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.

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.

Molecular characterization of type specimens is a powerful tool used in clarifying species identity/circumscription, as well as establishing the taxonomic and phylogenetic status of organisms in question. However, DNA sequencing of aged herbarium collections can be a challenge due to the quantity and quality of DNA still present in the specimens. Herein, we report a custom DNA isolation protocol suitable for processing minute quantities of old specimen tissue and its utilization via high-throughput sequencing technologies to obtain, for the first time, the genome assembly of the 134-year-old holotype of Boletus subvelutipes Peck, a North American fleshy pored mushroom of taxonomic and historical significance. A side-by-side evaluation of our DNA isolation method with that of a commercial "kit" by Qiagen is also presented. By relying on the type material, we have established the genetic identity of B. subvelutipes, as well as providing preliminary phylogenetic evidence for its generic affinities in Neoboletus within Boletaceae. The reference genome of the B. subvelutipes holotype provides a resource for future comparative genomic studies, taxonomic revisions in Boletaceae, and other evolutionary studies of fungi.

Микроводоросли накапливают в качестве основных запасных веществ крахмал и триацилглицерины (ТАГ). При этом соотношения этих запасных продуктов могут сильно варьироваться и в значительной степени оказываются видо- и даже штамм-специфичны. Кроме того, различные стрессовые воздействия могут способствовать запасанию крахмала или же ТАГ, открывая возможности для биотехнологического «управления» процессами запасания в клетках культивируемых микроводорослей Microalgae accumulate starch and triacylglycerols (TAG) as the main reserve substances. At the same time, the ratios of these reserve products can vary greatly and, to a large extent, turn out to be species- and even strain-specific. In addition, various stress effects can promote the storage of starch or TAG, opening up opportunities for biotechnological "management" of storage processes in the cells of cultivated microalgae.

Quenching of excess excitation energy is necessary for the photoprotection of light-harvesting complexes. In cyanobacteria, quenching of phycobilisome (PBS) excitation energy is induced by the Orange Carotenoid Protein (OCP), which becomes photoactivated under high light conditions. A decrease in energy transfer efficiency from the PBSs to the reaction centers decreases photosystem II (PS II) activity. However, quantitative analysis of OCP-induced photoprotection in vivo is complicated by similar effects of both photochemical and non-photochemical quenching on the quantum yield of the PBS fluorescence overlapping with the emission of chlorophyll. In the present study, we have analyzed chlorophyll a fluorescence induction to estimate the effective cross-section of PS II and compared the effects of reversible OCP-dependent quenching of PBS fluorescence with reduction of PBS content upon nitrogen starvation or mutations of key PBS components. This approach allowed us to estimate the dependency of the rate constant of PS II primary electron acceptor reduction on the amount of PBSs in the cell. We found that OCP-dependent quenching triggered by blue light affects approximately half of PBSs coupled to PS II, indicating that under normal conditions, the concentration of OCP is not sufficient for quenching of all PBSs coupled to PS II.

Cyanobacteria are model photosynthetic prokaryotic organisms often used in biotechnology to produce biofuels including alcohols. The effect of alcohols on cyanobacterial cell physiology and specifically on membrane fluidity is poorly understood. Previous research on various primary aliphatic alcohols found that alcohols with a short hydrocarbon chain (C1-C3) do not affect expression of genes related to membrane physical state. In addition, less water-soluble alcohols with a hydrocarbon chain longer than C8 are found to have a reduced ability to reach cellular membranes hence do not drastically change membrane physical state or induce expression of stress-responsive genes. Therefore, hexan-1-ol (C6) is suggested to have the most profound effect on cyanobacterial membrane physical state. Here, we studied the effects of hexan-1-ol on the cyanobacterium Synechocystis sp. PCC 6803 transcriptome. The transcriptome data obtained is compared to the previously reported analysis of gene expression induced by benzyl alcohol and butan-1-ol. The set of genes whose expression is induced after exposure to all three studied alcohols is identified. The expression under alcohol stress for several general stress response operons is analyzed, and examples of antisense interactions of RNA are investigated.

