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Histidine kinases are common sensory proteins used to detect environmental changes in bacteria. They respond to specific stimuli via a signal-input domain and alter gene expression through a cognate response regulator. The modulation/control of transcriptional regulation in cyanobacteria is important to reinforce the production of useful target compounds via photosynthesis without altering the growth profiles. For instance, heavy metal ions (Ni2+ and Cu2+), chemical inducers (IPTG), and a volatile compound (toluene) have been previously applied to regulate gene expression in cyanobacteria. However, most systems/regulators are only able to regulate gene expression once because it is impossible to eliminate them from the medium. To construct a reversible regulation system, a chimeric sensor, VanN_SphS, was developed by fusing the signal input domain of a quorum-sensing (QS) sensor, VanN, from Vibrio anguillarum, responding N-3-hydroxyhexanoyl-L-homoserine lactone (OHC6-HSL), with the kinase domain of SphS, a phosphate-deficiency sensor from the cyanobacterium Synechocystis sp. PCC 6803. After expression of the chimeric sensor in Synechocystis cells, responses to the various N-acyl-homoserine-lactones (AHLs) were evaluated by measuring the alkaline phosphatase (AP) activity, which is regulated by SphS. VanN_SphS responded only to OHC6-HSL and repressed AP activity. Then, the coexpression of the AHLs-degradation enzyme, Aii20J, a lactonase from Tenacibaculum sp. 20J, resumed the activity. This is the first report on the use of AHL-mediated transcriptional regulation in Synechocystis, which could be used in the future for the controlled production of useful compounds in the cyanobacterium.

Natural astaxanthin is known to be produced by green microalgae, a potent producer of the most powerful antioxidant. To increase the productivity of astaxanthin in microalgae, random mutagenesis has been extensively used to improve the yield of valuable substances. In the presented work, a newly isolated Coelastrum sp. was randomly mutagenized by exposure to ethyl methane sulfonate and further screened using two approaches; an approach for high growth mutant and an approach for high astaxanthin producing mutant with a high-throughput screening method using glufosinate. Among these, mutant G1-C1 that was selected using glufosinate showed the highest of total carotenoids (45.48±1.5 mg/L) and astaxanthin (28.32±2.5 mg/L) production, which was almost 2-fold higher than that of wild type. This study indicates that random mutagenesis via chemical mutation strategy and screening using glufosinate successfully expedited astaxanthin production in a mutated strain of a Coelastrum sp.

In the cultivation of microalgae for the production of useful compounds, cell disruption to extract the products of interest is a bottleneck process. To establish a cost-effective method to recover these cellular compounds, we developed a method to induce cell lysis via phosphate deficiency in the cyanobacterium Synechocystis sp. PCC 6803. In this system, the promoter from the phoA gene for alkaline phosphatase expressed bacteriophage genes encoding the lytic enzymes holin and endolysin, thus the cell lysis is induced under phosphate-deficient condition. We observed that 90% of the cells, introduced this bacteriophage genes, were lysed after 24 h of incubation under phosphate-deficient conditions. We also developed a method to induce cell lysis in highly concentrated cells for the efficient recovery of valuable cellular products and observed over 90% cell lysis after 16 h of incubation under these conditions. This inducible lysis system may contribute to decreased cell disruption costs in the algal biotechnology industry.

A newly isolated green microalga, Coelastrum sp. has the capability to produce and accumulate astaxanthin under various stress conditions. At present, a mutant G1-C1 of Coelastrum sp. obtained through chemical mutagenesis using ethyl methane sulfonate displayed an improvement in astaxanthin accumulation, which was 2-fold higher than that of the wild-type. However, lack of genomic information limits the understanding of the molecular mechanism that leads to a high level of astaxanthin in the mutant G1-C1. In this study, transcriptome sequencing was performed to compare the transcriptome of astaxanthin hyper-producing mutant G1-C1 and wild-type of Coelastrum sp. with respect to astaxanthin biosynthesis. This is to clarify why the mutant produced higher astaxanthin yield compared to the wild-type strain. Based on the transcriptomic analysis, the differentially expressed genes involved in astaxanthin biosynthesis were significantly upregulated in the mutant G1-C1 of Coelastrum sp. Genes coding phytoene synthase, phytoene desaturase, ζ-carotene desaturase, and lycopene β-cyclase involved in β-carotene biosynthesis in the mutant cells were upregulated by 10-, 9.2-, 8.4-, and 8.7-fold, respectively. Genes coding beta-carotene ketolase and beta-carotene 3-hydroxylase involved in converting β-carotene into astaxanthin were upregulated by 7.8- and 8.0-fold, respectively. In contrast, the lycopene ε-cyclase gene was downregulated by 9.7-fold in mutant G1-C1. Together, these results contribute to higher astaxanthin accumulation in mutant G1-C1. Overall, the data in this study provided molecular insight for a better understanding of the differences in astaxanthin biosynthesis between the wild-type and mutant G1-C1 strains.

Alteration of fatty-acid unsaturation is a universal response to temperature changes. Marine microalgae display the largest diversity of polyunsaturated fatty-acid (PUFA) whose content notably varies according to temperature. The physiological relevance and the molecular mechanisms underlying these changes are however, still poorly understood. The ancestral green picoalga Ostreococcus tauri displays original lipidic features that combines PUFAs from two distinctive microalgal lineages (Chlorophyceae, Chromista kingdom). In this study, optimized conditions were implemented to unveil early fatty-acid and desaturase transcriptional variations upon chilling and warming. We further functionally characterized the O. tauri ω3-desaturase which is closely related to ω3-desaturases from Chromista species. Our results show that the overall omega-3 to omega-6 ratio is swiftly and reversibly regulated by temperature variations. The proportion of the peculiar 18:5 fatty-acid and temperature are highly and inversely correlated pinpointing the importance of 18:5 temperature-dependent variations across kingdoms. Chilling rapidly and sustainably up-regulated most desaturase genes. Desaturases involved in the regulation of the C18-PUFA pool as well as the Δ5-desaturase appear to be major transcriptional targets. The only ω3-desaturase candidate, related to ω3-desaturases from Chromista species, is localized at chloroplasts in Nicotiana benthamiana and efficiently performs ω3-desaturation of C18-PUFAs in Synechocystis sp. PCC6803. Overexpression in the native host further unveils a broad impact on plastidial and non-plastidial glycerolipids illustrated by the alteration of omega-3/omega-6 ratio in C16-PUFA and VLC-PUFA pools. Global glycerolipid features of the overexpressor recall those of chilling acclimated cells.

Chionaster nivalis is frequently detected in thawing snowpacks and glaciers. However, the taxonomic position of this species above the genus level remains unclear. We herein conducted molecular analyses of C. nivalis using the ribosomal RNA operon sequences obtained from more than 200 cells of this species isolated from a field-collected material. Our molecular phylogenetic analyses revealed that C. nivalis is a sister to Bartheletia paradoxa, which is an orphan basal lineage of Agaricomycotina. We also showed that C. nivalis sequences were contained in several previously examined meta-amplicon sequence datasets from snowpacks and glaciers in the Northern Hemisphere and Antarctica.

Biomineralization by calcifying microalgae is a precisely controlled intracellular calcification process that produces delicate calcite scales (or coccoliths) in the coccolithophore Emiliania huxleyi (Haptophycea). Despite its importance in biogeochemical cycles and the marine environment globally, the underlying molecular mechanism of intracellular coccolith formation, which requires calcium, bicarbonate, and coccolith-polysaccharides, remains unclear. In E. huxleyi CCMP 371, we demonstrated that reducing the calcium concentration from 10 (ambient seawater) to 0.1 mM strongly restricted coccolith production, which was then recovered by adding 10 mM calcium, irrespective of inorganic phosphate conditions, indicating that coccolith production could be finely controlled by the calcium supply. Using this strain, we investigated the expression of differentially expressed genes (DEGs) to observe the cellular events induced by changes in calcium concentrations. Intriguingly, DEG analysis revealed that the phosphatidylinositol-specific phospholipase C (PI-PLC) gene was upregulated and coccolith production by cells was blocked by the PI-PLC inhibitor U73122 under conditions closely associated with calcium-induced calcification. These findings imply that PI-PLC plays an important role in the biomineralization process of the coccolithophore E. huxleyi.

