defective in ManS and ManR. These mutants were unable to grow on high concentrations of manganese. Furthermore, they were sensitive to high light intensity and unable to recover after short-term photoinhibition. Under standard illumination and Mn2+ concentration, mutant cells revealed the elevated levels of transcripts of genes involved in the formation of Photosystem II (psbA, psbD, psbC, pap-operon). This finding suggests that, in mutant cells, the PSII is sensitive to high concentrations of Mn2+ even at relatively low light intensity.

Ultradian metabolic rhythm in the diazotrophic cyanobacterium Cyanothece sp. ATCC 51142.

The unicellular cyanobacterium Cyanothece sp. American Type Culture Collection (ATCC) 51142 is capable of performing oxygenic photosynthesis during the day and microoxic nitrogen fixation at night. These mutually exclusive processes are possible only by temporal separation by circadian clock or another cellular program. We report identification of a temperature-dependent ultradian metabolic rhythm that controls the alternating oxygenic and microoxic processes of Cyanothece sp. ATCC 51142 under continuous high irradiance and in high CO2 concentration. During the oxygenic photosynthesis phase, nitrate deficiency limited protein synthesis and CO2 assimilation was directed toward glycogen synthesis. The carbohydrate accumulation reduced overexcitation of the photosynthetic reactions until a respiration burst initiated a transition to microoxic N2 fixation. In contrast to the circadian clock, this ultradian period is strongly temperature-dependent: 17 h at 27 °C, which continuously decreased to 10 h at 39 °C. The cycle was expressed by an oscillatory modulation of net O2 evolution, CO2 uptake, pH, fluorescence emission, glycogen content, cell division, and culture optical density. The corresponding ultradian modulation was also observed in the transcription of nitrogenase-related nifB and nifH genes and in nitrogenase activities. We propose that the control by the newly identified metabolic cycle adds another rhythmic component to the circadian clock that reflects the true metabolic state depending on the actual temperature, irradiance, and CO2 availability.

Cyanofuels: biofuels from cyanobacteria. Reality and perspectives.

Cyanobacteria are represented by a diverse group of microorganisms that, by virtue of being a part of marine and freshwater phytoplankton, significantly contribute to the fixation of atmospheric carbon via photosynthesis. It is assumed that ancient cyanobacteria participated in the formation of earth's oil deposits. Biomass of modern cyanobacteria may be converted into bio-oil by pyrolysis. Modern cyanobacteria grow fast; they do not compete for agricultural lands and resources; they efficiently convert excessive amounts of CO2 into biomass, thus participating in both carbon fixation and organic chemical production. Many cyanobacterial species are easier to genetically manipulate than eukaryotic algae and other photosynthetic organisms. Thus, the cyanobacterial photosynthesis may be directed to produce carbohydrates, fatty acids, or alcohols as renewable sources of biofuels. Here we review the recent achievements in the developments and production of cyanofuels-biofuels produced from cyanobacterial biomass.

Identification and functional role of the carbonic anhydrase Cah3 in thylakoid membranes of pyrenoid of Chlamydomonas reinhardtii.

The distribution of the luminal carbonic anhydrase Cah3 associated with thylakoid membranes in the chloroplast and pyrenoid was studied in wild-type cells of Chlamydomonas reinhardtii and in its cia3 mutant deficient in the activity of the Cah3 protein. In addition, the effect of CO(2) concentration on fatty acid composition of photosynthetic membranes was examined in wild-type cells and in the cia3 mutant. In the cia3 mutant, the rate of growth was lower as compared to wild-type, especially in the cells grown at 0.03% CO(2). This might indicate a participation of thylakoid Cah3 in the CO(2)-concentrating mechanism (CCM) of chloroplast and reflect the dysfunction of the CCM in the cia3 mutant. In both strains, a decrease in the CO(2) concentration from 2% to 0.03% caused an increase in the content of polyunsaturated fatty acids in membrane lipids. At the same time, in the cia3 mutant, the increase in the majority of polyunsaturated fatty acids was less pronounced as compared to wild-type cells, whereas the amount of 16:4ω3 did not increase at all. Immunoelectron microscopy demonstrated that luminal Cah3 is mostly located in the thylakoid membranes that pass through the pyrenoid. In the cells of CCM-mutant, cia3, the Cah3 protein was much less abundant, and it was evenly distributed throughout the pyrenoid matrix. The results support our hypothesis that CO(2) might be generated from HCO(3)(-) by Cah3 in the thylakoid lumen with the following CO(2) diffusion into the pyrenoid, where the CO(2) fixing Rubisco is located. This ensures the maintenance of active photosynthesis under CO(2)-limiting conditions, and, as a result, the active growth of cells. The relationships between the induction of CCM and restructuring of the photosynthetic membranes, as well as the involvement of the Cah3 of the pyrenoid in these events, are discussed. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

CO2-concentrating mechanism in cyanobacterial photosynthesis: organization, physiological role, and evolutionary origin.

