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.

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.

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.

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).

'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.

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.

'Candidatus Oscillochloris fontis': a novel mesophilic phototrophic Chloroflexota bacterium belonging to the ubiquitous Oscillochloris genus.

We present the results of a study of mesophilic anoxygenic phototrophic Chloroflexota bacteria from Mechigmen hot spring (the Chukotka Peninsula) and Siberia. According to 16S rRNA phylogenetic analysis, these bacteria belong to Oscillochloris trichoides. However, sequencing the draft genome of the bacterium from the Chukotka and analysis of the average nucleotide identity, as well as in silico DNA-DNA hybridization, reveal that this bacterium belongs to a novel species within the Oscillochloris genus. We, therefore, propose 'Candidatus Oscillochloris fontis' as a novel taxon to represent this mesophilic alkaliphilic anaerobic anoxygenic phototrophic bacterium. Spectrophotometry and high-performance liquid chromatography analysis show that the bacterium possesses bacteriochlorophylls c and a, as well as lycopene, ß-carotene and γ-carotene. In addition, transmission electron microscopy shows the presence of chlorosomes, polyhydroxyalkanoate- and polyphosphate-like granules. The genome of 'Ca. Oscillochloris fontis' and the Siberian strains of Oscillochloris sp. possess the key genes for nitrogenase complex (nifH) and ribulose-1,5-bisphosphate carboxylase/oxygenase (cbbL), as previously described for O. trichoides DG-6. The results presented here, and previously published data, show that Oscillochloris bacteria from different aquatic environments have the potential for CO2 and N2 fixation. Additionally, we describe a new primer system for the detection of RuBisCo form I.

Draft Genome Sequences of a Putative Prokaryotic Consortium (IPPAS B-1204) Consisting of a Cyanobacterium (Leptolyngbya sp.) and an Alphaproteobacterium (Porphyrobacter sp.).

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. The consortium has been deposited in the Collection of Microalgae and Cyanobacteria of the Institute of Plant Physiology, Moscow, Russia, under the accession number IPPAS B-1204.

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.

Spectrophotometric Determination of Phycobiliprotein Content in Cyanobacterium Synechocystis.

This is a simple protocol for the quantitative determination of phycobiliprotein content in the model cyanobacterium Synechocystis. Phycobiliproteins are the most important components of phycobilisomes, the major light-harvesting antennae in cyanobacteria and several algae taxa. The phycobilisomes of Synechocystis contain two phycobiliproteins: phycocyanin and allophycocyanin. This protocol describes a simple, efficient, and reliable method for the quantitative determination of both phycocyanin and allophycocyanin in this model cyanobacterium. We compared several methods of phycobiliprotein extraction and spectrophotometric quantification. The extraction procedure as described in this protocol was also successfully applied to other cyanobacteria strains such as Cyanothece sp., Synechococcuselongatus, Spirulina sp., Arthrospira sp., and Nostoc sp., as well as to red algae Porphyridium cruentum. However, the extinction coefficients of specific phycobiliproteins from various taxa can differ and it is, therefore, recommended to validate the spectrophotometric quantification method for every single strain individually. The protocol requires little time and can be performed in any standard life science laboratory since it requires only standard equipment.

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.

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.

Determination of Storage (Starch/Glycogen) and Total Saccharides Content in Algae and Cyanobacteria by a Phenol-Sulfuric Acid Method.

This is a protocol for quantitative determination of storage and total carbohydrates in algae and cyanobacteria. The protocol is simple, fast and sensitive and it requires only few standard chemicals. Great advantage of this protocol is that both storage and total saccharides can be determined in the cellular pellets that were already used for chlorophyll and carotenoids quantification. Since it is recommended to perform the pigments measurement in triplicates, each pigment analysis can generate samples for both total saccharide and glycogen/starch content quantification. The protocol was applied for quantification of both storage and total carbohydrates in cyanobacteria Synechocystis sp. PCC 6803, Cyanothece sp. ATCC 51142 and Cyanobacterium sp. IPPAS B-1200. It was also applied for estimation of storage polysaccharides in Galdieria (IPPAS P-500, IPPAS P-507, IPPAS P-508, IPPAS P-513), Cyanidium caldarium IPPAS P-510, in green algae Chlorella sp. IPPAS C-1 and C-1210, Parachlorella kessleri IPPAS C-9, Nannochloris sp. C-1509, Coelastrella sp. IPPAS H-626, Haematococcus sp. IPPAS H-629 and H-239, and in Eustigmatos sp. IPPAS H-242 and IPPAS C-70.

Polyphasic characterization of the thermotolerant cyanobacterium Desertifilum sp. strain IPPAS B-1220.

A cyanobacterial strain from Lake Shar-Nuur, a freshwater lake in Mongolia, was isolated and characterized by a polyphasic approach. According to the 16S ribosomal RNA gene sequence, this strain (IPPAS B-1220) belongs to a newly described genus Desertifilum. In general, strains of Desertifilum maintain their genetic stability, as seen from the analysis of the 16S rRNA gene and 16S-23S rRNA internal transcribed spacer sequences from strains collected at distant locations. The newly discovered strain is characterized by an unusual fatty acid composition (16:1Δ7 and 16:2Δ7,10). Analysis of its draft genomic sequence reveals the presence of six genes for the acyl-lipid desaturases: two Δ9-desaturases, desC1 and desC2; two Δ12-desaturases, desA1 and desA2; one desaturase of unknown specificity, desX; and one gene for the bacillary-type desaturase, desG, which supposedly encodes an ω9-desaturase. A scheme for a fatty acid desaturation pathway that describes the biosynthesis of 16:1Δ7 and 16:2Δ7,10 fatty acids in Desertifilum is proposed.

Draft Genome Sequence of the Thermotolerant Cyanobacterium Desertifilum sp. IPPAS B-1220.

Here, we report the draft genome of the filamentous cyanobacterium Desertifilum sp. strain IPPAS B-1220, isolated from Lake Shar-Nuur, Mongolia. The genome of 6.1 Mb codes for 5,113 genes. Genome mining revealed 10 clusters for the synthesis of bioactive compounds (nonribosomal peptides, polyketides, bacteriocins, and lantipeptides) with potential biotechnological or medical importance.

Draft Genome Sequence of Cyanobacterium sp. Strain IPPAS B-1200 with a Unique Fatty Acid Composition.

Here, we report the draft genome of Cyanobacterium sp. IPPAS strain B-1200, isolated from Lake Balkhash, Kazakhstan, and characterized by the unique fatty acid composition of its membrane lipids, which are enriched with myristic and myristoleic acids. The approximate genome size is 3.4 Mb, and the predicted number of coding sequences is 3,119.

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.