The main limiting factors for RNA-Seq analysis are quality and quantity of the isolated mRNA. In prokaryotes, the proportion of messenger RNA to total RNA is rather low. Therefore, the main strategy of library preparation for sequencing is mRNA enrichment. Ribosomal and transfer RNAs, both monophosphorylated at the 5'-ends, are the major fractions of total RNA, while the bulk of primary transcripts is triphosphorylated at the 5'-teminus. Due to its low molecular weight, transfer RNA could be easily removed by a quick precipitation in LiCl solution. Ribosomal RNA may be degraded enzymatically by 5'-end terminal exonuclease XRN-1. These steps allow enriching samples in mRNA during the first stages of RNA-Seq library preparation. The desired level of fragmentation of enriched mRNA necessary for the 2nd generation sequencing can be controlled by the duration of incubation at elevated temperatures in the presence of Mg2+-ions. Here, we describe a simple protocol for construction of the primary prokaryotic mRNA-saturated library without long depletion procedures. (C) 2020 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

Fatty acid desaturases (FADs) represent a class of oxygen-dependent enzymes that dehydrogenate C-C bonds in the fatty acids (FAs) producing unsaturated C = C double bonds that markedly change the properties of biological membranes. FADs are highly specific towards their acyl substrates, the position and configuration of the introduced double bonds. The double bond positioning of soluble acyl-carrier-protein Delta 9-FADs was determined relative to the carboxyl end of a FA. Similar mode was suggested for the acyl-lipid Delta 12-FADs (also known as omega 6-FADs), however, their exact counting order remain unknown. Here we used monounsaturated odd(17:1 Delta(10)) and even-chain (18:1 Delta(11)) FAs to show that acyl-lipid Delta 12-FADs of, at least, two cyanobacterial species, Gloeobacter violaceus and Synechocystis sp. strain PCC 6803, use neither end of the fatty acid (Delta or omega) as a counting reference point; but count three carbons toward the methyl end from an existing double bond in the monoene precursors irrespective of a FA chain length. (C) 2020 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