The cellular and molecular organization of the CO2-concentrating mechanism (CCM) of cyanobacteria is reviewed. The primary processes of uptake, translocation, and accumulation of inorganic carbon (Ci) near the active site of carbon assimilation by the enzyme ribulose-1,5-bisphosphate carboxylase in the C3 cycle in cyanobacteria are described as one of the specialized forms of CO2 concentration which occurs in some photoautotrophic cells. The existence of this form of CO2 concentration expands our understanding of photosynthetic Ci assimilation. The means of supplying Ci to the C3 cycle in cyanobacteria is not by simple diffusion into the cell, but it is the result of coordinated functions of high-affinity systems for the uptake of CO2 and bicarbonate, as well as intracellular CO2/HCO3 (-) interconversions by carbonic anhydrases. These biochemical events are under genetic control, and they serve to maintain cellular homeostasis and adaptation to CO2 limitation. Here we describe the organization of the CCM in cyanobacteria with a special focus on the CCM of relict halo- and alkaliphilic cyanobacteria of soda lakes. We also assess the role of the CCM at the levels of the organism, the biosphere, and evolution.

Plants, Cells, Algae, and Cyanobacteria In Vitro and Cryobank Collections at the Institute of Plant Physiology, Russian Academy of Sciences-A Platform for Research and Production Center.

Ex situ collections of algae, cyanobacteria, and plant materials (cell cultures, hairy and adventitious root cultures, shoots, etc.) maintained in vitro or in liquid nitrogen (-196 °C, LN) are valuable sources of strains with unique ecological and biotechnological traits. Such collections play a vital role in bioresource conservation, science, and industry development but are rarely covered in publications. Here, we provide an overview of five genetic collections maintained at the Institute of Plant Physiology of the Russian Academy of Sciences (IPPRAS) since the 1950-1970s using in vitro and cryopreservation approaches. These collections represent different levels of plant organization, from individual cells (cell culture collection) to organs (hairy and adventitious root cultures, shoot apices) to in vitro plants. The total collection holdings comprise more than 430 strains of algae and cyanobacteria, over 200 potato clones, 117 cell cultures, and 50 strains of hairy and adventitious root cultures of medicinal and model plant species. The IPPRAS plant cryobank preserves in LN over 1000 specimens of in vitro cultures and seeds of wild and cultivated plants belonging to 457 species and 74 families. Several algae and plant cell culture strains have been adapted for cultivation in bioreactors from laboratory (5-20-L) to pilot (75-L) to semi-industrial (150-630-L) scale for the production of biomass with high nutritive or pharmacological value. Some of the strains with proven biological activities are currently used to produce cosmetics and food supplements. Here, we provide an overview of the current collections' composition and major activities, their use in research, biotechnology, and commercial application. We also highlight the most interesting studies performed with collection strains and discuss strategies for the collections' future development and exploitation in view of current trends in biotechnology and genetic resources conservation.

A leader peptide of the extracellular cyanobacterial carbonic anhydrase ensures the efficient secretion of recombinant proteins in Escherichia coli.

Several forms of EcaA protein, correspondent to the extracellular α-class carbonic anhydrase (CA) of cyanobacterium Crocosphaera subtropica ATCC 51142 were expressed in Escherichia coli. The recombinant proteins with no leader peptide (EcaA and its fusion with thioredoxin or glutathione S-transferase) were allocated inside cells in a full-length form; these cells did not display any extracellular CA activity. Soluble proteins (including that of periplasmic space) of E. coli cells that expressed both ЕсаА equipped with its native leader peptide (L-EcaA) as well as L-EcaA fused with thioredoxin or glutathione S-transferase at N-terminus, mainly contained the processed EcaA. The appearance of mature ЕсаА in outer layers of E. coli cells expressed leader peptide-containing forms of recombinant proteins, has been directly confirmed by immunofluorescent microscopy. Those cells also displayed high extracellular CA activity. In addition, the mature EcaA protein was detected in the culture medium. This suggests that cyanobacterial signal peptide is recognized by the secretory machinery and by the leader peptidase of E. coli even as a part of a fusion protein. The efficiency of EcaA leader peptide was comparable to that of PelB and TorA signal peptides, commonly used for biotechnological production of extracellular recombinant proteins in E. coli.

