Plastoquinone Lipids: Their Synthesis via a Bifunctional Gene and Physiological Function in a Euryhaline Cyanobacterium, Synechococcus sp. PCC 7002.

Eukaryotic photosynthetic organisms synthesize triacylglycerols, which are crucial physiologically as major carbon and energy storage compounds and commercially as food oils and raw materials for carbon-neutral biofuel production. TLC analysis has revealed triacylglycerols are present in several cyanobacteria. However, mass spectrometric analysis has shown that freshwater cyanobacterium, Synechocystis sp. PCC 6803, contains plastoquinone-B and acyl plastoquinol with triacylglycerol-like TLC mobility, concomitantly with the absence of triacylglycerol. Synechocystis contains slr2103, which is responsible for the bifunctional synthesis of plastoquinone-B and acyl plastoquinol and also for NaCl-stress acclimatizing cell growth. However, information is limited on the taxonomical distribution of these plastoquinone lipids, and their synthesis genes and physiological roles in cyanobacteria. In this study, a euryhaline cyanobacterium, Synechococcus sp. PCC 7002, shows the same plastoquinone lipids as those in Synechocystis, although the levels are much lower than in Synechocystis, triacylglycerol being absent. Furthermore, through an analysis of a disruptant to the homolog of slr2103 in Synechococcus, it is found that the slr2103 homolog in Synechococcus, similar to slr2103 in Synechocystis, contributes bifunctionally to the synthesis of plastoquinone-B and acyl plastoquinol; however, the extent of the contribution of the homolog gene to NaCl acclimatization is smaller than that of slr2103 in Synechocystis. These observations suggest strain- or ecoregion-dependent development of the physiological roles of plastoquinone lipids in cyanobacteria and show the necessity to re-evaluate previously identified cyanobacterial triacylglycerol through TLC analysis with mass spectrometric techniques.

slr2103, a homolog of type-2 diacylglycerol acyltransferase genes, for plastoquinone-related neutral lipid synthesis and NaCl-stress acclimatization in a cyanobacterium, Synechocystis sp. PCC 6803.

A cyanobacterium, Synechocystis sp. PCC 6803, contains a lipid with triacylglycerol-like TLC mobility but its identity and physiological roles remain unknown. Here, on ESI-positive LC-MS2 analysis, it is shown that the triacylglycerol-like lipid (lipid X) is related to plastoquinone and can be grouped into two subclasses, Xa and Xb, the latter of which is esterified by 16:0 and 18:0. This study further shows that a Synechocystis homolog of type-2 diacylglycerol acyltransferase genes, slr2103, is essential for lipid X synthesis: lipid X disappears in a Synechocystis slr2103-disruptant whereas it appears in an slr2103-overexpressing transformant (OE) of Synechococcus elongatus PCC 7942 that intrinsically lacks lipid X. The slr2103 disruption causes Synechocystis cells to accumulate plastoquinone-C at an abnormally high level whereas slr2103 overexpression in Synechococcus causes the cells to almost completely lose it. It is thus deduced that slr2103 encodes a novel acyltransferase that esterifies 16:0 or 18:0 with plastoquinone-C for the synthesis of lipid Xb. Characterization of the slr2103-disruptant in Synechocystis shows that slr2103 contributes to sedimented-cell growth in a static culture, and to bloom-like structure formation and its expansion by promoting cell aggregation and floatation upon imposition of saline stress (0.3-0.6 M NaCl). These observations provide a basis for elucidation of the molecular mechanism of a novel cyanobacterial strategy to acclimatize to saline stress, and one for development of a system of seawater-utilization and economical harvesting of cyanobacterial cells with high-value added compounds, or blooming control of toxic cyanobacteria.

Diacylglyceryl-N,N,N-trimethylhomoserine-dependent lipid remodeling in a green alga, Chlorella kessleri.

Membrane lipid remodeling contributes to the environmental acclimation of plants. In the green lineage, a betaine lipid, diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), is included exclusively among green algae and nonflowering plants. Here, we show that the green alga Chlorella kessleri synthesizes DGTS under phosphorus-deficient conditions through the eukaryotic pathway via the ER. Simultaneously, phosphatidylcholine and phosphatidylethanolamine, which are similar to DGTS in their zwitterionic properties, are almost completely degraded to release 18.1% cellular phosphorus, and to provide diacylglycerol moieties for a part of DGTS synthesis. This lipid remodeling system that substitutes DGTS for extrachloroplast phospholipids to lower the P-quota operates through the expression induction of the BTA1 gene. Investigation of this lipid remodeling system is necessary in a wide range of lower green plants for a comprehensive understanding of their phosphorus deficiency acclimation strategies.