The problem of the survival of plants under low temperatures becomes more relevant in the light of global climate change and the growing needs of the population. In this regard, studies of the impact of hypothermia on plants are not only fundamental, but also applied. Potatoes are an important food crop, ranking fourth in the world in terms of growing. The actual yield of potatoes is significantly lower than its potential productivity, and one of the limiting factors is the lack of resistance of many modern varieties to spring frosts. Decoding of the potato genome made it possible to use advances in molecular biology to study the role of individual genes and identify key proteins that can increase resistance to low temperature. It is widely known that under the action of low temperatures, there is a phase transition of membrane lipids, which is accompanied by a decrease in membrane fluidity and loss of their barrier properties and, as a result, by inactivation of enzymes. In response to changes in physical properties of membranes, cells activate protection systems, among which an important role is played by the cold-induced increase in the degree of unsaturation of fatty acids of membrane lipids. Therefore, one of the main goals of adaptation is the stabilization of membranes, for example, due to the work of enzymes, fatty acid desaturase (encoded by genes FAD), catalyzing the conversion of saturated fatty acids (FA) into unsaturated. Among all plant cell membranes, chloroplast membranes play a special role in the formation of plant resistance to low temperatures, since it is in chloroplasts that photosynthesis, the main source of energy necessary for the restructuring of metabolism during the adaptation period, takes place. The aim of the research was to study the role of chloroplast localized Delta 9-, Delta 12 - and omega 3(Delta 15)-desaturases in adaptive transformations of the fatty acid composition of chloroplast membranes when forming potato plant cold resistance during hardening. The object of the study was potato plants (Solanum tuberosum L., cultivar Jubilee Zhukov), 3 weeks of age, grown in soil culture at a temperature of 22 degrees C, illumination of 100 mu mol/(m(2) c) and 16-h photoperiod. Hardening of plants was carried out in the climatic chamber KBW-240 "Binder" (Germany) under 16-h photoperiod and illumination of 100 mu mol/(m(2)c) at a temperature of 3 degrees C for 7 days. Controls were nonhardened plants. To assess the effectiveness of adaptation, whole plants were frozen at a temperature of 2 degrees C for 18 hours in the climatic chamber MIR-153 "Sanyo" (Japan), and then transferred to the growing conditions to determine survival. The following genes of FA desaturases were selected for the study: SAD (encodes one of the soluble Delta 9-ACP-), FAD6 (encodes membrane-bound acyl-lipid Delta 12-), FAD7 (encodes membrane- bound acyl-lipid Delta 15(omega 3)-desaturase). Protein products of these genes are localized in chloroplasts. Total RNA from leaves was isolated using Spectrum Plant Total RNA Kit "Sigma" (USA). The reverse transcription reaction was performed using a set of reagents and the Protocol MMLV RT Kit "Eurogen" (Russia). The resulting cDNA was used for real-time PCR (q-PCR) using the amplifier CFX96 Touch Real-Time PCR Detection System "Bio-Rad" (USA), using a set of reagents qPCRmix-HS SYBR kit "Eurogen" (Russia). The relative transcript content was calculated by calculating the normalized expression (Delta Delta C-T). Primers for the genes of FA desaturases were selected using the database NCBI and Internet resource Primer3Plus. Intact chloroplasts were isolated by centrifugation in a percol step gradient. Chloroplast lipids were methylated by boiling in a mixture of CH3OH and CH3COCl. The obtained LC methyl esters were analyzed by GL-MS using Agilent 7890A GC (USA). The experiments were conducted in 5-6 biological replicates and 3-4 analytical ones. Statistical data processing was performed using the program SigmaPlot 11. The data are presented as means and their standard errors. We showed that the hardened potato plants survived after -2 degrees C for 18 h, which indicates the successful hardening of S. tuberosum, Jubilee Zhukov cultivar (See Fig. 1). Among the studied genes Delta 9-, Delta 12- and omega 3-desaturases of chloroplasts, a short-term (after 2 h of adaptation) increase in the transcripts of the FAD6 gene encoding acyl-lipid Delta 12-desaturase was found. The relative content of FAD7 gene transcripts encoding omega 3-desaturase remained stable and maintained at the level of control. The character of SAD gene expression encoding Delta 9-ACP desaturase differed from the others: the relative transcript content decreased during adaptation (See Fig. 2). It should be noted that potatoes have 13 soluble Delta 9-ACP-desaturase genes forming the first double bond, whose proteins are localized in the stroma of chloroplasts. Perhaps, the studied gene is not cold-inducible. The total percentage of polyunsaturated fatty acids (PUFA) of lipids in chloroplasts of potato was high and constitutive in non-hardened plants accounted for almost 90% of the total content of all FA (See Table). Probably, therefore, in the process of adaptation there was no noticeable increase in the relative content of the transcripts of the studied genes Delta 12- and omega 3-desaturases of chloroplasts. During the period of low-temperature hardening, the part of PUFA, and especially alpha-linolenic acid, was maintained at a high level; there was an increase in the content of palmitic acid, which may indicate an increase in the intensity of synthesis of FA de novo. In addition, an increase in the content of C-16(:1) (Delta 7) acid was observed, which is also important for adaptation. It is known that the fluidity of membranes with decreasing temperature is determined not only by the content of FA with a larger number of double bonds, but also by the content of FA with a smaller number of carbon atoms. Maintaining a high amount of PUFA helped to maintain the thylakoid membranes of chloroplasts in a functional state during the hardening process, which, in turn, allowed the potato plants to realize other processes of adaptation.

A new presumably simple consortium of a Leptolyngbya sp. and a Porphyrobacter sp. was isolated from Tolbo Lake in Mongolia. The draft genome sequences of both species are reported.

Here, for the first time, we report the presence of highly active extracellular carbonic anhydrase (CA) of alpha-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 similar to 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 alpha- and beta-class, respectively, have been heterologously expressed in Escherichia coli. High specific activity (similar to 1.1 x 10(4) 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 alpha-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. (C) 2019 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

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.

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.

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.

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.

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.

The PepP protein has been purified in vitro and characterized for the first time. It is encoded by the sll0136 gene of the unicellular cyanobacterium Synechocystis sp. PCC6803. It is established that the PepP protein is a Mn2+-dependent Xaa-Pro-specific aminopeptidase. The protein in the reaction of hydrolysis of the fluorescent peptide Lys(N-Abz)-Pro-Pro-pNA has a maximal activity at pH 7.6 and 32°C.