Optimization of CO2 Supply for the Intensive Cultivation of Chlorella sorokiniana IPPAS C-1 in the Laboratory and Pilot-Scale Flat-Panel Photobioreactors.

Microalgae are increasingly being used for capturing carbon dioxide and converting it into valuable metabolites and biologically active compounds on an industrial scale. The efficient production of microalgae biomass requires the optimization of resources, including CO2. Here, we estimated the productivity of Chlorella sorokiniana IPPAS C-1 depending on CO2 concentrations and the ventilation coefficient of the gas-air mixture (GAM) in flat-panel photobioreactors (FP-PBRs) at laboratory (5 L) and pilot (18 L) scales. For the laboratory scale, the PBRs operated at 900 µmol quanta m-2 s-1 and 35.5 ± 0.5 °C; the optimal CO2 flow rate was estimated at 3 mL CO2 per 1 L of suspension per minute, which corresponds to 1.5% CO2 in the GAM and an aeration rate of 0.2 vvm. These parameters, being scaled up within the pilot PBRs, resulted in a high specific growth rate (µ ≈ 0.1 h-1) and high specific productivity (Psp ≈ 1 g dw L-1 d-1). The principles of increasing the efficiency of the intensive cultivation of C. sorokiniana IPPAS C-1 are discussed. These principles are relevant for the development of technological regimes for the industrial production of Chlorella in flat-panel PBRs of various sizes.

Cultivation of Chlorella sorokiniana IPPAS C-1 in Flat-Panel Photobioreactors: From a Laboratory to a Pilot Scale.

Flat-panel photobioreactors are effective systems for microalgae cultivation. This paper presents the growth characteristics of the microalgae Chlorella sorokiniana IPPAS C-1 as a result of three-stage scale-up cultivation in a specially designed cultivation system. First, C. sorokiniana was grown aseptically in 250 mL glass vessels; then, it was diluted and inoculated into a 5-liter flat-panel horizontal photobioreactor; and, at the last stage, the culture was diluted and inoculated into a 70-liter flat-panel vertical photobioreactor. In the presented cycle, the cultured biomass increased by 326 times in 13 days (from 0.6 to 195.6 g dw), with a final biomass concentration of 2.8 g dw L-1. The modes of semi-continuous cultivation were considered. The biomass harvest and dilution of the suspension were carried out either every day or every 3-4 days. For C. sorokiniana IPPAS C-1, a conversion coefficient of optical density values to dry biomass (g L-1) was refined through a factor of 0.33. The key parameters of the photobioreactors tested in this work are discussed.

Ecology and biogeography of the 'marine Geitlerinema' cluster and a description of Sodalinema orleanskyi sp. nov., Sodalinema gerasimenkoae sp. nov., Sodalinema stali sp. nov. and Baaleninema simplex gen. et sp. nov. (Oscillatoriales, Cyanobacteria).

Filamentous cyanobacteria belonging to the 'marine Geitlerinema' cluster are spread worldwide in saline environments and considered to play an important ecological role. However, the taxonomy of this group remains unclear. Here, we analyzed the phylogeny, ecology and biogeography of the 'marine Geitlerinema' cluster representatives and revealed two subclusters: (1) an 'oceanic' subcluster containing PCC7105 clade and black band disease (BBD) clade with free-living and pathogenic strains distributed in Atlantic, Indian and Pacific Ocean-related localities, and (2) a Sodalinema subcluster containing free-living strains from marine, hypersaline, saline-alkaline and soda lake habitats from the Eurasian and African continents. Polyphasic analysis using genetic and phenotypic criteria demonstrated that these two groups represent separate genera. Representatives of Sodalinema subcluster were phylogenetically attributed to the genus Sodalinema. Our data expand the ecological and geographical distribution of this genus. We emended the description of the genus Sodalinema and proposed three new species differing in phylogenetic, geographic and ecological criteria: Sodalinema orleanskyi sp. nov., Sodalinema gerasimenkoae sp. nov. and Sodalinema stali sp. nov. Additionally, a new genus and species Baaleninema simplex gen. et sp. nov. was discribed within the PCC7105 clade. By this, we put in order the current confusion of the 'marine Geitlerinema' group and highlight its ecological diversity.

Effect of salt stress on physiological parameters of microalgae Vischeria punctata strain IPPAS H-242, a superproducer of eicosapentaenoic acid.

