The Specificities of Lysophosphatidic Acid Acyltransferase and Fatty Acid Desaturase Determine the High Content of Myristic and Myristoleic Acids in Cyanobacterium sp. IPPAS B-1200.

The cyanobacterial strain Cyanobacterium sp. IPPAS B-1200 isolated from Lake Balkhash is characterized by high relative amounts of myristic (30%) and myristoleic (10%) acids. The remaining fatty acids (FAs) are represented mainly by palmitic (20%) and palmitoleic (40%) acids. We expressed the genes for lysophosphatidic acid acyltransferase (LPAAT; EC 2.3.1.51) and Δ9 fatty acid desaturase (FAD; EC 1.14.19.1) from Cyanobacterium sp. IPPAS B-1200 in Synechococcus elongatus PCC 7942, which synthesizes myristic and myristoleic acids at the level of 0.5-1% and produces mainly palmitic (~60%) and palmitoleic (35%) acids. S. elongatus cells that expressed foreign LPAAT synthesized myristic acid at 26%, but did not produce myristoleic acid, suggesting that Δ9-FAD of S. elongatus cannot desaturate FAs with chain lengths less than C16. Synechococcus cells that co-expressed LPAAT and Δ9-FAD of Cyanobacterium synthesized up to 45% palmitoleic and 9% myristoleic acid, suggesting that Δ9-FAD of Cyanobacterium is capable of desaturating saturated acyl chains of any length.

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.

Membrane physical state and stress regulation in Synechocystis: fluidizing alcohols repress fatty acid desaturation.

Cyanobacteria are prokaryotic photosynthetic organisms widely used in biotechnology, photosynthesis and abiotic stress research. There are several cyanobacterial strains modified to produce biofuels, but the influence of alcohols on cyanobacterial cell physiology is poorly understood. Here, we conducted a systematic study of the effects of nine primary aliphatic alcohols and an aromatic benzyl alcohol on both membrane physical state and the expression of genes for fatty acid desaturases (FADs) in a model cyanobacterium Synechocystis sp. strain PCC 6803. Hexan-1-ol was found to have the most membrane fluidizing action among all alcohols studied, with its efficiency correlating with both duration of treatment and alcohol concentration. A prolonged exposure to alcohol results in a continuous loss of unsaturated fatty acids (FAs) followed by cell death, an undesired challenge that should be considered in cyanobacterial biotechnology. We suggest that membrane fluidization is the key component in alcohol stress causing inactivation of FADs and resulting in a lethal depletion of unsaturated FAs. Due to the most pronounced effects of alcohol- and heat-induced membrane fluidization on desB encoding a terminal ω3-FAD, we propose to call desB a 'viscosity gene' in analogy to heat-induced 'fluidity gene' hspA.

The impact of the phytochromes on photosynthetic processes.

This review describes the phytochrome system in higher plants and cyanobacteria and its role in regulation of photosynthetic processes and stress protection of the photosynthetic apparatus. A relationship between the content of the different phytochromes, the changes in the ratios of the physiologically active forms of phytochromes to their total pool and the resulting influence on photosynthetic processes is reviewed. The role of the phytochromes in the regulation of the expression of genes encoding key photosynthetic proteins, antioxidant enzymes and other components involved in stress signaling is elucidated.

Hydrogen Peroxide Participates in Perception and Transduction of Cold Stress Signal in Synechocystis.

The double mutant ΔkatG/tpx of cyanobacterium Synechocystis sp. strain PCC 6803, defective in the anti-oxidative enzymes catalase (KatG) and thioredoxin peroxidase (Tpx), is unable to grow in the presence of exogenous H2O2. The ΔkatG/tpx mutant is shown to be extremely sensitive to very low concentrations of H2O2, especially when intensified with cold stress. Analysis of gene expression in both wild-type and ΔkatG/tpx mutant cells treated by combined cold/oxidative stress revealed that H2O2 participates in regulation of expression of cold-responsive genes, affecting either signal perception or transduction. The central role of a transmembrane stress-sensing histidine kinase Hik33 in the cold/oxidative signal transduction pathway is discussed.

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.

The Unique Protein-to-Protein Carotenoid Transfer Mechanism.

