Transcriptome and proteome analysis of nitrogen starvation responses in Synechocystis 6803 ΔglgC, a mutant incapable of glycogen storage (original) (raw)
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Plant Physiology, 2007
Concerted changes in the transcriptional pattern and physiological traits that result from long-term (here defined as up to 24 h) limitation of inorganic carbon (C i) have been investigated for the cyanobacterium Synechocystis sp. strain PCC 6803. Results from reverse transcription-polymerase chain reaction and genome-wide DNA microarray analyses indicated stable up-regulation of genes for inducible CO 2 and HCO 3 2 uptake systems and of the rfb cluster that encodes enzymes involved in outer cell wall polysaccharide synthesis. Coordinated up-regulation of photosystem I genes was further found and supported by a higher photosystem I content and activity under low C i (LC) conditions. Bacterial-type glycerate pathway genes were induced by LC conditions, in contrast to the genes for the plant-like photorespiratory C2 cycle. Down-regulation was observed for nitrate assimilation genes and surprisingly also for almost all carboxysomal proteins. However, for the latter the observed elongation of the half-life time of the large subunit of Rubisco protein may render compensation. Mutants defective in glycolate turnover (DglcD and DgcvT) showed some transcriptional changes under high C i conditions that are characteristic for LC conditions in wild-type cells, like a modest down-regulation of carboxysomal genes. Properties under LC conditions were comparable to LC wild type, including the strong response of genes encoding inducible high-affinity C i uptake systems. Electron microscopy revealed a conspicuous increase in number of carboxysomes per cell in mutant DglcD already under high C i conditions. These data indicate that an increased level of photorespiratory intermediates may affect carboxysomal components but does not intervene with the expression of majority of LC inducible genes.
PLANT PHYSIOLOGY, 2007
Concerted changes in the transcriptional pattern and physiological traits that result from long-term (here defined as up to 24 h) limitation of inorganic carbon (C i ) have been investigated for the cyanobacterium Synechocystis sp. strain PCC 6803. Results from reverse transcription-polymerase chain reaction and genome-wide DNA microarray analyses indicated stable up-regulation of genes for inducible CO 2 and HCO 3 2 uptake systems and of the rfb cluster that encodes enzymes involved in outer cell wall polysaccharide synthesis. Coordinated up-regulation of photosystem I genes was further found and supported by a higher photosystem I content and activity under low C i (LC) conditions. Bacterial-type glycerate pathway genes were induced by LC conditions, in contrast to the genes for the plant-like photorespiratory C2 cycle. Down-regulation was observed for nitrate assimilation genes and surprisingly also for almost all carboxysomal proteins. However, for the latter the observed elongation of the half-life time of the large subunit of Rubisco protein may render compensation. Mutants defective in glycolate turnover (DglcD and DgcvT) showed some transcriptional changes under high C i conditions that are characteristic for LC conditions in wild-type cells, like a modest down-regulation of carboxysomal genes. Properties under LC conditions were comparable to LC wild type, including the strong response of genes encoding inducible high-affinity C i uptake systems. Electron microscopy revealed a conspicuous increase in number of carboxysomes per cell in mutant DglcD already under high C i conditions. These data indicate that an increased level of photorespiratory intermediates may affect carboxysomal components but does not intervene with the expression of majority of LC inducible genes.
PLANT PHYSIOLOGY, 2011
The amount of inorganic carbon is one of the main limiting environmental factors for photosynthetic organisms such as cyanobacteria. Using Synechococcus elongatus PCC 7942, we characterized metabolic and transcriptomic changes in cells that had been shifted from high to low CO 2 levels. Metabolic phenotyping indicated an activation of glycolysis, the oxidative pentose phosphate cycle, and glycolate metabolism at lowered CO 2 levels. The metabolic changes coincided with a general reprogramming of gene expression, which included not only increased transcription of inorganic carbon transporter genes but also genes for enzymes involved in glycolytic and photorespiratory metabolism. In contrast, the mRNA content for genes from nitrogen assimilatory pathways decreased. These observations indicated that cyanobacteria control the homeostasis of the carbon-nitrogen ratio. Therefore, results obtained from the wild type were compared with the MP2 mutant of Synechococcus 7942, which is defective for the carbon-nitrogen ratio-regulating PII protein. Metabolites and genes linked to nitrogen assimilation were differentially regulated, whereas the changes in metabolite concentrations and gene expression for processes related to central carbon metabolism were mostly similar in mutant and wild-type cells after shifts to low-CO 2 conditions. The PII signaling appears to down-regulate the nitrogen metabolism at lowered CO 2 , whereas the specific shortage of inorganic carbon is recognized by different mechanisms.