MITOL deletion in the brain impairs mitochondrial structure and ER tethering leading to oxidative stress.

Mitochondrial abnormalities are associated with developmental disorders, although a causal relationship remains largely unknown. Here, we report that increased oxidative stress in neurons by deletion of mitochondrial ubiquitin ligase MITOL causes a potential neuroinflammation including aberrant astrogliosis and microglial activation, indicating that mitochondrial abnormalities might confer a risk for inflammatory diseases in brain such as psychiatric disorders. A role of MITOL in both mitochondrial dynamics and ER-mitochondria tethering prompted us to characterize three-dimensional structures of mitochondria in vivo. In MITOL-deficient neurons, we observed a significant reduction in the ER-mitochondria contact sites, which might lead to perturbation of phospholipids transfer, consequently reduce cardiolipin biogenesis. We also found that branched large mitochondria disappeared by deletion of MITOL. These morphological abnormalities of mitochondria resulted in enhanced oxidative stress in brain, which led to astrogliosis and microglial activation partly causing abnormal behavior. In conclusion, the reduced ER-mitochondria tethering and excessive mitochondrial fission may trigger neuroinflammation through oxidative stress.

Short-lived long noncoding RNAs as surrogate indicators for chemical stress in HepG2 cells and their degradation by nuclear RNases.

Long noncoding RNAs (lncRNAs) are non-protein-coding transcripts >200 nucleotides in length that have been shown to play important roles in various biological processes. The mechanisms underlying the induction of lncRNA expression by chemical exposure remain to be determined. We identified a novel class of short-lived lncRNAs with half-lives (t1/2) ≤4 hours in human HeLa Tet-off cells, which have been suggested to express many lncRNAs with regulatory functions. As they may affect various human biological processes, short-lived lncRNAs may be useful indicators of the degree of stress on chemical exposure. In the present study, we identified four short-lived lncRNAs, designated as OIP5-AS1, FLJ46906, LINC01137, and GABPB1-AS1, which showed significantly upregulated expression following exposure to hydrogen peroxide (oxidative stress), mercury II chloride (heavy metal stress), and etoposide (DNA damage stress) in human HepG2 cells. These lncRNAs may be useful indicators of chemical stress responses. The levels of these lncRNAs in the cells were increased because of chemical stress-induced prolongation of their decay. These lncRNAs were degraded by nuclear RNases, which are components of the exosome and XRN2, and chemical exposure inhibited the RNase activities within the cells.

Electrochemically active bacteria sense electrode potentials for regulating catabolic pathways.

Electrochemically active bacteria (EAB) receive considerable attention for their utility in bioelectrochemical processes. Although electrode potentials are known to affect the metabolic activity of EAB, it is unclear whether EAB are able to sense and respond to electrode potentials. Here, we show that, in the presence of a high-potential electrode, a model EAB Shewanella oneidensis MR-1 can utilize NADH-dependent catabolic pathways and a background formate-dependent pathway to achieve high growth yield. We also show that an Arc regulatory system is involved in sensing electrode potentials and regulating the expression of catabolic genes, including those for NADH dehydrogenase. We suggest that these findings may facilitate the use of EAB in biotechnological processes and offer the molecular bases for their ecological strategies in natural habitats.

Fatty Acid Content and Composition of Triacylglycerols of Chlorella kessleri.

Triacylglycerols (TAGs) are esters formed from one glycerol and three fatty acids. TAGs are induced to accumulate in algal cells under environmental stress conditions including nutrient-limitation, hyperosmosis, and low temperature, for the storage of metabolic energy and carbon, and also for the consumption of excess energy (e.g., Hirai et al., 2016 ; Hayashi et al., 2017 ). Beside their physiological significance, the commercial utilization of algal TAG has been expected for the production of biodiesel, the methyl esters of fatty acids, from the aspect of carbon-neutral conception. The amounts of TAGs can be determined through quantitative measurement of their constituent fatty acids. This protocol consists of the following three parts: the first is the extraction of total lipids from algal cells with the use of organic solvents, chloroform and methanol, according to the method of Bligh and Dyer (1959), the second is the separation of TAG from the other lipid classes by thin-layer chromatography (TLC), and the third is the production of methyl-esterified derivatives of their constitutive fatty acids and subsequent quantitation of them by capillary gas-liquid chromatography (GLC). This protocol adapted from Sato and Tsuzuki (2011) is used for TAG analysis in a green alga, Chlorella kessleri.