Orange Carotenoid Protein (OCP) is known as an effector and regulator of cyanobacterial photoprotection. This 35 kDa water-soluble protein provides specific environment for blue-green light absorbing keto-carotenoids, which excitation causes dramatic but fully reversible rearrangements of the OCP structure, including carotenoid translocation and separation of C- and N-terminal domains upon transition from the basic orange to photoactivated red OCP form. Although recent studies greatly improved our understanding of the OCP photocycle and interaction with phycobilisomes and the fluorescence recovery protein, the mechanism of OCP assembly remains unclear. Apparently, this process requires targeted delivery and incorporation of a highly hydrophobic carotenoid molecule into the water-soluble apoprotein of OCP. Recently, we introduced, to our knowledge, a novel carotenoid carrier protein, COCP, which consists of dimerized C-domain(s) of OCP and can combine with the isolated N-domain to form transient OCP-like species. Here, we demonstrate that in vitro COCP efficiently transfers otherwise tightly bound carotenoid to the full-length OCP apoprotein, resulting in formation of photoactive OCP from completely photoinactive species. We accurately analyze the peculiarities of this process that, to the best of our knowledge, appears unique, a previously uncharacterized protein-to-protein carotenoid transfer mechanism. We hypothesize that a similar OCP assembly can occur in vivo, substantiating specific roles of the COCP carotenoid carrier in cyanobacterial photoprotection.

Fluorescent Labeling Preserving OCP Photoactivity Reveals Its Reorganization during the Photocycle.

Orange carotenoid protein (OCP), responsible for the photoprotection of the cyanobacterial photosynthetic apparatus under excessive light conditions, undergoes significant rearrangements upon photoconversion and transits from the stable orange to the signaling red state. This is thought to involve a 12-Å translocation of the carotenoid cofactor and separation of the N- and C-terminal protein domains. Despite clear recent progress, the detailed mechanism of the OCP photoconversion and associated photoprotection remains elusive. Here, we labeled the OCP of Synechocystis with tetramethylrhodamine-maleimide (TMR) and obtained a photoactive OCP-TMR complex, the fluorescence of which was highly sensitive to the protein state, showing unprecedented contrast between the orange and red states and reflecting changes in protein conformation and the distances from TMR to the carotenoid throughout the photocycle. The OCP-TMR complex was sensitive to the light intensity, temperature, and viscosity of the solvent. Based on the observed Förster resonance energy transfer, we determined that upon photoconversion, the distance between TMR (donor) bound to a cysteine in the C-terminal domain and the carotenoid (acceptor) increased by 18 Å, with simultaneous translocation of the carotenoid into the N-terminal domain. Time-resolved fluorescence anisotropy revealed a significant decrease of the OCP rotation rate in the red state, indicating that the light-triggered conversion of the protein is accompanied by an increase of its hydrodynamic radius. Thus, our results support the idea of significant structural rearrangements of OCP, providing, to our knowledge, new insights into the structural rearrangements of OCP throughout the photocycle and a completely novel approach to the study of its photocycle and non-photochemical quenching. We suggest that this approach can be generally applied to other photoactive proteins.

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.

Membrane fluidity controls redox-regulated cold stress responses in cyanobacteria.

Membrane fluidity is the important regulator of cellular responses to changing ambient temperature. Bacteria perceive cold by the transmembrane histidine kinases that sense changes in thickness of the cytoplasmic membrane due to its rigidification. In the cyanobacterium Synechocystis, about a half of cold-responsive genes is controlled by the light-dependent transmembrane histidine kinase Hik33, which also partially controls the responses to osmotic, salt, and oxidative stress. This implies the existence of some universal, but yet unknown signal that triggers adaptive gene expression in response to various stressors. Here we selectively probed the components of photosynthetic machinery and functionally characterized the thermodynamics of cyanobacterial photosynthetic membranes with genetically altered fluidity. We show that the rate of oxidation of the quinone pool (PQ), which interacts with both photosynthetic and respiratory electron transport chains, depends on membrane fluidity. Inhibitor-induced stimulation of redox changes in PQ triggers cold-induced gene expression. Thus, the fluidity-dependent changes in the redox state of PQ may universally trigger cellular responses to stressors that affect membrane properties.

The complete genome of a cyanobacterium from a soda lake reveals the presence of the components of CO2-concentrating mechanism.