Lipid droplets are ubiquitous cellular compartments that store neutral lipids and specific proteins localize on their surface. These proteins work as a scaffold in maintaining the lipid droplet structure or as regulators of lipogenesis or lipolysis. Previously, the most abundant lipid droplet protein, namely stramenopile-type lipid droplet protein (StLDP), was identified in the marine diatom Phaeodactylum tricornutum; however, its function remains unclear because StLDP does not reveal homology with known lipid droplet proteins and lacks a predictable domain. In this study, P. tricornutum was transformed to express a homologous StLDP gene under an fcpA promoter in order to determine its function. StLDP expression was strongly enhanced in the mutant (H8), especially in nitrogen-sufficient conditions; however, it was attenuated in nitrogen-deficient conditions. Despite the strong expression, no significant difference was observed in the lipid composition between the wild type (WT) and H8 under nitrogen-sufficient conditions. After cultivation in nitrogen-free medium for 6 days, neutral lipid content significantly increased in H8 than in WT. After 2 days of cultivation in nitrogen-free medium, 97.0% of single cells in WT formed one or two lipid droplets, whereas in H8, this proportion decreased to 78.8%, and the proportion of cells forming three or four lipid droplets increased. Thus, the function of StLDP was speculated to sequester triacylglycerol on the initial lipid droplet formation.

Recently, microalgae have attracted attention as sources of biomass energy. However, fatty acids from the microalgae are mainly unsaturated and show low stability in oxygenated environments, due to oxidation of the double bonds. The branched-chain fatty acid, 10-methyl stearic acid, is synthesized from oleic acid in certain bacteria; the fatty acid is saturated, but melting point is low. Thus, it is stable in the presence of oxygen and is highly fluid. We previously demonstrated that BfaA and BfaB in Mycobacterium chlorophenolicum are involved in the synthesis of 10-methyl stearic acid from oleic acid. In this study, as a consequence of the introduction of bfaA and bfaB into the cyanobacterium, Synechocystis sp. PCC 6803, we succeeded in producing 10-methyl stearic acid, with yields up to 4.1% of the total fatty acid content. The synthesis of 10-methyl stearic acid in Synechocystis cells did not show a significant effect on photosynthetic activity, but the growth of the cells was retarded at 34 °C. We observed that the synthesis of 10-methylene stearic acid, a precursor of 10-methyl stearic acid, had an inhibitory effect on the growth of the transformants, which was mitigated under microoxic conditions. Eventually, the amount of 10-methyl stearic acid present in the sulfoquinovosyl diacylglycerol and phosphatidylglycerol of the transformants was remarkably higher than that in the monogalactosyldiacylglycerol and digalactosyldiacylglycerol. Overall, we successfully synthesized 10-methyl stearic acid in the phototroph, Synechocystis, demonstrating that it is possible to synthesize unique modified fatty acids via photosynthesis that are not naturally produced in photosynthetic organisms.

The cosmopolitan marine haptophyte alga Emiliania huxleyi accumulates very long-chain (C37-C40) alkyl ketones with two to four trans-type carbon-carbon double bonds (alkenones). These compounds are used as biomarkers of haptophytes and as palaeothermometers for estimating sea-surface temperatures in biogeochemistry. However, the biosynthetic pathway of alkenones in algal cells remains enigmatic, although it is well known that the C37 tri-unsaturated alkenone (K37:3) becomes dominant at low temperatures, either by desaturation of K37:2 or by a separate pathway involving the elongation of tri-unsaturated alkenone precursors. Here, we present experimental evidence regarding K37:3 synthesis. Using the well-known cosmopolitan alkenone producer E. huxleyi, we labelled K37:2 with 13C by incubating cells with 13C-bicarbonate in the light at 25 °C under conditions of little if any K37:3 production. After stabilisation of the 13C-K37:2 level by depleting 13C-bicarbonate from the medium, the temperature was suddenly reduced to 15 °C. The 13C-K37:2 level rapidly decreased, and the 13C-K37:3 level increased, whereas the total 13C-K37 level—namely [K37:2 + K37:3]—remained constant. These 13C-pulse-chase-like experimental results indicate that 13C-K37:2 is converted directly to 13C-K37:3 by a desaturation reaction that is promoted by a cold signal. This clear-cut experimental evidence is indicative of the existence of a cold-signal-triggered desaturation reaction in alkenone biosynthesis.

Transport systems, which perform a pivotal function in regulating the import and export of various substances via cellular membranes, are important machinery in all living organisms. The ATP-binding cassette (ABC) transporter, one of such transporters, has a crucial role in the high-affinity uptake of various nutrients in bacteria. In the cyanobacterium Synechocystis sp. PCC 6803, a model cyanobacterium, its response to phosphate (Pi)-depleted conditions and the consequential regulation of the Pi transport system have been well-studied. However, its responses and the Pi uptake under Pi-repleted conditions are not comprehended yet. This study observed the peculiar stimulation of the Pi uptake following severe chlorosis in the Synechocystis cells grown under Pi-repleted conditions. The Synechocystis cells grown under high Pi conditions (approximately 4 mM) showed the rapid Pi uptake after 3 d of cultivation, and significantly severe degradation of chlorophyll was observed. Remarkably, the Synechocystis cells showed more than 16-fold higher Pi uptake at that time. Furthermore, we revealed that sulfate (S) deficiency-induced Pi uptake and that magnesium deficiency caused chlorosis and released the intracellularly accumulated Pi. Moreover, reverse genetics revealed that the rapid Pi uptake occurred via an ABC transporter induced by S deficiency, namely, a putative sulfate transporter, SbpA-CysTWA. Our findings provide a new perspective on the Pi uptake under high Pi conditions in cyanobacteria and bacteria.

Microalgae have been used to produce useful compounds. In many cases in which target compounds are produced from CO2 via photosynthesis, there is a trade-off between the proliferation of cells and the production of the target compounds. This is due to carbon partitioning between the synthesis of cellular components and the target compounds, and/or the toxicity of the target products. Therefore, there is a requirement to develop a gene switch that can modulate the metabolism from the growth phase of cells to the production phase of the target molecules. In this study, we attempted to develop an artificial sensory protein that can respond to insoluble gaseous compounds and regulate gene expression in the model cyanobacterium Synechocystis sp. PCC 6803. We constructed a chimeric histidine kinase by fusing the signal input domain of TodS, a toluene sensor from Pseudomonas putida, and the kinase domain of SphS, a phosphate-deficient sensor from Synechocystis. Because TodS contains two kinase domains, we substituted either of the kinase domains of TodS with the kinase domain of SphS to obtain TodSS_SphS and TodSL_SphS. We introduced a gene for the chimeric sensor to Synechocystis cells and evaluated their response to toluene by measuring alkaline phosphatase (AP) activity and phoA mRNA expression. Although the cells harboring TodSL_SphS did not exhibit AP activity, both in the presence or absence of toluene, the cells harboring TodSS_SphS accumulated the transcript of the phoA gene after 5-h exposure to toluene, and only exhibited AP activity after exposure to toluene.

Alkenones are long-chain methyl/ethyl ketones (mainly in length of C37-C39) with two to four trans-unsaturated bonds produced by several kinds of marine haptophytes such as Emiliania huxleyi (coccolithophore). The physiological functions and metabolic profile of alkenones are not well known yet. In this study, we focused on elucidating how alkenones contribute to energy storage and cellular carbon partitioning in relation to other cellular components. For the purpose, we analyzed the changes in carbon allocation among various cell components like lipids, alkenones, proteins, and polysaccharides between cells exposed to N-sufficient (+N) and N-limited conditions (−N) in E. huxleyi CCMP 2090. Finally, the alkenones were found to function as main storage lipids and their accumulation was clearly increased by −N, whereas triacylglycerols (TAGs) were barely detected under any N conditions. The mobilization of carbons into alkenones was stimulated by −N from 15% under +N to 27% under −N. However, photosynthetic C allocation into other components was suppressed by −N, showing that percent C allocation into fatty acids, proteins, and polysaccharides was decreased from 9, 46, and 6.8% under +N to 7, 25, and 4.5% under −N, respectively. In addition, fatty acids such as 16:0, 18:0, 18:1, and 18:2 became dominant under −N while 18:5 became dominant under +N conditions, with no significant change in 22:6. This study revealed that alkenones function as primary carbon storage pools especially under −N condition in E. huxleyi CCMP 2090 and that N supply triggers a dynamic change in carbon metabolism by modifying membrane lipid composition and regulating carbon allocation preferences.