Microbiology, 2012
Using metabolic and transcriptomic phenotyping, we studied acclimation of cyanobacteria to low inorganic carbon (LC) conditions and the requirements for coordinated alteration of metabolism and gene expression. To analyse possible metabolic signals for LC sensing and compensating reactions, the carboxysome-less mutant DccmM and the photorespiratory mutant DglcD1/D2 were compared with wild-type (WT) Synechocystis. Metabolic phenotyping revealed accumulation of 2-phosphoglycolate (2PG) in DccmM and of glycolate in DglcD1/D2 in LC-but also in high inorganic carbon (HC)-grown mutant cells. The accumulation of photorespiratory metabolites provided evidence for the oxygenase activity of RubisCO at HC. The global gene expression patterns of HC-grown DccmM and DglcD1/D2 showed differential expression of many genes involved in photosynthesis, high-light stress and N assimilation. In contrast, the transcripts of LC-specific genes, such as those for inorganic carbon transporters and components of the carbon-concentrating mechanism (CCM), remained unchanged in HC cells. After a shift to LC, DglcD1/D2 and WT cells displayed induction of many of the LC-inducible genes, whereas DccmM lacked similar changes in expression. From the coincidence of the presence of 2PG in DccmM without CCM induction and of glycolate in DglcD1/D2 with CCM induction, we regard a direct role for 2PG as a metabolic signal for the induction of CCM during LC acclimation as less likely. Instead, our data suggest a potential role for glycolate as a signal molecule for enhanced expression of CCM genes. 3Present address: Two supplementary figures, showing mutant genotypes and growth and global changes in transcriptomic patterns, and three supplementary tables listing the complete metabolomic and transcriptomic datasets, and the distribution of differentially regulated genes in different metabolic categories in cells of the WT and DccmM and DglcD1/D2 mutants, are available with the online version of this paper.
PLANT PHYSIOLOGY, 2008
The amount of inorganic carbon represents one of the main environmental factors determining productivity of photoautotrophic organisms. Using the model cyanobacterium Synechocystis sp. PCC 6803, we performed a first metabolome study with cyanobacterial cells shifted from high CO 2 (5% in air) into conditions of low CO 2 (LC; ambient air with 0.035% CO 2 ). Using gas chromatography-mass spectrometry, 74 metabolites were reproducibly identified under different growth conditions. Shifting wild-type cells into LC conditions resulted in a global metabolic reprogramming and involved increases of, for example, 2-oxoglutarate (2OG) and phosphoenolpyruvate, and reductions of, for example, sucrose and fructose-1,6-bisphosphate. A decrease in Calvin-Benson cycle activity and increased usage of associated carbon cycling routes, including photorespiratory metabolism, was indicated by synergistic accumulation of the fumarate, malate, and 2-phosphoglycolate pools and a transient increase of 3-phosphoglycerate. The unexpected accumulation of 2OG with a concomitant decrease of glutamine pointed toward reduced nitrogen availability when cells are confronted with LC. Despite the increase in 2OG and low amino acid pools, we found a complete dephosphorylation of the PII regulatory protein at LC characteristic for nitrogen-replete conditions. Moreover, mutants with defined blocks in the photorespiratory metabolism leading to the accumulation of glycolate and glycine, respectively, exhibited features of LC-treated wild-type cells such as the changed 2OG to glutamine ratio and PII phosphorylation state already under high CO 2 conditions. Thus, metabolome profiling demonstrated that acclimation to LC involves coordinated changes of carbon and interacting nitrogen metabolism. We hypothesize that Synechocystis has a temporal lag of acclimating carbon versus nitrogen metabolism with carbon leading.
PLANT PHYSIOLOGY, 2008
Light drives the production of chemical energy and reducing equivalents in photosynthetic organisms required for the assimilation of essential nutrients. This process also generates strong oxidants and reductants that can be damaging to the cellular processes, especially during absorption of excess excitation energy. Cyanobacteria, like other oxygenic photosynthetic organisms, respond to increases in the excitation energy, such as during exposure of cells to high light (HL) by the reduction of antenna size and photosystem content. However, the mechanism of how Synechocystis sp. PCC 6803, a cyanobacterium, maintains redox homeostasis and coordinates various metabolic processes under HL stress remains poorly understood. In this study, we have utilized time series transcriptome data to elucidate the global responses of Synechocystis to HL. Identification of differentially regulated genes involved in the regulation, protection, and maintenance of redox homeostasis has offered important insights into the optimized response of Synechocystis to HL. Our results indicate a comprehensive integrated homeostatic interaction between energy production (photosynthesis) and energy consumption (assimilation of carbon and nitrogen). In addition, measurements of physiological parameters under different growth conditions showed that integration between the two processes is not a consequence of limitations in the external carbon and nitrogen levels available to the cells. We have also discovered the existence of a novel glycosylation pathway, to date known as an important nutrient sensor only in eukaryotes. Up-regulation of a gene encoding the rate-limiting enzyme in the hexosamine pathway suggests a regulatory role for protein glycosylation in Synechocystis under HL.