The strain IPPAS H-242 is an eustigmatophycean alga with good growth characteristics and high content of the long chain polyunsaturated eicosapentaenoic fatty acid (EPA) - a very-long-chain fatty acid with high nutraceutical value. In this study, based on 18S rRNA gene and ITS1-5.8S-ITS2 sequences the strain IPPAS H-242 was identified as an authentic strain of Vischeria punctata. The effect of salt stress (0.5 M NaCl) on growth, cell morphology, ultrastructure, and biochemical composition with the emphasis on the fatty acid (FA) profile was investigated in batch cultures. Under salt stress, biomass accumulation and cell division were severely inhibited; cells were bigger, with higher chloroplast volume and numerous mitochondria, they had more proteins (73 % from the initial concentration as compared to 23 % in control) and their lipids had higher EPA proportion (13.6 % of total FA as compared to 6.4 % of total FA in control). In salt-stressed cells, thylakoid organization and photosynthetic activity were impaired, and D1 protein content decreased to trace amounts. In spite of an increase in EPA proportion in total FA, salt stress causes a decrease in total EPA productivity (49 mg/L as compared to 130 mg/L in control).

Stress sensors and signal transducers in cyanobacteria.

In living cells, the perception of environmental stress and the subsequent transduction of stress signals are primary events in the acclimation to changes in the environment. Some molecular sensors and transducers of environmental stress cannot be identified by traditional and conventional methods. Based on genomic information, a systematic approach has been applied to the solution of this problem in cyanobacteria, involving mutagenesis of potential sensors and signal transducers in combination with DNA microarray analyses for the genome-wide expression of genes. Forty-five genes for the histidine kinases (Hiks), 12 genes for serine-threonine protein kinases (Spks), 42 genes for response regulators (Rres), seven genes for RNA polymerase sigma factors, and nearly 70 genes for transcription factors have been successfully inactivated by targeted mutagenesis in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Screening of mutant libraries by genome-wide DNA microarray analysis under various stress and non-stress conditions has allowed identification of proteins that perceive and transduce signals of environmental stress. Here we summarize recent progress in the identification of sensory and regulatory systems, including Hiks, Rres, Spks, sigma factors, transcription factors, and the role of genomic DNA supercoiling in the regulation of the responses of cyanobacterial cells to various types of stress.

'Candidatus Oscillochloris kuznetsovii' a novel mesophilic filamentous anoxygenic phototrophic Chloroflexales bacterium from Arctic coastal environments.

Chloroflexales bacteria are mostly known as filamentous anoxygenic phototrophs that thrive as members of the microbial communities of hot spring cyanobacterial mats. Recently, we described many new Chloroflexales species from non-thermal environments and showed that mesophilic Chloroflexales are more diverse than previously expected. Most of these species were isolated from aquatic environments of mid-latitudes. Here, we present the comprehensive characterization of a new filamentous multicellular anoxygenic phototrophic Chloroflexales bacterium from an Arctic coastal environment (Kandalaksha Gulf, the White Sea). Phylogenomic analysis and 16S rRNA phylogeny indicated that this bacterium belongs to the Oscillochloridaceae family as a new species. We propose that this species be named 'Candidatus Oscillochloris kuznetsovii'. The genomes of this species possessed genes encoding sulfide:quinone reductase, the nitrogenase complex and the Calvin cycle, which indicate potential for photoautotrophic metabolism. We observed only mesophilic anaerobic anoxygenic phototrophic growth of this novel bacterium. Electron microphotography showed the presence of chlorosomes, polyhydroxyalkanoate-like granules and polyphosphate-like granules in the cells. High-performance liquid chromatography also revealed the presence of bacteriochlorophylls a, c and d as well as carotenoids. In addition, we found that this bacterium is present in benthic microbial communities of various coastal environments of the Kandalaksha Gulf.

Polyphosphate accumulation dynamics in a population of Synechocystis sp. PCC 6803 cells under phosphate overplus.

In this study, a simple and rapid DAPI-based protocol was developed and optimized to visualize polyphosphates (polyPs) in the cyanobacterium Synechocystis sp. PCC 6803. The optimum dye concentration and incubation time were determined, and formaldehyde fixation was shown to significantly improve polyP detection in Synechocystis cells. Using the developed protocol, for the first time, it was shown that 80% of Synechocystis cells under phosphate overplus were able to accumulate phosphorus as polyP 3 min after the addition of K2HPO4. After 1 h, the number of cells with polyP began to decrease, and after 24 h, polyP granules were detected in only 30% of the cells. Thus, the Synechocystis cells appeared to be heterogeneous in their ability to accumulate and mobilize polyP. Like other photosynthetic organisms, Synechocystis synthesized less polyP in the dark than in the light. The accumulation of polyP was not inhibited under conditions of cold and heat stresses, and some cells were even able to synthesize polyP at a temperature of approximately 0 °C.