Eukaryotic Δ6-desaturases are microsomal enzymes which balance the synthesis of ω-3 and ω-6 C18-polyunsaturated-fatty-acids (PUFA) accordingly to their specificity. In several microalgae, includingO. tauri, plastidic C18-PUFA are specifically regulated by environmental cues suggesting an autonomous control of Δ6-desaturation of plastidic PUFA. Sequence retrieval fromO. tauridesaturases, highlighted two putative Δ6/Δ8-desaturases sequences clustering, with other microalgal homologs, apart from other characterized Δ-6 desaturases. Their overexpression in heterologous hosts, includingN. benthamianaandSynechocystis, unveiled their Δ6-desaturation activity and plastid localization.O. taurilines overexpressing these Δ6-desaturases no longer adjusted their plastidic C18-PUFA amount under phosphate starvation but didn’t show any obvious physiological alterations. Detailed lipid analyses from the various overexpressing hosts, unravelled that the substrate features involved in the Δ6-desaturase specificity importantly involved the lipid head-group and likely the non-substrate acyl-chain, in addition to the overall preference for the ω-class of the substrate acyl-chain. The most active desaturase displayed a broad range substrate specificity for plastidic lipids and a preference for ω-3 substrates, while the other was selective for ω-6 substrates, phosphatidylglycerol and 16:4-galactolipid species specific to the native host. The distribution of plastidial Δ6-desaturase products in eukaryotic hosts suggested the occurrence of C18-PUFA export from the plastid.One sentence summaryOsteococcus tauri plastidic lipid C18-PUFA remodelling involves two plastid-located cytochrome-b5 fused Δ6-desaturases with distinct preferences for both head-group and acyl-chain.

International audience; Eukaryotic Δ6-desaturases are microsomal enzymes that balance the synthesis of ω-3 and ω-6 C18-polyunsaturated fatty acids (C18-PUFAs) according to their specificity. In several microalgae, including Ostreococcus tauri, plastidic C18-PUFAs are strictly regulated by environmental cues suggesting an autonomous control of Δ6-desaturation of plastidic PUFAs. Here, we identified two putative front-end Δ6/Δ8-desaturases from Otauri that, together with putative homologs, cluster apart from other characterized Δ6-desaturases. Both were plastid-located and unambiguously displayed a Δ6-desaturation activity when overexpressed in the heterologous hosts Nicotiana benthamiana and Synechocystis sp. PCC6803, as in the native host. Detailed lipid analyses of overexpressing lines unveiled distinctive ω-class specificities, and most interestingly pointed to the importance of the lipid head-group and the nonsubstrate acyl-chain for the desaturase efficiency. One desaturase displayed a broad specificity for plastidic lipids and a preference for ω-3 substrates, while the other was more selective for ω-6 substrates and for lipid classes including phosphatidylglycerol as well as the peculiar 16:4-galactolipid species occurring in the native host. Overexpression of both Δ6-desaturases in Otauri prevented the regulation of C18-PUFA under phosphate deprivation and triggered glycerolipid fatty-acid remodeling, without causing any obvious alteration in growth or photosynthesis. Tracking fatty-acid modifications in eukaryotic hosts further suggested the export of plastidic lipids to extraplastidic compartments.

Microalgae have received much attention as a next-generation source of biomass energy. However, most of the fatty acids (FAs) from microalgae are multiply unsaturated; thus, the biofuels derived from them are fluid, but vulnerable to oxidation. In this study, we attempted to synthesize cyclopropane FAs in the cyanobacterium Synechocystis sp. PCC 6803 by expressing the cfa gene for cyclopropane FA synthase from Escherichia coli with the aim of producing FAs that are fluid and stable in response to oxidization. We successfully synthesized cyclopropane FAs in Synechocystis with a yield of ~30% of total FAs. Growth of the transformants was altered, particularly at low temperatures, but photosynthesis and respiration were not significantly affected. C16:1(∆9) synthesis in the desA(-)/desD(-) strain by expression of the desC2 gene for sn-2 specific ∆9 desaturase positively affected growth at low temperatures via promotion of various cellular processes, with the exceptions of photosynthesis and respiration. Estimation of the apparent activities of desaturases suggested that some acyl-lipid desaturases might recognize the lipid side chain.

Strains of the coccolithophore Emiliania huxleyi (Haptophyta) collected from the subarctic North Pacific and Arctic oceans in 2010 were established as clone cultures and have been maintained in the laboratory at 15 °C and 32 ‰ salinity. To study the physiological responses of coccolith formation to changes in temperature and salinity, growth experiments and morphometric investigations were performed on two strains, namely MR57N isolated from the northern Bering Sea and MR70N at the Chukchi Sea. This is the first report of a detailed morphometric and morphological investigation of Arctic Ocean coccolithophore strains. The specific growth rates at the logarithmic growth phases in both strains markedly increased as temperature was elevated from 5 to 20 °C, although coccolith productivity (estimated as the percentage of calcified cells) was similar at 10–20 % at all temperatures. On the other hand, the specific growth rate of MR70N was affected less by changes in salinity in the range 26–35 ‰, but the proportion of calcified cells decreased at high and low salinities. According to scanning electron microscopy (SEM) observations, coccolith morphotypes can be categorized into Type B/C on the basis of their biometrical parameters. The central area elements of coccoliths varied from thin lath type to well-calcified lath type when temperature was increased or salinity was decreased, and coccolith size decreased simultaneously. Coccolithophore cell size also decreased with increasing temperature, although the variation in cell size was slightly greater at the lower salinity level. This indicates that subarctic and arctic coccolithophore strains can survive in a wide range of seawater temperatures and at lower salinities with change in their morphology. Because all coccolith biometric parameters followed the scaling law, the decrease in coccolith size was caused simply by the reduced calcification. Taken together, our results suggest that calcification productivity may be used to predict future oceanic environmental conditions in the polar regions.

Microalgal lipids and fatty acids are important components for achieving biofuel because of their potential high productivity. Although fatty acids that have a hydroxy group adjacent to the end of the acyl chain might be an important chemical feedstock, most algae do not accumulate it. To produce (ω−1)-hydroxy fatty acids from 3-hydroxybutylyl-CoA, an intermediate for polyhydroxybutylate biosynthesis in the cyanobacterium Synechocystis sp. PCC 6803, we expressed a gene for the promiscuous 3-ketoacyl-ACP synthase III from Alicyclobacillus acidocalderius (aaKASIII) by the cpc560 promoter. To supply 3-hydroxybutyryl-CoAs for aaKASIII, the phaC gene for polyhydroxybutylate polymerase was deleted, and the phaAB genes for 3-hydroxybutyryl-CoA synthesis from Cupriavidus necator were overexpressed. The genetically modified strain synthesized 15-hydroxyhexadecanoic acid, 17-hydroxyoctadecanoic acid, and 17-hydroxyoctadec-9-enoic acid, and accumulated approximately 2.1 mol% of (ω−1)-hydroxy fatty acids in total fatty acids under illumination with 70 μmol photons m−2 s−1, although its growth was severely retarded. Under weak light (35 μmol photons m−2 s−1) conditions, the strain grew as well as the wild-type and showed lower hydroxy fatty acids (0.04 mol%) accumulation than that at higher illumination levels. The photosynthetic activity of this strain was lower than that of wild-type cells, suggesting that high light conditions enhanced hydroxy fatty acids production and inhibited photosynthesis. (ω−1)-Hydroxy fatty acids were not predominantly observed in the galactolipids from thin-layer chromatography, which are the major lipid classes in cyanobacteria. To the best of our knowledge, this is the first report on photoautotrophic production of fatty acids possessing a functional group near the end of the acyl chain in cyanobacteria.