Short-lived long non-coding RNAs as surrogate indicators for chemical exposure and LINC00152 and MALAT1 modulate their neighboring genes.

Whole transcriptome analyses have revealed a large number of novel long non-coding RNAs (lncRNAs). Although accumulating evidence demonstrates that lncRNAs play important roles in regulating gene expression, the detailed mechanisms of action of most lncRNAs remain unclear. We previously reported that a novel class of lncRNAs with a short half-life (t1/2 < 4 h) in HeLa cells, termed short-lived non-coding transcripts (SLiTs), are closely associated with physiological and pathological functions. In this study, we focused on 26 SLiTs and nuclear-enriched abundant lncRNA, MALAT1(t1/2 of 7.6 h in HeLa cells) in neural stem cells (NSCs) derived from human induced pluripotent stem cells, and identified four SLiTs (TUG1, GAS5, FAM222-AS1, and SNHG15) that were affected by the following typical chemical stresses (oxidative stress, heavy metal stress and protein synthesis stress). We also found the expression levels of LINC00152 (t1/2 of 2.1 h in NSCs), MALAT1 (t1/2 of 1.8 h in NSCs), and their neighboring genes were elevated proportionally to the chemical doses. Moreover, we confirmed that the overexpression of LINC00152 or MALAT1 upregulated the expressions of their neighboring genes even in the absence of chemical stress. These results reveal that LINC00152 and MALAT1 modulate their neighboring genes, and thus provide a deeper understanding of the functions of lncRNAs.

Identification of genes for sulfolipid synthesis in primitive red alga Cyanidioschyzon merolae.

Sulfoquinovosyl diacylglycerol is one of the lipids that construct thylakoid membranes, and is distributed from cyanobacteria to plastids in plants including a red lineage. One of the most primitive red algae, Cyanidioschyzon melorae, similar to cyanobacteria and green plants, possesses homologs of the SQD1 and SQD2 genes that code for UDP-sulfoquinovose and sulfoquinovosyl diacylglycerol synthases, respectively, for the synthesis of sulfoquinovosyl diacylglycerol. We here revealed the structural properties of SQD1 and SQD2 homologs in C. melorae intrinsic to those of the authentic proteins, and verified their enzymatic functions through heterologous expression in cyanobacterial disruptants as to the corresponding genes. The results demonstrated that the system of sulfoquinovosyl diacylglycerol synthesis could have been conserved through evolution of cyanobacteria to plastids in a red lineage, which is compatible with the monophyletic origin of plastids.

Profiling of lipid and glycogen accumulations under different growth conditions in the sulfothermophilic red alga Galdieria sulphuraria.

The unicellular red alga Galdieria sulphuraria grows efficiently and produces a large amount of biomass in acidic conditions at high temperatures. It has great potential to produce biofuels and other beneficial compounds without becoming contaminated with other organisms. In G. sulphuraria, biomass measurements and glycogen and lipid analyses demonstrated that the amounts and compositions of glycogen and lipids differed when cells were grown under autotrophic, mixotrophic, and heterotrophic conditions. Maximum biomass production was obtained in the mixotrophic culture. High amounts of glycogen were obtained in the mixotrophic cultures, while the amounts of neutral lipids were similar between mixotrophic and heterotrophic cultures. The amounts of neutral lipids were highest in red algae, including thermophiles. Glycogen structure and fatty acids compositions largely depended on the growth conditions.

Protein cohesion induced by metal ions observed with fluorescence correlation spectroscopy.