Algal Research, 2018
Nitrogen deprivation increases the triacylglycerol (TAG) content in microalgae but also severely decreases the growth rate. Most approaches that attempted to increase TAG productivity by overexpression or knockdown of specific genes related to the regulation of the lipid synthesis have reported only little success. More insight into the molecular mechanisms related to lipid accumulation and impaired growth rate is needed to find targets for improving TAG productivity. By using the emerging "omics" approach, we comprehensively profiled the physiology, transcriptome, proteome and metabolome of the diatom Phaeodactylum tricornutum during steady state growth at both nitrogen limited and replete levels during light:dark cycles. Under nitrogen limited conditions, 22% (2699) of the total identified transcripts, 17% (543) of the proteins and 44% (345) of the metabolites were significantly differentially regulated compared to nitrogen replete growth conditions. Although nitrogen limitation was responsible for the majority of significant differential transcript, protein and metabolite accumulation, we also observed differential expression over a diurnal cycle. Nitrogen limitation mainly induced an upregulation of nitrogen fixation, central carbon metabolism and TCA cycle, while photosynthetic and ribosomal protein synthesis are mainly downregulated. Regulation of the lipid metabolism and the expression of predicted proteins involved in lipid processes suggest that lipid rearrangements may substantially contribute to TAG distribution. However, TAG synthesis is also limited by the reduced carbon flux through central metabolism. Future strain improvements should therefore focus on understanding and improving the carbon flux through central carbon metabolism, selectivity and activity of DGAT isoforms and lipase enzymes.
Algal Research, 2019
Euglena gracilis can use a wide range of organic carbon sources, as well as CO 2 from the atmosphere. This metabolic versatility is owed to the genome of E. gracilis that can encode a wide range of enzymes. Many of these enzymes are regulated post-transcriptionally, allowing the cells to adapt quickly to changes in their surroundings. Here we investigated the effect of predominantly phototrophic (PT), mixotrophic (MT) and heterotrophic (HT) cultivation on central carbon metabolism in E. gracilis Z using label-free shotgun proteomics. Differential expression between isozymes was observed based on the cultivation condition. A hexokinase enzyme identified in the published transcriptome was not detected in the proteome. Instead, a high-specificity glucokinase appeared to conduct the first step of glycolysis. Two candidates for paramylon synthase were identified (EgGSL1 and EgGSL2), of which the predominant EgGSL2 protein was detected across all growth conditions, while EgGSL1 was only detected in the presence of light (PT and MT cultivations). Proteomic analysis revealed that the oxidative pentose phosphate pathway also plays a key role in glucose metabolism under MT and HT cultivation. Some chloroplast-encoded proteins and enzymes of the Calvin pathway were detected under HT cultivation indicating regulation at the post-translational level. The carbon metabolic pathways investigated here in terms of proteomic changes provide new information, as well as validate data presented elsewhere with quantitative proteomics, adding to the existing knowledge of metabolism in E. gracilis. Putative functional annotations of several proteins that were previously unidentified are also provided.
FEBS Letters, 2013
Rre37 (sll1330) in a cyanobacterium Synechocystis sp. PCC 6803 acts as a regulatory protein for sugar catabolic genes during nitrogen starvation. Low glycogen accumulation in Drre37 was due to low expression of glycogen anabolic genes. In addition to low 2-oxoglutarate accumulation, normal upregulated expression of genes encoding glutamate synthases (gltD and gltB) as well as accumulation of metabolites in glycolysis (fructose-6-phosphate, fructose-1,6-bisphosphate, and glyceraldehyde-3-phosphate) and tricarboxylic acid (TCA) cycle (oxaloacetate, fumarate, succinate, and aconitate) were abolished by rre37 knockout. Rre37 regulates 2-oxoglutarate accumulation, glycogen accumulation through expression of glycogen anabolic genes, and TCA cycle metabolites accumulation.