Microalgae are known to be a potential resource of high-value metabolites that can be used in the growing field of biotechnology. These metabolites constitute valuable compounds with a wide range of applications that strongly enhance a bio-based economy. Among these metabolites, astaxanthin is considered the most important secondary metabolite, having superior antioxidant properties. For commercial feasibility, microalgae with enhanced astaxanthin production need to be developed. In this study, the tropical green microalgae strain, Coelastrum sp., isolated from the environment in Malaysia, was incubated with methyl viologen, a reactive oxygen species (ROS) reagent that generates superoxide anion radicals (O2-) as an enhancer to improve the accumulation of astaxanthin. The effect of different concentrations of methyl viologen on astaxanthin accumulation was investigated. The results suggested that the supplementation of methyl viologen at low concentration (0.001 mM) was successfully used as a ROS reagent in facilitating and thereby increasing the production of astaxanthin in Coelastrum sp. at a rate 1.3 times higher than in the control.

Alkenone-producing species have been recently found in diverse lacustrine environments, albeit with taxonomic information derived indirectly from environmental genomic techniques. In this study, we isolated alkenone-producing algal species from Canadian saline lakes and established unialgal cultures of individual strains to identify their taxonomical and molecular biological characteristics. Water and sediments collected from the lakes were first enriched in artificial seawater medium over a range of salinities (5–40 g/L) to cultivate taxa in vitro. Unialgal cultures of seven haptophyte strains were isolated and categorized in the Isochrysis clade using SSU and LSU rRNA gene analysis. The alkenone distributions within isolated strains were determined to be novel compared with other previously reported alkenone-producing haptophytes. While all strains produced the typical C37 and C38 range of isomers, one strain isolated from Canadian salt lakes also produced novel C41 and C42 alkenones that are temperature sensitive. In addition, we showed that all alkenone unsaturation indices (e.g., U37 K and U37 K′) are temperature-dependent in culture experiments, and that alkenoate indices (e.g., U37 A, U38 A, RIA38 and A37/A38) provide alternative options for temperature calibration based on these new lacustrine algal strains. Importantly, these indices show temperature dependence in culture experiments at temperatures below 10 °C, where traditional alkenone proxies were not as sensitive. We hypothesize that this suite of calibrations may be used for reconstructions of past water temperature in a broad range of lakes in the Canadian prairies.

Alkenones are unusual long-chain neutral lipids that were first identified in oceanic sediments. Currently they are regarded as reliable palaeothermometers, since their unsaturation status changes depending on temperature. These molecules are synthesised by specific haptophyte algae and are stored in the lipid body as the main energy storage molecules. However, the molecular mechanisms that regulate the alkenone biosynthetic pathway, especially the low temperature-dependent desaturation reaction, have not been elucidated. Here, using an alkenone-producing haptophyte alga, Tisochrysis lutea, we show that the alkenone desaturation reaction is catalysed by a newly identified desaturase. We first isolated two candidate desaturase genes and found that one of these genes was drastically upregulated in response to cold stress. Gas chromatographic analysis revealed that the overexpression of this gene, named as Akd1 finally, increased the conversion of di-unsaturated C37-alkenone to tri-unsaturated molecule by alkenone desaturation, even at a high temperature when endogenous desaturation is efficiently suppressed. We anticipate that the Akd1 gene will be of great help for elucidating more detailed mechanisms of temperature response of alkenone desaturation, and identification of active species contributing alkenone production in metagenomic and/or metatranscriptomic studies in the field of oceanic biogeochemistry.

Various kinds of organisms, including microalgae, accumulate neutral lipids in distinct intracellular compartments called lipid droplets. Generally, lipid droplets are generated from the endoplasmic reticulum, and particular proteins localize on their surface. Some of these proteins function as structural proteins to prevent fusion between the lipid droplets, and the others could have an enzymatic role or might be involved in intracellular membrane trafficking. However, information about lipid droplet proteins in microalgae is scarce as compared with that in animals and land plants. We focused on the oil-producing, marine, pennate diatom Phaeodactylum tricornutum that forms lipid droplets during nitrogen deprivation and we investigated the proteins located on the lipid droplets. After 6 d of cultivation in a nitrate-deficient medium, the mature lipid droplets were isolated by sucrose density gradient centrifugation. Proteomic analyses revealed five proteins, with Stramenopile-type lipid droplet protein (StLDP) being the most abundant protein in the lipid droplet fraction. Although the primary sequence of StLDP did not have homology to any known lipid droplet proteins, StLDP had a central hydrophobic domain. This structural feature is also detected in oleosin of land plants and in lipid droplet surface protein (LDSP) of Nannochloropsis species. As a proline knot motif of oleosin, conservative proline residues existed in the hydrophobic domain. StLDP was up-regulated during nitrate deprivation, and fluctuations of StLDP expression levels corresponded to the size of the lipid droplets.

Cyanobacterium is an attractive host for the production of various chemicals and alternative fuels using solar energy and carbon dioxide. In previous study, we succeeded to produce isopropanol using engineered Synechococcus elongatus PCC 7942 under dark and anaerobic conditions (0.43 mM, 26.5 mg/l). In the present study, we report the further optimization of this isopropanol producing condition. We then optimized growth conditions for production of isopropanol by the engineered cyanobacteria, including the use of cells in early stationary phase and buffering of the production medium to neutral pH. We observed that shifting of cultures from dark and anaerobic to light and aerobic conditions during the production phase dramatically increased isopropanol production by conversion to isopropanol from acetate, byproduct under dark and anaerobic condition. Under the optimized production conditions, the titer of isopropanol was elevated 6-fold, to 2.42 mM (146 mg/l).

The bloom-forming coccolithophore Emiliania huxleyi (Haptophyta) is a dominant marine phytoplankton, cells of which are covered with calcareous plates (coccoliths). E. huxleyi produces unique lipids of C37–C40 long-chain ketones (alkenones) with two to four trans-unsaturated bonds, β-glucan (but not α-glucan) and acid polysaccharide (AP) associated with the morphogenesis of CaCO3 crystals in coccoliths. Despite such unique features, there is no detailed information on the patterns of carbon allocation into these compounds. Therefore, we performed quantitative estimation of carbon flow into various macromolecular products by conducting 14C-radiotracer experiments using NaH14CO3 as a substrate. Photosynthetic 14C incorporation into low molecular-mass compounds (LMC), extracellular AP, alkenones, and total lipids except alkenones was estimated to be 35, 13, 17, and 25 % of total 14C fixation in logarithmic growth phase cells and 33, 19, 18, and 18 % in stationary growth phase cells, respectively. However, less than 1 % of 14C was incorporated into β-glucan in both cells. 14C-mannitol occupied ca. 5 % of total fixed 14C as the most dominant LMC product. Levels of all 14C compounds decreased in the dark. Therefore, alkenones and LMC (including mannitol), but not β-glucan, function in carbon/energy storage in E. huxleyi, irrespective of the growth phase. Compared with other algae, the low carbon flux into β-glucan is a unique feature of carbon metabolism in E. huxelyi. Electronic supplementary material The online version of this article (doi:10.1007/s10126-015-9632-1) contains supplementary material, which is available to authorized users.

The hydrocarbons in cultures of marine haptophytes Emiliania huxleyi NIES837 and Gephyrocapsa oceanica NIES1315 were analyzed, and nonacosadienes and hentriacontadienes were detected as the major compounds in both strains. C29 and C31 monoenes and di-, tri- and tetra-unsaturated C33 alkenes were also detected as minor compounds but not C37 and C38 alkenes. The positions of the double bonds in the C29 and C31 alkenes were determined by mass spectrometry of their dimethyl disulfide (DMDS) adducts. Among the four C29 alkenes identified, the most abundant isomer was 2,20-nonacosadiene, and the other three compounds were 1,20-nonacosadiene, 3,20-nonacosadiene and 9-nonacosene, respectively. Hitherto, 2,20-nonacosadiene and 3,20-nonacosadiene were unknown to be natural products. The double bond at the n-9 (ω9) position in these C29 alkenes is hypothesized to be derived from precursors of unsaturated fatty acids possessing an n-9 double bond, such as (9Z)-9-octadecenoic acid. Nonacosadienes have the potential for being used as distinct haptophyte biomarkers.