Nine metal ions were evaluated in the point of denaturating action of proteins. When some metal ions were added to the diluted protein solutions, aggregates appear: stronger denaturation causes the appearance of the larger-size aggregate. The size of the aggregatates are determined by fluorescence correlation spectroscopy (FCS). Green fluorescent protein (ZsGreen) and PE(phycoerythrin)-conjugated human-antibody monoclonal protein were employed as the target protein, of which solution was diluted 100-500 times and mixed with metal ions. According to this process, the denaturation power of metal ions is in the order of Mn(2+)≈ Fe(2+)< Co(2+)< Ni(2+)< Tl(+)< Cd(2+)< Cu(+)< Cu(2+)< Pb(2+)for ZsGreen, and Tl(+)≈ Ni(2+)< Cd(2+)< Fe(2+)< Cr(3+)≪ Pb(2+)for PE-conjugated antibody protein. Pb(2+)exhibits the strongest power of denaturation. In the case of ZsGreen, the denaturation power of metal ions is on the order of the Irving-Williams series, which provide the coordination tendency against ligands possessing nitrogen and oxygen. The present method with FCS is effective to evaluate the denaturation power of metal ions against proteins.

Involvement of sulfoquinovosyl diacylglycerol in DNA synthesis in Synechocystis sp. PCC 6803.

BACKGROUND: Sulfoquinovosyl diacylglycerol (SQDG) is present in the membranes of cyanobacteria and their postulated progeny, plastids, in plants. A cyanobacterium, Synechocystis sp. PCC 6803, requires SQDG for growth: its mutant (SD1) with the sqdB gene for SQDG synthesis disrupted can grow with external supplementation of SQDG. However, upon removal of SQDG from the medium, its growth is retarded, with a decrease in the cellular content of SQDG throughout cell division, and finally ceases. Concomitantly with the decrease in SQDG, the maximal activity of photosynthesis at high-light intensity is repressed by 40%. FINDINGS: We investigated effects of SQDG-defect on physiological aspects in Synechocystis with the use of SD1. SD1 cells defective in SQDG exhibited normal photosynthesis at low-light intensity as on culturing. Meanwhile, SD1 cells defective in SQDG were impaired in light-activated heterotrophic growth as well as in photoautotrophic growth. Flow cytometric analysis of the photoautotrophically growing cells gave similar cell size histograms for the wild type and SD1 supplemented with SQDG. However, the profile of SD1 defective in SQDG changed such that large part of the cell population was increased in size. Of particular interest was the microscopic observation that the mitotic index, i.e., population of dumbbell-like cells with a septum, increased from 14 to 29% in the SD1 culture without SQDG. Flow cytometric analysis also showed that the enlarged cells of SD1 defective in SQDG contained high levels of Chl, however, the DNA content was low. CONCLUSIONS: Our experiments strongly support the idea that photosynthesis is not the limiting factor for the growth of SD1 defective in SQDG, and that SQDG is responsible for some physiologically fundamental process common to both photoautotrophic and light-activated heterotrophic growth. Our findings suggest that the SQDG-defect allows construction of the photosynthetic machinery at an elevated level for an increase in cell mass, but represses DNA synthesis. SQDG may be essential for normal replication of chromosomal DNA for completion of the cell cycle.

Comparison of fluorescence correlation spectrometry with the ordinary fluorescence optical configuration with flow cytometry as a tool for micrometer-level size determination.

As a tool for micrometer-level size determination, fluorescence correlation spectroscopy (FCS), performed based on our previous report, was compared to flow cytometry (FCM). For this purpose, standard fluorescent beads were subjected to both methods. And hence, it was found that our FCS is a useful method with satisfactory precision for size determinations of individual particles at micrometer size levels, while providing the average size for a mixture of two kinds of particles with different sizes.

Size-dependent toxicity of silica nano-particles to Chlorella kessleri.

SiO(2) nano-particles were found to exhibit size-dependent toxicity toward the alga, Chlorella kessleri. Small SiO(2) nano-particles exhibit stronger toxicity: 50% inhibitory concentrations (IC(50)) value for 5 nm = 0.8 +/- 0.6%, 26 nm = 7.1 +/- 2.8%, and 78 nm = 9.1 +/- 4.7%. Enlargement of the cell body was observed by flow cytometry, which is due to the presence of structures that obstructed cell division. Optical and transmission microscopes were used to observe coagulated cells with incomplete division. Although the physiological effect of SiO(2) nano-particles was not clear, SiO(2) nano-particles are toxic, at least for algae in aquatic media. Under the transmission electron microscope, several amorphous structures appeared in the cells that were exposed to 5-nm silica nano-particles.

Optical parameter dependence of fluorescence correlation spectrometry without using magnification tools.