Universal Molecular Triggers of Stress Responses in Cyanobacterium Synechocystis.

Systemic analysis of stress-induced transcription in the cyanobacterium Synechocystis sp. strain PCC 6803 identifies a number of genes as being induced in response to most abiotic stressors (heat, osmotic, saline, acid stress, strong light, and ultraviolet radiation). Genes for heat-shock proteins (HSPs) are activated by all these stresses and form a group that universally responds to all environmental changes. The functions of universal triggers of stress responses in cyanobacteria can be performed by reactive oxygen species (ROS), in particular H2O2, as well as changes in the redox potential of the components of the photosynthetic electron transport chain. The double mutant of Synechocystis sp. PCC 6803 (katG/tpx, or sll1987/sll0755), which is defective in antioxidant enzymes catalase (KatG) and thioredoxin peroxidase (Tpx), cannot grow in the presence of exogenous hydrogen peroxide (H2O2); and it is extremely sensitive to low concentrations of H2O2, especially under conditions of cold stress. Experiments on this mutant demonstrate that H2O2 is involved in regulation of gene expression that responds to a decrease in ambient temperature, and affects both the perception and the signal transduction of cold stress. In addition, they suggest that formation of ROS largely depends on the physical state of the membranes such as fluidity or viscosity. In cyanobacteria, an increase in membrane turnover leads to a decrease in the formation of ROS and an increase in resistance to cold stress. Therefore: (1) H2O2 is the universal trigger of stress responses in cyanobacterial cells; (2) ROS formation (in particular, H2O2) depends on the physical properties of both cytoplasmic and thylakoid membranes; (3) The destructive effect of H2O2 is reduced by increasing of fluidity of biological membranes.

Quantitative insights into the cyanobacterial cell economy.

Phototrophic microorganisms are promising resources for green biotechnology. Compared to heterotrophic microorganisms, however, the cellular economy of phototrophic growth is still insufficiently understood. We provide a quantitative analysis of light-limited, light-saturated, and light-inhibited growth of the cyanobacterium Synechocystis sp. PCC 6803 using a reproducible cultivation setup. We report key physiological parameters, including growth rate, cell size, and photosynthetic activity over a wide range of light intensities. Intracellular proteins were quantified to monitor proteome allocation as a function of growth rate. Among other physiological acclimations, we identify an upregulation of the translational machinery and downregulation of light harvesting components with increasing light intensity and growth rate. The resulting growth laws are discussed in the context of a coarse-grained model of phototrophic growth and available data obtained by a comprehensive literature search. Our insights into quantitative aspects of cyanobacterial acclimations to different growth rates have implications to understand and optimize photosynthetic productivity.

Highly active extracellular α-class carbonic anhydrase of Cyanothece sp. ATCC 51142.

Here, for the first time, we report the presence of highly active extracellular carbonic anhydrase (CA) of α-class in cyanobacterial cells. The enzyme activity was confirmed both in vivo in intact cells and in vitro, using the recombinant protein. CA activity in intact cells of Cyanothece sp. ATCC 51142 reached ∼0.6 Wilbur-Anderson units (WAU) per 1 mg of total cell protein, and it was inhibited by a specific CAs inhibitor, ethoxyzolamide. The genes cce_4328 (ecaA) and cce_0871 (ecaB), encoding two potential extracellular CAs of Cyanothece have been cloned, and the corresponding proteins EcaA and EcaB, representing CAs of α- and ß-class, respectively, have been heterologously expressed in Escherichia coli. High specific activity (∼1.1 × 104 WAU per 1 mg of target protein) was detected for the recombinant EcaA only. The presence of EcaA in the outer cellular layers of Cyanothece was confirmed by immunological analysis with antibodies raised against the recombinant protein. The absence of redox regulation of EcaA activity indicates that this protein does not possess a disulfide bond essential for some α-class CAs. The content and activity of EcaA in a fraction of periplasmic proteins was higher in Cyanothece cells grown at ambient concentration of CO2 (0.04%) compared to those grown at an elevated CO2 concentration (1.7%). At the same time, the level of ecaA gene mRNA varied insignificantly in response to changes in CO2 supply. Our results indicate that EcaA is responsible for CA activity of intact Cyanothece cells and point to its possible physiological role under low-CO2 conditions.