Photoautotrophic bacteria have developed mechanisms to maintain K + homeostasis under conditions of changing ionic concentrations in the environment. Synechocystis sp. strain PCC 6803 contains genes encoding a well-characterized Ktr-type K + uptake transporter (Ktr) and a putative ATP-dependent transporter specific for K + (Kdp). The contributions of each of these K + transport systems to cellular K + homeostasis have not yet been defined conclusively. To verify the functionality of Kdp, kdp genes were expressed in Escherichia coli , where Kdp conferred K + uptake, albeit with lower rates than were conferred by Ktr. An on-chip microfluidic device enabled monitoring of the biphasic initial volume recovery of single Synechocystis cells after hyperosmotic shock. Here, Ktr functioned as the primary K + uptake system during the first recovery phase, whereas Kdp did not contribute significantly. The expression of the kdp operon in Synechocystis was induced by extracellular K + depletion. Correspondingly, Kdp-mediated K + uptake supported Synechocystis cell growth with trace amounts of external potassium. This induction of kdp expression depended on two adjacent genes, hik20 and rre19 , encoding a putative two-component system. The circadian expression of kdp and ktr peaked at subjective dawn, which may support the acquisition of K + required for the regular diurnal photosynthetic metabolism. These results indicate that Kdp contributes to the maintenance of a basal intracellular K + concentration under conditions of limited K + in natural environments, whereas Ktr mediates fast potassium movements in the presence of greater K + availability. Through their distinct activities, both Ktr and Kdp coordinate the responses of Synechocystis to changes in K + levels under fluctuating environmental conditions.

Cyanobacteria possess circadian clocks consisting of KaiABC proteins, and circadian rhythm must closely relate to the primary metabolism. A histidine kinase, SasA, interacts with KaiC to transduce circadian signals and widely regulates transcription in Synechococcus sp. PCC 7942, although the involvement of SasA in primary metabolism has not been demonstrated at metabolite levels. Here, we generated a strain overexpressing hik8 (HOX80), an orthologue of SasA in Synechocystis sp. PCC 6803. HOX80 grew slowly under light conditions and lost viability under continuous dark conditions. Transcript levels of genes related to sugar catabolism remained higher in HOX80 under dark conditions. Metabolomic analysis revealed that under light conditions, glycogen was undetectable in HOX80, and there were decreased levels of metabolites of sugar catabolism and increased levels of amino acids, compared with those in the wild-type strain. HOX80 exhibited aberrant degradation of SigE proteins after a light-to-dark transition and immunoprecipitation analysis revealed that Hik8 directly interacts with KaiC1. The results of this study demonstrate that overexpression of hik8 widely alters sugar and amino acid metabolism, revealing the involvement of Hik8 in primary metabolism under both light and dark conditions in this cyanobacterium.

The coccolithophorid Emiliania huxleyi is a bloom-forming marine phytoplankton thought to play a key role as a biological pump that transfers carbon from the surface to the bottom of the ocean, thus contributing to the global carbon cycle. This alga is also known to accumulate a variety of polyunsaturated fatty acids. At 25 °C, E. huxleyi produces mainly 14:0, 18:4n − 3, 18:5n − 3 and 22:6n − 3. When the cells were transferred from 25 °C to 15 °C, the amount of unsaturated fatty acids, i.e. 18:1n − 9, 18:3n − 3 and 18:5n − 3, gradually increased. Among the predicted desaturase genes whose expression levels were up-regulated at low temperature, we identified a gene encoding novel ∆15 fatty acid desaturase, EhDES15, involved in the production of n − 3 polyunsaturated fatty acids in E. huxleyi. This desaturase contains a putative transit sequence for localization in chloroplasts and a ∆6 desaturase-like domain, but it does not contain a cytochrome b5 domain nor typical His-boxes found in ∆15 desaturases. Heterologous expression of EhDES15 cDNA in cyanobacterium Synechocystis sp. PCC 6803 cells increased the level of n − 3 fatty acid species, which are produced at low levels in wild-type cells grown at 30 °C. The orthologous genes are only conserved in the genomes of prasinophytes and cryptophytes. The His-boxes conserved in orthologues varied from that of the canonical ∆15 desaturases. These results suggested the gene encodes a novel ∆15 desaturase responsible for the synthesis of 18:3n − 3 from 18:2n − 6 in E. huxleyi.

Thraustochytrids are osmoheterotrophic protists that are emerging as a promising group of oleaginous microorganisms due to their high DHA, EPA and palmitic acid production. Squalene, a triterpene hydrocarbon known for its antioxidant, antitumorigenic and emollient properties, is accumulated in some strains of thraustochytrids especially in the genus Aurantiochytrium. Thraustochytrids can produce extracellular enzymes to degrade organic matter as their means of nutrition. Many studies attempt to grow thraustochytrids in cost-effective substrates such as lignocellulosic biomass. As the first step towards cellulose utilization, we genetically modified Aurantiochytrium sp. strain 18W-13a to express and secrete a highly active cellulase, β-glucosidase from Aspergillus aculeatus (AaBgl). The transformant strain, AaBgl+, exhibited increasing growth and enzyme activity under cellobiose as the sole carbon source. Native PAGE zymogram showed an active β-glucosidase enzyme in the supernatant of the AaBgl+ strain. Both wild-type and AaBgl+ strains can produce squalene and fatty acids DHA and DPA. This work is one of the first reports in targeted secretion of a functional enzyme in thraustochytrids. Moreover, enhanced expression of cellulases has never been done in thraustochytrids. We hope that this study could pave way for establishing consolidated bioprocessing thraustochytrid strains.

The compositions of long chain alkenes, alkenones and alkenoates in a cultured strain of the haptophyte Chrysotila lamellosa CCMP1307, isolated from a salt marsh, were investigated. The biomarker patterns were distinctive and showed a high proportion of tetraunsaturated alkenones and alkenoates, with a pronounced proportion of C 40 alkenones and a lack of C 38 methyl and C 39 ethyl alkenones. Linear regression of the alkenone unsaturation degree ( U 37 K ) with growth temperature ( T ) was obtained over the range of possible CCMP1307 growth temperature values (4–20 °C): U 37 K = 0.045 × T (°C) − 1.016, ( n = 13, r 2 0.96), while the U 37 K ′ values were weakly correlated with T : U 37 K ′ = 0.0035 T (°C) + 0.0511 ( n = 18, r 2 0.70). The U 37 K calibration exhibited a low y-intercept in comparison with that of a Chinese inland lake strain reported previously. The data show significant intraspecific variation in U 37 K for C. lamellosa between strains from different geographic origins. C. lamellosa CCMP1307 reproduced typical U 37 K values observed in C 37:4 rich lakes, especially at lower temperature ( 29:2 , C 31:1 , C 31:2 and C 31:3 alkenes, and found that the unsaturation degree of C 31 alkenes ( U 31 en ), calculated with C 31:1 and C 31:2 alkenes, might also be a useful index of growth temperature for the haptophyte C. lamellosa .

A conserved hypothetical protein, Sll1130, is a novel transcription factor that regulates the expression of major heat-responsive genes in Synechocystis sp. PCC6803. Synechocystis exhibited an increased thermotolerance due to disruption of sll1130. Δsll1130 cells recovered much faster than wild-type cells after they were subjected to heat shock (50°C) for 30 min followed by recovery at 34°C for 48 h. In Δsll1130 cultures, 70% of the cells were viable compared with the wild-type culture in which only 30% of the cells were viable. DNA microarray analysis revealed that in Δsll1130, expression of the heat-responsive genes such as htpG, hspA, isiA, isiB and several hypothetical genes were up-regulated. Sll1130 binds to a conserved inverted-repeat (GGCGATCGCC) located in the upstream region of the above genes. In addition, both the transcript and protein levels of sll1130 were immediately down-regulated upon shift of wild-type cells from 34 to 42°C. Collectively the results of the present study suggest that Sll1130 is a heat-responsive transcriptional regulator that represses the expression of certain heat-inducible genes at optimum growth temperatures. Upon heat shock, a quick drop in the Sll1130 levels leads to de-repression of the heat-shock genes and subsequent thermal acclimation. On the basis of the findings of the present study, we present a model which describes the heat-shock response involving Sll1130.

