Mutations in hik26 and slr1916 lead to high-light stress tolerance in Synechocystis sp. PCC6803.
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ABSTRACT:
SUBMITTER: Yoshikawa K
PROVIDER: S-EPMC7966805 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Polyunsaturated lipids are important components of photosynthetic membranes. Xanthophylls are the main photoprotective agents, can assist in protection against light stress, and are crucial in the recovery from photoinhibition. We generated the xanthophyll- and polyunsaturated lipid-deficient ROAD mutant of Synechocystis sp. PCC6803 (Synechocystis) in order to study the little-known cooperative effects of lipids and carotenoids (Cars). Electron microscopic investigations confirmed that in the absence of xanthophylls the S-layer of the cellular envelope is missing. In wild-type (WT) cells, as well as the xanthophyll-less (RO), polyunsaturated lipid-less (AD), and the newly constructed ROAD mutants the lipid and Car compositions were determined by MS and HPLC, respectively. We found that, relative to the WT, the lipid composition of the mutants was remodeled and the Car content changed accordingly. In the mutants the ratio of non-bilayer-forming (NBL) to bilayer-forming (BL) lipids was found considerably lower. Xanthophyll to ?-carotene ratio increased in the AD mutant. In vitro and in vivo methods demonstrated that saturated, monounsaturated lipids and xanthophylls may stabilize the trimerization of Photosystem I (PSI). Fluorescence induction and oxygen-evolving activity measurements revealed increased light sensitivity of RO cells compared to those of the WT. ROAD showed a robust increase in light susceptibility and reduced recovery capability, especially at moderate low (ML) and moderate high (MH) temperatures, indicating a cooperative effect of xanthophylls and polyunsaturated lipids. We suggest that both lipid unsaturation and xanthophylls are required for providing the proper structure and functioning of the membrane environment that protects against light and temperature stress.
| S-EPMC5877414 | biostudies-literature
Project description:we obtained a HL tolerant (Tol) strain Synechocystis sp. PCC 6803 by adaptive evolution experiment that the cells were repeatedly subcultured for prolonged periods of time (52 days) under high light stress condition (7000 to 9000 μmol m-2 s-1). Although the growth of the parental strain almost stopped under 9000 μmol m-2 s-1, no growth inhibition was observed in the Tol strain. Furthermore, the growth rate was identical to that of parental strain under low light condition (40 μmol m-2 s-1). To further investigate the high light tolerant mechanisms in the Tol strain, the transcriptome was performed. The transcriptome data suggests the increase of isiA expression in the Tol strain under HL condition. The overexpression of isiA successfully enhanced the HL stress tolerance in the parental strain. The HL tolerant mechanism was different from previous reported mechanisms, such as a reduction of the light-harvesting antenna size. The tolerant strain would be an attractive host for bio-production under high light conditions.
2020-05-06 | GSE149892 | GEO
Project description:Random mutagenesis is a useful tool to genetically modify organisms for various purposes, such as adaptation to cultivation conditions, the induction of tolerances, or increased yield of valuable substances. This is especially attractive for systems where it is not obvious which genes require modifications. Random mutagenesis has been extensively used to modify crop plants, but even with the renewed interest in microalgae and cyanobacteria for biofuel applications, there is relatively limited current research available on the application of random mutagenesis for these organisms, especially for cyanobacteria. In the presented work we characterized the lethality and rate of non-lethal point mutations for ultraviolet radiation and methyl methanesulphonate on the model cyanobacteria Synechocystis sp. PCC6803. Based on these results an optimal dosage of 10-50 J/m(2) for UV and either 0.1 or 1 v% for MMS was determined. A Synechocystis wildtype culture was then mutagenized and selected for increased temperature tolerance in vivo. During the second round of mutagenesis the viability of the culture was monitored on a cell by cell level from the treatment of the cells up to the growth at an increased temperature. After four distinct rounds of treatment (two with each mutagen) the temperature tolerance of the strain was effectively raised by about 2°C. Coupled with an appropriate in vivo screening, the described methods should be applicable to induce a variety of desirable characteristics in various strains. Coupling random mutagenesis with high-throughput screening methods would additionally allow to select for important characteristics for biofuel production, which do not yield a higher fitness and can not be selected for in vivo, such as fatty acid concentration. In a combined approach with full genome sequencing random mutagenesis could be used to determine suitable target-genes for more focused methods.