At present geological epoch, the carbon concentrating mechanism (CCM) of cyanobacteria represents the obligatory tool for adaptation to low content of CO2 in the atmosphere and for the maintenance of sufficient photosynthetic activity. Functional CCM was found in modern cyanobacteria from different ecological niches. However, the presence of such mechanism in species that inhabit soda lakes is not obvious due to high content of inorganic carbon (C i) in the environment. Here we analyze CCM components that have been identified by sequencing of the whole genome of the alkaliphilic cyanobacterium Microcoleus sp. IPPAS B-353. The composition of the CCM components of Microcoleus is similar to that of 'model' ß-cyanobacteria, freshwater and marine Synechococcus or Synechocystis spp. However, CahB1 protein of Microcoleus, which is the homolog of CcaA, the carboxysomal ß-type carbonic anhydrase (CA) of ß-cyanobacteria, appeared to be the only active CA located in cell envelopes. The conservative regions of CcmM, CahG (a homolog of archeal γ-CAs, Cam/CamH), and ChpX of Microcoleus possess single amino acid substitutions that may cause a lack of CA activities. Unlike model cyanobacteria, Microcoleus induces only one BicA-type bicarbonate transporter in response to C i limitation. The differences in the appearance of CCM components and in their characteristics between alkaliphilic Microcoleus and freshwater or marine cyanobacteria are described. The possible reasons for the maintenance of CCM components in cyanobacteria, which permanently live at high concentrations of C i in soda lakes, are discussed.

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.

Reactive oxygen species: re-evaluation of generation, monitoring and role in stress-signaling in phototrophic organisms.

This review provides an overview about recent developments and current knowledge about monitoring, generation and the functional role of reactive oxygen species (ROS) - H2O2, HO2, HO, OH(-), (1)O2 and O2(-) - in both oxidative degradation and signal transduction in photosynthetic organisms including microscopic techniques for ROS detection and controlled generation. Reaction schemes elucidating formation, decay and signaling of ROS in cyanobacteria as well as from chloroplasts to the nuclear genome in eukaryotes during exposure of oxygen-evolving photosynthetic organisms to oxidative stress are discussed that target the rapidly growing field of regulatory effects of ROS on nuclear gene expression.

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.

Redox potentials of primary electron acceptor quinone molecule (QA)- and conserved energetics of photosystem II in cyanobacteria with chlorophyll a and chlorophyll d.

In a previous study, we measured the redox potential of the primary electron acceptor pheophytin (Phe) a of photosystem (PS) II in the chlorophyll d-dominated cyanobacterium Acaryochloris marina and a chlorophyll a-containing cyanobacterium, Synechocystis. We obtained the midpoint redox potential (E(m)) values of -478 mV for A. marina and -536 mV for Synechocystis. In this study, we measured the redox potentials of the primary electron acceptor quinone molecule (Q(A)), i.e., E(m)(Q(A)/Q(A)(-)), of PS II and the energy difference between [P680·Phe a(-)·Q(A)] and [P680·Phe a·Q(A)(-)], i.e., ΔG(PhQ). The E(m)(Q(A)/Q(A)(-)) of A. marina was determined to be +64 mV without the Mn cluster and was estimated to be -66 to -86 mV with a Mn-depletion shift (130-150 mV), as observed with other organisms. The E(m)(Phe a/Phe a(-)) in Synechocystis was measured to be -525 mV with the Mn cluster, which is consistent with our previous report. The Mn-depleted downshift of the potential was measured to be approximately -77 mV in Synechocystis, and this value was applied to A. marina (-478 mV); the E(m)(Phe a/Phe a(-)) was estimated to be approximately -401 mV. These values gave rise to a ΔG(PhQ) of -325 mV for A. marina and -383 mV for Synechocystis. In the two cyanobacteria, the energetics in PS II were conserved, even though the potentials of Q(A)(-) and Phe a(-) were relatively shifted depending on the special pair, indicating a common strategy for electron transfer in oxygenic photosynthetic organisms.

Transmembrane and PAS domains of the histidine kinase Hik33 as regulators of cold and light responses in the cyanobacterium Synechocystis sp. PCC 6803.

The PAS (Per-ARNT-Sim) domain is a sensory protein regulatory module found in archaea, prokaryotes, and eukaryotes. Histidine and serine/threonine protein kinases, chemo- and photoreceptors, circadian rhythm regulators, ion channels, phosphodiesterases, and other cellular response regulators are among these proteins. Hik33 is a multifunctional sensory histidine kinase that is implicated in cyanobacterial responses to cold, salt, hyperosmotic, and oxidative stressors. The functional roles of individual Hik33 domains in signal transduction were investigated in this study. Synechocystis Hik33 deletion variants were developed, in which either both or a portion of the transmembrane domains and/or the PAS domain were deleted. Cold stress was applied to the mutant strains either under illumination or in the dark. The findings show that the transmembrane domains govern temperature responses, whereas PAS domain may be involved in regulation of downstream gene expression in light-dependent manner.

Spirulina/Arthrospira/Limnospira-Three Names of the Single Organism.