Histidine kinase Hik33 responds to a variety of stress conditions and regulates the expression of stress-inducible genes in the cyanobacterium Synechocystis sp. PCC 6803. However, the mechanisms of response and regulation remain unknown. Generally, a histidine kinase perceives a specific signal via its N-terminal region. Hik33 has two transmembrane helices, a periplasmic loop, and HAMP and PAS domains in its N-terminal region, all of which might be involved in signal perception. To investigate the functions of these subdomains in vivo, we expressed a chimeric histidine kinase (Hik33n-SphSc) by fusing the N-terminal region of Hik33 with the C-terminal region of a sensory histidine kinase that is activated under phosphate-deficient conditions, SphS. Hik33n-SphSc responded to several stimuli that are perceived by intact Hik33 and regulated expression of the phoA gene for alkaline phosphatase, which is normally regulated under phosphate-deficient conditions by SphS. We introduced genes for modified versions of Hik33n-SphSc into Synechocystis and monitored expression of phoA under standard and stress conditions. Hik33n-SphSc lacking either the transmembrane helices or both the HAMP and PAS domains had no kinase activity, whereas Hik33n-SphSc lacking the HAMP or the PAS domain enhanced expression of phoA. Moreover, variants of Hik33n-SphSc, in which the membrane-localizing region was replaced by those of other histidine kinases, also responded to stress conditions. Thus, transmembrane helices, regardless of sequence, appear to be essential for the function of Hik33, while the HAMP and PAS domains play important roles in regulating kinase activity in vivo.

Optimization of the light conditions for biofuel production by the microalga Botryococcus braunii BOT-22 (race B) was performed using monochromatic red light. The lipid and sugar contents were approximately 40% and 20-30% of the cell dry weight, respectively, and about half of the lipids were liquid hydrocarbons. The half-saturation intensities for the production rate of lipids, hydrocarbons, and sugars were 63, 49, and 44μmolm(-2)s(-1), respectively. Fluorescence microscopic images of Nile Red-stained cells showed an increased number of intracellular neutral lipid granules due to increased light intensity. After 16days of incubation in the dark, lipid and sugar, but not hydrocarbon content decreased. Growth, metabolite production, and photosynthesis were saturated at 100, 200 and 1000μmolm(-2)s(-1), respectively. These results indicate that photosynthetically captured energy is not used efficiently for metabolite production; thus, improvements in metabolic regulation may increase hydrocarbon production.

Cyanobacteria are the only prokaryotes possessing plasma, thylakoid, and outer membranes. The plasma membrane of a cyanobacterial cell is essential for the biogenesis of cyanobacterial photosystems and serves as a barrier against environmental stress. We previously identified dozens of salt-responsive proteins in the plasma membrane of Synechocystis sp. PCC 6803. Five histidine kinases (Hiks) including Hik33 were also proposed to be involved in the perception of salt stress in Synechocystis. In this study, we analyzed proteomic profiles of the plasma membrane from a hik33-knockout mutant (ΔHik33) under normal and salt-stress conditions. Using 2D-DIGE followed by mass spectrometry analysis, we identified 26 differentially expressed proteins in ΔHik33 mutant cells. Major changes, due to the Hik33 mutation, included the substrate-binding proteins of ABC transporters, such as GgtB and FutA1, regulatory proteins including MorR and Rre13, as well as several hypothetical proteins. Under salt-stress conditions, the Hik33 mutation reduced levels of 7 additional proteins, such as NrtA, nitrate/sulfonate/bicarbonate-binding protein and LexA, and enhanced levels of 9 additional proteins including SphX. These observations suggest a substantial rearrangement in the plasma membrane proteome of Synechocystis due to the loss of hik33. Furthermore, a comprehensive molecular network was revealed in ΔHik33 mutant coping with salt stress.

The unicellular green alga Chlamydomonas reinhardtii can acclimate to a wide range of CO(2) concentrations through the regulation of a CO(2)-concentrating mechanism (CCM). By proteomic analysis, here we identified the proteins which were specifically accumulated under high-CO(2) conditions in a cell wall-less strain of C. reinhardtii which release their extracellular matrix into the medium. When the CO(2) concentration was elevated from the ambient air level to 3% during culture, the algal growth rate increased 1.5-fold and the composition of extracellular proteins, but not intracellular soluble and insoluble proteins, clearly changed. Proteomic analysis data showed that the levels of 22 of 129 extracellular proteins increased for 1 and 3 d and such multiple high-CO(2)-inducible proteins include gametogenesis-related proteins and hydroxyproline-rich glycoproteins. However, we could not prove the induction of gametogenesis under high-CO(2) conditions, suggesting that the inductive signal might be incomplete, not strong enough or that only high-CO(2) conditions might be not sufficient for the cell stage to proceed to the formation of sexually active gametes. However, these gametogenesis-related proteins and/or hydroxyproline-rich glycoproteins may have novel roles outside the cell under high-CO(2) conditions.

The marine coccolithophore Emiliania huxleyi (Haptophyta) requires selenium as an essential element for growth, and the active species absorbed is selenite, not selenate. This study characterized the selenite uptake mechanism using ⁷⁵Se as a tracer. Kinetic analysis of selenite uptake showed the involvement of both active and passive transport processes. The active transport was suppressed by 0.5 mM vanadate, a membrane-permeable inhibitor of H⁺-ATPase, at pH 8.3. When the pH was lowered from 8.3 to 5.3, the selenite uptake activity greatly increased, even in the presence of vanadate, suggesting that the H⁺ concentration gradient may be a motive force for selenite transport. [⁷⁵Se]Selenite uptake at selenite-limiting concentrations was hardly affected by selenate, sulfate and sulfite, even at 100 μM. In contrast, 3 μM orthophosphate increased the K(m) 5-fold. These data showed that HSeO₃⁻, a dominant selenite species at acidic pH, is the active species for transport through the plasma membrane and transport is driven by ΔpH energized by H⁺-ATPase. Kinetic analysis showed that the selenite uptake activity was competitively inhibited by orthophosphate. Furthermore, the active selenite transport mechanism was shown to be induced de novo under Se-deficient conditions and induction was suppressed by the addition of either sufficient selenite or cycloheximide, an inhibitor of de novo protein synthesis. These results indicate that E. huxleyi cells developed an active selenite uptake mechanism to overcome the disadvantages of Se limitation in ecosystems, maintaining selenium metabolism and selenoproteins for high viability.

Serine/threonine protein kinases (STPKs) are the major participants in intracellular signal transduction in eukaryotes, such as yeasts, fungi, plants, and animals. Genome sequences indicate that these kinases are also present in prokaryotes, such as cyanobacteria. However, their roles in signal transduction in prokaryotes remain poorly understood. We have attempted to identify the roles of STPKs in response to heat stress in the prokaryotic cyanobacterium Synechocystis sp. PCC 6803, which has 12 genes for STPKs. Each gene was individually inactivated to generate a gene-knockout library of STPKs. We applied in vitro Ser/Thr protein phosphorylation and phosphoproteomics and identified the methionyl-tRNA synthetase, large subunit of RuBisCO, 6-phosphogluconate dehydrogenase, translation elongation factor Tu, heat-shock protein GrpE, and small chaperonin GroES as the putative targets for Ser/Thr phosphorylation. The expressed and purified GroES was used as an external substrate to screen the protein extracts of the individual mutants for their Ser/Thr kinase activities. The mutants that lack one of the three protein kinases, SpkC, SpkF, and SpkK, were unable to phosphorylate GroES in vitro, suggesting possible interactions between them towards their substrate. Complementation of the mutated SpkC, SpkF, and SpkK leads to the restoration of the ability of cells to phosphorylate the GroES. This suggests that these three STPKs are organized in a sequential order or a cascade and they work one after another to finally phosphorylate the GroES.