| S-EPMC3504032 | biostudies-literature
Project description:In order to construct a green-light-regulated gene expression system for cyanobacteria, we characterized a green-light sensing system derived from Synechocystis sp. PCC6803, consisting of the green-light sensing histidine kinase CcaS, the cognate response regulator CcaR, and the promoter of cpcG2 (PcpcG 2 ). CcaS and CcaR act as a genetic controller and activate gene expression from PcpcG 2 with green-light illumination. The green-light induction level of the native PcpcG 2 was investigated using GFPuv as a reporter gene inserted in a broad-host-range vector. A clear induction of protein expression from native PcpcG 2 under green-light illumination was observed; however, the expression level was very low compared with Ptrc , which was reported to act as a constitutive promoter in cyanobacteria. Therefore, a Shine-Dalgarno-like sequence derived from the cpcB gene was inserted in the 5' untranslated region of the cpcG2 gene, and the expression level of CcaR was increased. Thus, constructed engineered green-light sensing system resulted in about 40-fold higher protein expression than with the wild-type promoter with a high ON/OFF ratio under green-light illumination. The engineered green-light gene expression system would be a useful genetic tool for controlling gene expression in the emergent cyanobacterial bioprocesses.
| S-EPMC3937721 | biostudies-literature
Project description:BackgroundMannitol is a C(6) polyol that is used in the food and medical sector as a sweetener and antioxidant, respectively. The sustainable production of mannitol, especially via the direct conversion of CO2 by photosynthetic cyanobacteria, has become increasingly appealing. However, previous work aiming to achieve mannitol production in the marine Synechococcus sp. PCC7002 via heterologous expression of mannitol-1-phosphate-5-dehydrogenase (mtlD) and mannitol-1-phosphatase (m1p, in short: a 'mannitol cassette'), proved to be genetically unstable. In this study, we aim to overcome this genetic instability by conceiving a strategy to stabilize mannitol production using Synechocystis sp. PCC6803 as a model cyanobacterium.ResultsHere, we explore the stabilizing effect that mannitol production may have on cells faced with osmotic stress, in the freshwater cyanobacterium Synechocystis sp. PCC6803. We first validated that mannitol can function as a compatible solute in Synechocystis sp. PCC6803, and in derivative strains in which the ability to produce one or both of the native compatible solutes was impaired. Wild-type Synechocystis, complemented with a mannitol cassette, indeed showed increased salt tolerance, which was even more evident in Synechocystis strains in which the ability to synthesize the endogenous compatible solutes was impaired. Next we tested the genetic stability of all these strains with respect to their mannitol productivity, with and without salt stress, during prolonged turbidostat cultivations. The obtained results show that mannitol production under salt stress conditions in the Synechocystis strain that cannot synthesize its endogenous compatible solutes is remarkably stable, while the control strain completely loses this ability in only 6 days. DNA sequencing results of the control groups that lost the ability to synthesize mannitol revealed that multiple types of mutation occurred in the mtlD gene that can explain the disruption of mannitol production.ConclusionsMannitol production in freshwater Synechocsytis sp. PCC6803 confers it with increased salt tolerance. Under this strategy, genetically instability which was the major challenge for mannitol production in cyanobacteria is tackled. This paper marks the first report of utilization of the response to salt stress as a factor that can increase the stability of mannitol production in a cyanobacterial cell factory.
| S-EPMC7331161 | biostudies-literature
Project description:This SuperSeries is composed of the SubSeries listed below.
2005-12-07 | GSE3755 | GEO
Project description:Cyanobacteria are unicellular prokaryotic algae that perform oxygenic photosynthesis, similar to plants. The cells harbor thylakoid membranes composed of lipids related to those of chloroplasts in plants to accommodate the complexes of photosynthesis. The occurrence of storage lipids, including triacylglycerol or wax esters, which are found in plants, animals, and some bacteria, nevertheless remained unclear in cyanobacteria. We show here that the cyanobacterium Synechocystis sp. PCC6803 accumulates both triacylglycerol and wax esters (fatty acid phytyl esters). Phytyl esters accumulate in higher levels under abiotic stress conditions. The analysis of an insertional mutant revealed that the acyltransferase slr2103, with sequence similarity to plant esterase/lipase/thioesterase (ELT) proteins, is essential for triacylglycerol and phytyl ester synthesis in Synechocystis The recombinant slr2103 enzyme showed acyltransferase activity with phytol and diacylglycerol, thus producing phytyl esters and triacylglycerol. Acyl-CoA thioesters were the preferred acyl donors, while acyl-ACP (acyl carrier protein), free fatty acids, or galactolipid-bound fatty acids were poor substrates. The slr2103 protein sequence is unrelated to acyltransferases from bacteria (AtfA) or plants (DGAT1, DGAT2, PDAT), and therefore establishes an independent group of bacterial acyltransferases involved in triacylglycerol and wax ester synthesis. The identification of the gene slr2103 responsible for triacylglycerol synthesis in cyanobacteria opens the possibility of using prokaryotic photosynthetic cells in biotechnological applications.