The performance of fluorescence correlation spectrometry (FCS) was examined for studying the solutions suspended with the fluorescent particles of various sizes from 50 nm to 10 microm in diameter and for different sizes of pinholes: the particles were made to move by simply stirring the solution in the quartz fluorescence cuvette. Without using any magnification tool for the optical image, this FCS system successfully distinguishes images with a size of smaller than 1 microm. This system was applied for determination of the sizes of microalgae.

Effects of derivatization on solar-induced decomposition of polycyclic aromatic hydrocarbons in aqueous media.

The solar-induced decomposition of 10 polycyclic aromatic hydrocarbons (PAHs) was observed in aqueous media. All 10 PAHs observed were half-decomposed within 120 min. Among anthracene derivatives, the decomposition rates were: anthracene = 1-methylanthracene < 2-methylanthracene < 9-methylanthracene < 9,10-dimethylanthracene approximately 2-aminoanthracene. The addition of commercial humic acid had no effect on the decomposition rates of these PAHs. Deuterium water also hastened the decomposition of PAH. The products obtained by the solar radiation of PAH after extraction to DCM were mainly ketone and hydroxyl derivatives. To explain these results, reactivities and electron charges at the constituent carbon atoms in each anthracene derivative were examined by an ab initio molecular orbital calculation method.

Differing involvement of sulfoquinovosyl diacylglycerol in photosystem II in two species of unicellular cyanobacteria.

Sulfoquinovosyl diacylglycerol (SQDG) is involved in the maintenance of photosystem II (PSII) activity in Chlamydomonas reinhardtii[Minoda, A., Sato, N., Nozaki, H., Okada, K., Takahashi, H., Sonoike, K. & Tsuzuki, M. et al. (2002) Eur. J. Biochem.269, 2353-2358]. To understand the spread of the taxa in which PSII interacts with SQDG, especially in cyanobacteria, we produced a mutant defective in the putative sqdB gene responsible for SQDG synthesis from two cyanobacteria, Synechocystis sp. PCC6803 and Synechococcus sp. PCC7942. The mutant of PCC6803, designated SD1, lacked SQDG synthetic ability and required SQDG supplementation for its growth. After transfer from SQDG-supplemented to SQDG-free conditions, SD1 showed decreased net photosynthetic and PSII activities on a chlorophyll (Chl) basis with a decrease in the SQDG content. Moreover, the sensitivity of PSII activity to 3-(3,4-dichlorophenyl)-1,1-dimethylurea and atrazine was increased in SD1. However, SD1 maintained normal amounts of cytochrome b559 and D1 protein (the subunits comprising the PSII complex) on a Chl basis, indicating that the PSII complex content changed little, irrespective of a decrease in the SQDG content. These results suggest that the role of SQDG is the conservation of the PSII properties in PCC6803, consistent with the results obtained with C. reinhardtii. In contrast, the SQDG-null mutant of PCC7942 showed the normal level of PSII activity with little effect on its sensitivity to PSII herbicides. Therefore, the difference in the SQDG requirement for PSII is species-specific in cyanobacteria; this could be of use when investigating the molecular evolution of the PSII complex.

Involvement of sulfoquinovosyl diacylglycerol in the structural integrity and heat-tolerance of photosystem II.

To examine the role of sulfoquinovosyl diacylglycerol (SQDG) in thylakoid membranes, we compared the structural and functional properties of photosystem II (PSII) between a mutant of Chlamydomonas reinhardtii defective in SQDG ( hf-2) and the wild type. The PSII core complex of hf-2, as compared with that of the wild type, showed structural fragility when solubilized with a detergent, dodecyl beta- d-maltoside, suggesting that the physical properties of the PSII complex were altered by the loss of SQDG. On the other hand, exposure of the cells to 41 degrees C for 120 min in the dark decreased the PSII activity to 70% and 50% of the initial levels in the wild type and hf-2, respectively, which implies that the PSII activity, in the absence of SQDG, becomes less stable under heat-stress conditions. PSII inactivated to 60% of the initial level by dark incubation at 41 degrees C was reactivated by following illumination even at 41 degrees C to more than 90% in the wild type, but only to 70% in hf-2. These results suggest that PSII inactivated by heat recovers through some mechanism dependent on light, and that SQDG participates in functioning of the mechanism. The conformational disorder of PSII caused by the defect in SQDG might be correlated with the increased susceptibility of its activity to heat-stress.