InSynechocystissp. PCC 6803 the histidine kinase SphS (sll0337) is involved in transcriptional activation of the phosphate (Pi)-acquisition system which includes alkaline phosphatase (AP). The N-terminal region of SphS contains both a hydrophobic region and a Per-Arnt-Sim (PAS) domain. The C-terminal region has a highly conserved transmitter domain. Immunological localization studies on heterologously expressed SphS inEscherichia coliindicate that the hydrophobic region is important for membrane localization. In order to evaluate the function of the N-terminal region of SphS, deletion mutants under the control of the native promoter were analysed forin vivoAP activity. Deletion of the N-terminal hydrophobic region resulted in loss of AP activity under both Pi-deficient and Pi-sufficient conditions. Substitution of the hydrophobic region of SphS with that from the Ni2+-sensing histidine kinase, NrsS, resulted in the same induction characteristics as SphS. Deletion of the PAS domain resulted in the constitutive induction of AP activity regardless of Piavailability. To characterize the PAS domain in more in detail, four amino acid residues conserved in the PAS domain were substituted with Ala. Among the mutants R121A constitutively expressed AP activity, suggesting that R121 is important for the function of the PAS domain. Our observations indicated that the presence of a transmembrane helix in the N-terminal region of SphS is critical for activity and that the PAS domain is involved in perception of Piavailability.

In the cyanobacterium Synechocystis sp. PCC 6803, the histidine kinase Hik33 regulates the expression of several stress-inducible genes. Recently, a yeast two-hybrid screen revealed a specific interaction between Hik33 and a small protein, Ssl3451. To investigate the function of Ssl3451, we developed an assay to monitor the autophosphorylation of Hik33 in vitro. Addition of Ssl3451 to the reaction mixture dramatically enhanced the autophosphorylation activity of Hik33. Pulse-chase experiments revealed that Ssl3451 stimulated the autophosphorylation of Hik33 but did not affect its dephosphorylation. These findings indicated that Ssl3451 might be an activator of Hik33. When the amount of Hik33 was kept constant and the amount of Ssl3451 was increased in the reaction mixture, the extent of autophosphorylation of Hik33 reached a plateau when equimolar concentrations were present, suggesting that Ssl3451 enhances the activity of Hik33 by associating with it with a 1 : 1 stoichiometry. Disruption of the gene for Ssl3451 resulted in increased expression of the hliB gene, which is induced by Hik33 under standard growth conditions, but it did not affect the levels of the hliB mRNA at low temperature. Together, these results suggest that Ssl3451 might enhance the activity of Hik33 both in vitro and in vivo.

application/pdf, Intracellular calcification by the coccolith-producing haptophyte Emiliania huxleyi (NIES 837) is regulated by various environmental factors. This study focused on the relationship between cold and phosphate-deficient stresses to elucidate how those factors control coccolith production. 45Ca incorporation into coccoliths was more than 97% of the total 45Ca incorporation by whole cells. In a batch culture, orthophosphate in the medium (final concentration, 28.7 μM) was rapidly depleted within 3 days, and then extracellular alkaline phosphatase (AP) activity, an indicator of phosphate deprivation, increased during the stationary growth phase. The increase in AP activity was slightly higher at 20°C than at 12°C. The calcification started to increase earlier than AP activity, and the increase was much higher at 12°C than at 20°C. Such enhancement of calcification was suppressed by the addition of phosphate, while AP activity was also suppressed after a transient increase. These results suggest that phosphate deprivation is a trigger for calcification and that a rather long induction period is needed for calcification compared to the increase in AP activity. While calcification was greatly stimulated by cold stress, other cellular activities such as growth, phosphate utilization, and the induction of AP activity were suppressed. The stimulation of coccolith production by cold stress was minimal under phosphate-sufficient conditions. The high calcification activity estimated by 45Ca incorporation was confirmed by morphological observations of coccoliths on the cell surface under bright-field and polarization microscopy. These results indicate that phosphate deprivation is the primary factor for stimulating coccolith production, and cold stress is a secondary acceleration factor that stimulates calcification under conditions of phosphate deprivation.

Several haptophyte algae produce unique neutral long-chain (C-37-C-40) ketones (alkenones) with two to four trans-carbon-carbon double (C=C) bonds and a keto-group. These molecules are of great biological interest and may have substantial commercial relevance as biofuel sources. Unfortunately, their detection and quantification, involving extraction from cells with organic solvents and fractionation of lipids through columns, are rather cumbersome. Hence, we developed a method for the rapid and reliable quantification of alkenones in intact cells using Fourier transform infrared spectroscopy (FTIR). The method is based on the absorption band at 962.5 cm(-1), which corresponds to a trans-C-C bond that is unequivocally attributable to alkenones. This peak was exclusively observed in alkenone-producing haptophytes such as Emiliania huxleyi NIES-837, Emiliania huxleyi CCMP 2090, Tisochrysis lutea (former Isochrysis galbana T-iso), Tisochrysis lutea CCMP 463, and Chrysotila lamellosa CCMP 1307, as well as in appropriate standards. We compared our FTIR method with a typical GC analysis method. The alkenone quantity determined by these two methods showed similar values. Nevertheless, the FTIR method developed in this study does not require complex extraction procedures and is therefore a much easier and rapid quantification method. This FTIR method can also be used for the screening of strains and the optimization of culture conditions for alkenone production. (C) 2016 Elsevier B.V. All rights reserved.

Strains of the coccolithophore Emiliania huxleyi (Haptophyta) collected from the subarctic North Pacific and Arctic Oceans during the R/V MIRAI cruise in 2010 (MR10-05) were established as clone cultures and have been maintained in the laboratory at 15 °C and 32 ‰ salinity. To study the physiological responses of coccolith formation to changes in temperature and salinity, growth experiments and morphometric investigations were performed on two strains of MR57N isolated from the northern Bering Sea (56°58' N, 167°11' W) and MR70N at the Chukchi Sea (69°99' N, 168° W). This is the first report of a detailed morphometric and morphological investigation of Arctic Ocean coccolithophore strains. The specific growth rates at the logarithmic growth phases in both strains markedly increased as temperature was elevated from 5 to 20 °C, although coccolith productivity (the percentage of calcified cells) was similar at 10–20 % at all temperatures. On the other hand, the specific growth rate of strain MR70N was affected less by changes in salinity in the range 26–35 ‰, but the proportion of calcified cells decreased at high and low salinities. According to scanning electron microscopy (SEM) observations, coccolith morphotypes can be categorized into Type B/C on the basis of their biometrical parameters, such as length of the distal shield (LDS), length of the inner central area (LICA), and the thickness of distal shield elements. The central area elements of coccoliths varied from grilled type to closed type when temperature was increased or salinity was decreased, and coccolith size decreased simultaneously. Coccolithophore cell size also decreased with increasing temperature, although the variation in cell size was slightly greater at the lower salinity level. This indicates that subarctic and arctic coccolithophore strains can survive in a wide range of seawater temperatures and at lower salinities due to their marked morphometric adaptation ability. Because all coccolith biometric parameters followed the scaling law, the decrease in coccolith size was caused simply by the reduced calcification. Taken together, our results suggest that calcification productivity may be used to predict future oceanic environmental conditions in the Polar Regions.

Histidine kinases (Hiks) in Synechocystis sp. PCC 6803 are involved in the transduction of signals associated with various kinds of environmental stress. To examine the potential role in thermotolerance of Hiks, we used genome microarray analysis to screen a Hik knockout library for mutations that affected the expression of genes for heat shock proteins. Mutation of the hik34 gene enhanced the levels of transcripts of a number of heat shock genes, including htpG and groESL1. Overexpression of the hik34 gene repressed the expression of these heat shock genes. In addition, the cells with a mutant gene for Hik34 (ΔHik34 cells) survived incubation at 48°C for 3 h, while wild-type cells and cells with mutations in other Hiks were killed. However, mutation of the hik34 gene had only an insignificant effect on the global expression of genes upon incubation of the mutant cells at 44°C for 20 min. Quantitative two-dimensional gel electrophoresis revealed that levels of GroES and HspA were elevated in ΔHik34 cells after incubation of cells at 42°C for 60 min. We overexpressed recombinant Hik34 protein in Escherichia coli and purified it. We found that the protein was autophosphorylated in vitro at physiological temperatures, but not at elevated temperatures, such as 44°C. These results suggest that Hik34 might negatively regulate the expression of certain heat shock genes that might be related to thermotolerance in Synechocystis.