| S-EPMC7084069 | biostudies-literature
Project description:The cyanobacterial circadian clock has been well-studied and shown to be both robust and a dominant factor in the control of gene expression in Synechococcus elongatus PCC7942. In Synechocystis sp. PCC6803, the circadian clock is assumed to function similarly, yet appears to control transcription to a far lesser extent and its circadian rhythm was reported to not be sustained, or at least rapidly damped, under continuous illumination. One of the feedback loops that governs the clock in S. elongatus in addition to the core oscillator, i.e., the transcriptional-translation regulation loop hinging on KaiC-dependent expression of kaiBC, appears to be missing in Synechocystis, which would account for this difference. Here, we show that the clock in Synechocystis fulfills all criteria of a circadian clock: 1) a free-running period of approximately 24 h, 2) temperature compensation, and 3) being able to be entrained. A remarkably stable rhythm is generated despite the fact that the organism grows with a doubling time of less than 24 h in a photobioreactor run in turbidostat mode. No damping of the free-running circadian oscillation was observed in 2 weeks, suggesting that the clock in individual cells stays synchronized within a culture despite the apparent lack of a transcriptional-translation regulation loop. Furthermore, the dependence of chlorophyll synthesis on the presence of O2 was demonstrated.
| S-EPMC4452363 | biostudies-literature
Project description:BackgroundNADPH is used as a reductant in various biosynthetic reactions. Cell-free bio-systems have gained considerable attention owing to their high energy utilization and time efficiency. Efforts have been made to continuously supply reducing power to the reaction mixture in a cyclical manner. The thylakoid membrane (TM) is a promising molecular energy generator, producing NADPH under light. Thus, TM sustainability is of major relevance for its in vitro utilization.ResultsOver 70% of TMs prepared from Synechocystis sp. PCC6803 existed in a sealed vesicular structure, with the F1 complex of ATP synthase facing outward (right-side-out), producing NADPH and ATP under light. The NADPH generation activity of TM increased approximately two-fold with the addition of carbonyl cyanide-p-(trifluoromethoxy) phenylhydrazone (FCCP) or removal of the F1 complex using EDTA. Thus, the uncoupling of proton translocation from the electron transport chain or proton leakage through the Fo complex resulted in greater NADPH generation. Biosilicified TM retained more than 80% of its NADPH generation activity after a week at 30°C in the dark. However, activity declined sharply to below 30% after two days in light. The introduction of engineered water-forming NADPH oxidase (Noxm) to keep the electron transport chain of TM working resulted in the improved sustainability of NADPH generation activity in a ratio (Noxm to TM)-dependent manner, which correlated with the decrease of singlet oxygen generation. Removal of reactive oxygen species (ROS) by catalase further highlighted the sustainable NADPH generation activity of up to 80% in two days under light.ConclusionReducing power generated by light energy has to be consumed for TM sustainability. Otherwise, TM can generate singlet oxygen, causing oxidative damage. Thus, TMs should be kept in the dark when not in use. Although NADPH generation activity by TM can be extended via silica encapsulation, further removal of hydrogen peroxide results in an improvement of TM sustainability. Therefore, as long as ROS formation by TM in light is properly handled, it can be used as a promising source of reducing power for in vitro biochemical reactions.
| S-EPMC9148488 | biostudies-literature
Project description:BackgroundInorganic phosphate (Pi) is a critical nutrient for all life and is periodically limiting in marine and freshwater provinces, yet little is understood how organisms acclimate to fluctuations in Pi within their environment. To investigate whole cell adaptation, we grew Synechocystis sp. PCC6803, a model freshwater cyanobacterium, in 3%, and 0.3% inorganic phosphate (Pi) media. The cells were allowed to acclimate over 60 days, and cells were harvested for quantitative high throughput mass spectrometry-based proteomics using the iTRAQ™ labelling technology.ResultsIn total, 120 proteins were identified, and 52 proteins were considered differentially abundant compared to the control. Alkaline phosphatase (APase) activities correlated significantly (p?<?0.05) with observed relative PhoA abundances. PstS1 and PstS2 were both observed, yet PstS1 was not differentially more abundant than the control. Phycobilisome protein abundances appeared to be coordinated, and are significantly less abundant in 0.3% Pi than 3% Pi cultures. Also, the central metabolic cell function appears to have shifted towards the production of (NADPH) reducing energy and nucleotide sugars.ConclusionsThis acclimation response bears strong similarity to the previously reported response to nitrogen deprivation within Synechocystis sp. PCC 6803. However, it also demonstrates some characteristics of desiccation stress, such as the regulation of fatty acids and increased abundance of rehydrin in the 3% Pi culture.
| S-EPMC3600050 | biostudies-literature