Vladimir S. Bedbenov

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.

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.

Cold-induced gene expression and ω3 fatty acid unsaturation is controlled by red light in Synechocystis

The expression of cold-induced genes, which are controlled by the cold sensor histidine kinase Hik33, and the formation of ω(3) polyunsaturated fatty acids are controlled by light in the cyanobacterium Synechocystis sp. PCC 6803. Cold-induced Hik33-dependent gene expression is initiated by red light (∼700nm), but not by blue or green light. Red light also turns on the ω(3) fatty acid desaturation. Different combinations of other wavelengths in red spectral region (635 and 726nm) had no effect on the red-light-activated cold-induced transcription or fatty acid desaturation. Therefore, the involvement of phytochrome-like photoreceptor(s), similar to phytochromes of higher plants, in this regulation was not confirmed. The absence of light-dependence of gene expression in the mutant cells deficient in Hik33 suggests the involvement of this histidine kinase in direct or mediated with red light regulation of cold responses in Synechocystis.

Involvement of serine/threonine protein kinases in the cold stress response in the cyanobacterium Synechocystis sp. PCC 6803: Functional characterization of SpkE protein kinase

Stress responses of the unicellular cyanobacterium Synechocystis involve several regulatory systems, including two-component ones, and negative supercoiling of genomic DNA. The role of serine/threonine protein kinases (STPKs) in the cold response was studied in Synechocystis. A screening of a collection of STPK mutants identified four enzymes—SpkB, SpkD, SpkE, and SpkG—as possible transcriptional regulators at lower temperatures. A proteome analysis in a SpkE Synechocystis mutant implicated SpkE in the formation of the protein pattern. In vitro phosphorylation assays of recombinant SpkE confirmed that the STPK was functionally active and utilized basic proteins as preferable substrates.

Optimization of Prochlorothrix hollandica cyanobacteria culturing for obtaining myristoleic acid

Plankton filament cyanobacteria Prochlorothrix hollandica is characterized by a very high content of C14 and C16 fatty acids (FA) in the lipid membranes. Depending on culturing conditions of the cyanobacteria, total concentrations of myristic and myristoleic acids can reach 35% and those of palmitic and palmitoleic acids can reach 60% of all esterified FA cells. In P. hollandica, a variety of monounsaturated FA is represented by myristoleic and palmitic acids, and by hexadecenoic (C16:1) acid with olefin bond of cis-configuration, located in the Δ4 position. The process of intensive culturing for P. hollandica cells to yield a maximal biomass in order to isolate the pure drug of myristoleic acid derivative has been optimized. The use of a threestage purification gives 30 mg of chromatographically pure myristoleic acid methyl ester from 17 g of P. hollandica raw biomass (dry mass is 3 g), which is 1% of dry cell mass.

Substrate Specificity of Acyl-Lipid Δ9-Desaturase from Cyanobacterium sp. IPPAS B-1200, a Cyanobacterium with Unique Fatty Acid Composition

Cyanobacterium sp. IPPAS B-1200 is characterized by a high content of rare fatty acids (FAs), both myristic (14:0–30%) and myristoleic (14:1Δ9–10%) in the membrane lipids. Thus, short-chain FAs reach 40% of the sum of all FAs in cells, which is unusual for Cyanobacteria. Monounsaturated palmitoleic acids (16:1Δ9) also reach 40% of the sum of the FAs. We determined the complete nucleotide sequence of the genome of this cyanobacterium and found the only gene for the acyl-lipid Δ9-desaturase, desC1. We cloned this gene and characterized its specificity to the length of the substrate using heterologous expression in Escheriсhia coli. The results show that DesC1 nonspecifically generates olefin bond in FAs with a length of 14, 16, and 18 carbon atoms. This finding confirms that all monoesterifed FAs in Cyanobacterium sp. IPPAS B-1200 are generated by one acyl-lipid Δ9-desaturase.

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.

Draft Genome Sequences of Two Thermotolerant Cyanobacterial Strains Isolated from Hot Springs

ABSTRACT We report here two draft cyanobacterial genome sequences, those of Cyanobacterium aponinum IPPAS B-1201, isolated from a hot spring in the Turgen Gorge (Kazakhstan), and the uncharacterized cyanobacterium IPPAS B-1203, isolated from a hot spring in Karlovy Vary (Czech Republic). These two strains were deposited at the Collection of Microalgae (IPPAS) of the Timiryazev Institute of Plant Physiology.