Recent advances in research techniques have enabled rapid progress in the study of spirulina, an ancient edible cyanobacteria. Nowadays, spirulina species are classified into three genera: Spirulina, Arthrospira, and Limnospira. The latter now refers to industrially manufactured spirulina strains. Whole-genome sequencing revealed gene clusters involved in metabolite production, and the physiology of spirulina. Omics technologies demonstrated the absence of hazardous compounds in spirulina cells, confirming the safety of this biomass as a food product. Spirulina is a good source of different chemicals used in food manufacturing, food supplements, and pharmaceuticals. Spirulina's enrichment with inherent biologically active substances makes it a potential supplier of natural products for dietary and pharmaceutical applications. Spirulina is also a prospective component of both terrestrial and space-based life support systems. Here, we review current breakthroughs in spirulina research and clarify fallacies that can be found in both professional literature and public media.

The Freshwater Cyanobacterium Synechococcus elongatus PCC 7942 Does Not Require an Active External Carbonic Anhydrase.

Under standard laboratory conditions, Synechococcus elongatus PCC 7942 lacks EcaASyn, a periplasmic carbonic anhydrase (CA). In this study, a S. elongatus transformant was created that expressed the homologous EcaACya from Cyanothece sp. ATCC 51142. This additional external CA had no discernible effect on the adaptive responses and physiology of cells exposed to changes similar to those found in S. elongatus natural habitats, such as fluctuating CO2 and HCO3- concentrations and ratios, oxidative or light stress, and high CO2. The transformant had a disadvantage over wild-type cells under certain conditions (Na+ depletion, a reduction in CO2). S. elongatus cells lacked their own EcaASyn in all experimental conditions. The results suggest the presence in S. elongatus of mechanisms that limit the appearance of EcaASyn in the periplasm. For the first time, we offer data on the expression pattern of CCM-associated genes during S. elongatus adaptation to CO2 replacement with HCO3-, as well as cell transfer to high CO2 levels (up to 100%). An increase in CO2 concentration coincides with the suppression of the NDH-14 system, which was previously thought to function constitutively.

Light-dependent cold-induced fatty acid unsaturation, changes in membrane fluidity, and alterations in gene expression in Synechocystis.

Cold stress causes unsaturation of the membrane lipids. This leads to adjustment of the membrane fluidity, which is necessary for cold acclimation of cells. Here we demonstrate that the cold-induced accumulation of PUFAs in the cyanobacterium Synechocystis is light-dependent. The desA(-)/desD(-) mutant, that lacks the genes for Δ12 and Δ6 desaturases, is still able to adjust the fluidity of its membranes in spite of its inability to synthesize PUFAs and modulate the fatty acid composition of the membrane lipids under cold stress. The expression of cold-induced genes, which are controlled by the cold sensor histidine kinase Hik33, depends on the fluidity of cell membranes and it is regulated by light, though it does not require the activity of the photosynthetic apparatus. The expression of cold-induced genes, which are not controlled by Hik33, does not depend on the membrane fluidity or light. Thus, membrane fluidity determines the temperature dependence of the expression of cold-induced genes that are under control of the Hik33, which might be the sensor of changes in the membrane fluidity. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.

Quantitative structure-activity relationship analysis of perfluoroiso-propyldinitrobenzene derivatives known as photosystem II electron transfer inhibitors.

Quantitative structure-activity relationship (QSAR) analysis of the twenty-six perfluoroisopropyl-dinitrobenzene (PFIPDNB) derivatives was performed to explain their ability to suppress photochemical activity of the plants photosystem II using chloroplasts and subchloroplast thylakoid membranes enriched in photosystem II, called DT-20. Compounds were optimized by semi-empirical PM3 and DFT/B3LYP/6-31G methods. The Heuristic and the Best Multi-Linear Regression (BMLR) method in CODESSA were used to select the most appropriate molecular descriptors and to develop a linear QSAR model between experimental pI(50) values and the most significant set of the descriptors. The obtained models were validated by cross-validation (R(2)(cv)) and internal validation to confirm the stability and good predictive ability. The obtained eight models with five-parameter show that: (a) coefficient (R(2)) value of the chloroplast samples are slightly higher than that of the DT-20 samples both of Heuristic and BMLR models; (b) the coefficients of the BMLR models are slightly higher than that of Heuristic models both of chloroplasts and DT-20 samples; (c) The YZ shadow parameter and the indicator parameter, for presence of NO(2) substituent in the ring, are the most important descriptor at PM3-based and DFT-based QSAR models, respectively. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.