In previous studies, we characterized five histidine kinases (Hiks) and the cognate response regulators (Rres) that control the expression of approximately 70% of the hyperosmotic stress-inducible genes in the cyanobacterium Synechocystis sp. PCC 6803. In the present study, we screened a gene knock-out library of Rres by RNA slot-blot hybridization and with a genome-wide DNA microarray and identified three Hik-Rre systems, namely, Hik33-Rre31, Hik10-Rre3, and Hik16-Hik41-Rre17, as well as another system that included Rre1, that were involved in perception of salt stress and transduction of the signal. We found that these Hik-Rre systems were identical to those that were involved in perception and transduction of the hyperosmotic stress signal. We compared the induction factors of the salt stress- and hyperosmotic stress-inducible genes that are located downstream of each system and found that these genes responded to the two kinds of stress to different respective extents. In addition, the Hik33-Rre31 system regulated the expression of genes that were specifically induced by hyperosmotic stress, whereas the system that included Rre1 regulated the expression of one or two genes that were specifically induced either by salt stress or by hyperosmotic stress. Our observations suggest that the perception of salt and hyperosmotic stress by the Hik-Rre systems is complex and that salt stress and hyperosmotic stress are perceived as distinct signals by the Hik-Rre systems.

The sigE gene of Synechocystis sp. PCC 6803 encodes a group 2 sigma factor for RNA polymerase and has been proposed to function in transcriptional regulation of nitrogen metabolism. By using microarray and Northern analyses, we demonstrated that the abundance of transcripts derived from genes important for glycolysis, the oxidative pentose phosphate pathway, and glycogen catabolism is reduced in a sigE mutant of Synechocystis maintained under the normal growth condition. Furthermore, the activities of the two key enzymes of the oxidative pentose phosphate pathway, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, encoded by the zwf and gnd genes were also reduced in the sigE mutant. The dark enhancements in both enzyme activity and transcript abundance apparent in the wild type were eliminated by the mutation. In addition, the sigE mutant showed a reduced rate of glucose uptake and an increased intracellular level of glycogen. Moreover, it was unable to proliferate under the light-activated heterotrophic growth conditions. These results indicate that SigE functions in the transcriptional activation of sugar catabolic pathways in Synechocystis sp. PCC 6803.

The photosynthetic machinery and, in particular, the photosystem II (PSII) complex are susceptible to strong light, and the effects of strong light are referred to as photodamage or photoinhibition. In living organisms, photodamaged PSII is rapidly repaired and, as a result, the extent of photoinhibition represents a balance between rates of photodamage and the repair of PSII. In this study, we examined the roles of electron transport and ATP synthesis in these two processes by monitoring them separately and systematically in the cyanobacterium Synechocystis sp. PCC 6803. We found that the rate of photodamage, which was proportional to light intensity, was unaffected by inhibition of the electron transport in PSII, by acceleration of electron transport in PSI, and by inhibition of ATP synthesis. By contrast, the rate of repair was reduced upon inhibition of the synthesis of ATP either via PSI or PSII. Northern blotting and radiolabeling analysis with [35S]Met revealed that synthesis of the D1 protein was enhanced by the synthesis of ATP. Our observations suggest that ATP synthesis might regulate the repair of PSII, in particular, at the level of translation of the psbA genes for the precursor to the D1 protein, whereas neither electron transport nor the synthesis of ATP affects the extent of photodamage.

The kinetics of genome-wide responses of gene expression during the acclimation of cells of Synechocystis sp. PCC 6803 to salt stress were followed by DNA-microarray technique and compared to changes in main physiological parameters. During the first 30 min of salt stress, about 240 genes became induced higher than 3-fold, while about 140 genes were repressed. However, most changes in gene expression were only transient and observed among genes for hypothetical proteins. At 24 h after onset of salt stress conditions, the expression of only 39 genes remained significantly enhanced. Among them, many genes that encode proteins essential for salt acclimation were detected, while only a small number of genes for hypothetical proteins remained activated. Following the expression of genes for main functions of the cyanobacterial cell, i.e. PSI, PSII, phycobilisomes, and synthesis of compatible solutes, such as ion homeostasis, distinct kinetic patterns were found. While most of the genes for basal physiological functions were transiently repressed during the 1st h after the onset of salt stress, genes for proteins specifically related to salt acclimation were activated. This gene expression pattern reflects well the changes in main physiological processes in salt-stressed cells, i.e. transient inhibition of photosynthesis and pigment synthesis as well as immediate activation of synthesis of compatible solutes. The results clearly document that following the kinetics of genome-wide expression, profiling can be used to envisage physiological changes in the cyanobacterial cell after certain changes in growth conditions.

Linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3) are polyunsaturated fatty acids that are essential for mammalian nutrition, because mammals lack the desaturases required for synthesis of Δ12 (n-6) and n-3 fatty acids. Many plants can synthesize these fatty acids and, therefore, to examine the effects of a plant desaturase in mammals, we generated transgenic pigs that carried the fatty acid desaturation 2 gene for a Δ12 fatty acid desaturase from spinach. Levels of linoleic acid (18:2n-6) in adipocytes that had differentiated in vitro from cells derived from the transgenic pigs were ≈10 times higher than those from wild-type pigs. In addition, the white adipose tissue of transgenic pigs contained ≈20% more linoleic acid (18:2n-6) than that of wild-type pigs. These results demonstrate the functional expression of a plant gene for a fatty acid desaturase in mammals, opening up the possibility of modifying the fatty acid composition of products from domestic animals by transgenic technology, using plant genes for fatty acid desaturases.

The cyanobacterium Synechocystis sp. PCC 6803 accumulates the compatible solute glucosylglycerol (GG) and sucrose under salt stress. Although the molecular mechanisms for GG synthesis including regulation of the GG-phosphate synthase (ggpS) gene, which encodes GgpS, has been intensively investigated, the role of GG in protection against salt stress remains poorly understood. In our study of the role of GG in the tolerance to salt stress, we found that salt stress due to 450 mm NaCl inhibited cell division and significantly increased cell size in ΔggpS mutant cells, whereas the inhibition of cell division and increase in cell size were observed in wild-type cells at high concentrations of NaCl, such as 800 mm. Electron microscopy revealed that, in ΔggpS cells, separation of daughter cells was incomplete, and aborted division could be recognized by the presence of a structure that resembled a division ring. The addition of GG to the culture medium protected ΔggpS cells against salt stress and reversed the adverse effects of NaCl on cell division and cell size. These observations suggest that GG is important for salt tolerance and thus for the proper division of cells under salt stress conditions.

Lipid body (LB) is recognized as the cellular carbon and energy storage organelle in many organisms. LBs have been observed in the marine haptophyte alga Tisochrysis lutea that produces special lipids such as long-chain (C37 -C40) ketones (alkenones) with 2-4 trans-type double bonds. In this study, we succeeded in developing a modified method to isolate LB from T. lutea. Purity of isolated LBs was confirmed by the absence of chlorophyll auto-fluorescence and no contamination of the most abundant cellular protein ribulose-1,5-bisphosphate carboxylase/oxygenase. As alkenones predominated in the LB by GC-MS analysis, the LB can be more appropriately named as "alkenone body (AB)." Extracted AB-containing proteins were analyzed by the combination of 1DE (SDS-PAGE) and MS/MS for confident protein identification and annotated using BLAST tools at National Center for Biotechnology Information. Totally 514 proteins were identified at the maximum. The homology search identified three major proteins, V-ATPase, a hypothetical protein EMIHUDRAFT_465517 found in other alkenone-producing haptophytes, and a lipid raft-associated SPFH domain-containing protein. Our data suggest that AB of T. lutera is surrounded by a lipid membrane originating from either the ER or the ER-derived four layer-envelopes chloroplast and function as the storage site of alkenones and alkenes.