Capillary electrophoresis-mass spectrometry reveals the distribution of carbon metabolites during nitrogen starvation in Synechocystis sp. PCC 6803.

Nitrogen availability is one of the most important factors for the survival of cyanobacteria. Previous studies on Synechocystis revealed a contradictory situation with regard to metabolism during nitrogen starvation; that is, glycogen accumulated even though the expressions of sugar catabolic genes were widely upregulated. Here, we conducted transcript and metabolomic analyses using capillary electrophoresis-mass spectrometry on Synechocystis sp. PCC 6803 under nitrogen starvation. The levels of some tricarboxylic acid cycle intermediates (succinate, malate and fumarate) were greatly increased by nitrogen deprivation. Purine and pyrimidine nucleotides were markedly downregulated under nitrogen depletion. The levels of 19 amino acids changed under nitrogen deprivation, especially those of amino acids synthesized from pyruvate and phosphoenolpyruvate, which showed marked increases. Liquid chromatography-mass spectrometry analysis demonstrated that the amount of NADPH and the NADPH/NADH ratio decreased under nitrogen depletion. These data demonstrate that there are increases in not only glycogen but also in metabolites downstream of sugar catabolism in Synechocystis sp. PCC 6803 under nitrogen starvation, resolving the contradiction between glycogen accumulation and induction of sugar catabolic gene expression in this unicellular cyanobacterium.

[1]  K. Forchhammer,et al.  Nitrogen-starvation-induced chlorosis in Synechococcus PCC 7942: adaptation to long-term survival. , 1998, Microbiology.

[2]  M. Ikeuchi,et al.  Genetic Engineering of Group 2 σ Factor SigE Widely Activates Expressions of Sugar Catabolic Genes in Synechocystis Species PCC 6803* , 2011, The Journal of Biological Chemistry.

[3]  K. Forchhammer,et al.  Requirement of the Nitrogen Starvation-Induced Protein Sll0783 for Polyhydroxybutyrate Accumulation in Synechocystis sp. Strain PCC 6803 , 2010, Applied and Environmental Microbiology.

[4]  Yves Gibon,et al.  Steps towards an integrated view of nitrogen metabolism. , 2002, Journal of experimental botany.

[5]  M. Stitt,et al.  Adjustment of growth and central metabolism to a mild but sustained nitrogen-limitation in Arabidopsis. , 2009, Plant, cell & environment.

[6]  B. Daignan-Fornier,et al.  Metabolic intermediates selectively stimulate transcription factor interaction and modulate phosphate and purine pathways. , 2009, Genes & development.

[7]  John G. K. Williams.  [85] Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803 , 1988 .

[8]  Kan Tanaka,et al.  Sugar catabolism regulated by light- and nitrogen-status in the cyanobacterium Synechocystis sp. PCC 6803 , 2007, Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.

[9]  Alisdair R Fernie,et al.  Unusual cyanobacterial TCA cycles: not broken just different. , 2012, Trends in plant science.

[10]  Joachim Kopka,et al.  Metabolome Phenotyping of Inorganic Carbon Limitation in Cells of the Wild Type and Photorespiratory Mutants of the Cyanobacterium Synechocystis sp. Strain PCC 68031[W] , 2008, Plant Physiology.

[11]  Masaru Tomita,et al.  Development of Bottom-Fermenting Saccharomyces Strains That Produce High SO2 Levels, Using Integrated Metabolome and Transcriptome Analysis , 2008, Applied and Environmental Microbiology.

[12]  M. Asayama,et al.  Nitrogen induction of sugar catabolic gene expression in Synechocystis sp. PCC 6803. , 2006, DNA research : an international journal for rapid publication of reports on genes and genomes.

[13]  Ralf Takors,et al.  Simultaneous determination of multiple intracellular metabolites in glycolysis, pentose phosphate pathway and tricarboxylic acid cycle by liquid chromatography-mass spectrometry. , 2007, Journal of chromatography. A.

[14]  Marianne Gründel,et al.  Impaired glycogen synthesis causes metabolic overflow reactions and affects stress responses in the cyanobacterium Synechocystis sp. PCC 6803. , 2012, Microbiology.

[15]  W. Vermaas,et al.  Succinate Dehydrogenase and Other Respiratory Pathways in Thylakoid Membranes of Synechocystis sp. Strain PCC 6803: Capacity Comparisons and Physiological Function , 2001, Journal of bacteriology.

[16]  Y. Hihara,et al.  Difference in metabolite levels between photoautotrophic and photomixotrophic cultures of Synechocystis sp. PCC 6803 examined by capillary electrophoresis electrospray ionization mass spectrometry , 2008, Journal of Experimental Botany.

[17]  Ji-Long Liu,et al.  Only One Isoform of Drosophila melanogaster CTP Synthase Forms the Cytoophidium , 2013, PLoS genetics.

[18]  M. Kanehisa,et al.  Positive Regulation of Sugar Catabolic Pathways in the Cyanobacterium Synechocystis sp. PCC 6803 by the Group 2 σ Factor SigE* , 2005, Journal of Biological Chemistry.

[19]  Satoru Watanabe,et al.  Identification of Substrain-Specific Mutations by Massively Parallel Whole-Genome Resequencing of Synechocystis sp. PCC 6803 , 2011, DNA research : an international journal for rapid publication of reports on genes and genomes.

[20]  J. Huisman,et al.  Concerted Changes in Gene Expression and Cell Physiology of the Cyanobacterium Synechocystis sp. Strain PCC 6803 during Transitions between Nitrogen and Light-Limited Growth1[W][OA] , 2011, Plant Physiology.

[21]  S. Tabata,et al.  Identification of PamA as a PII-binding Membrane Protein Important in Nitrogen-related and Sugar-catabolic Gene Expression in Synechocystis sp. PCC 6803* , 2005, Journal of Biological Chemistry.

[22]  W. Vermaas,et al.  Succinate:Quinol Oxidoreductases in the Cyanobacterium Synechocystis sp. Strain PCC 6803: Presence and Function in Metabolism and Electron Transport , 2000, Journal of bacteriology.

[23]  M. M. Allen,et al.  Nitrogen chlorosis in blue-green algae , 2004, Archiv für Mikrobiologie.

[24]  J. Reyes,et al.  Cyanobacteria perceive nitrogen status by sensing intracellular 2-oxoglutarate levels. , 2001, The Journal of biological chemistry.

[25]  R. Rippka.  Isolation and purification of cyanobacteria. , 1988, Methods in enzymology.

[26]  S. Gibson,et al.  Fumaric acid: an overlooked form of fixed carbon in Arabidopsis and other plant species , 2000, Planta.

[27]  O. Koksharova,et al.  Genetic and biochemical evidence for distinct key functions of two highly divergent GAPDH genes in catabolic and anabolic carbon flow of the cyanobacterium Synechocystis sp. PCC 6803 , 2004, Plant Molecular Biology.

[28]  Akira Oikawa,et al.  Metabolomics of a Single Vacuole Reveals Metabolic Dynamism in an Alga Chara australis1[W][OA] , 2011, Plant Physiology.

[29]  D. Bryant,et al.  The Tricarboxylic Acid Cycle in Cyanobacteria , 2011, Science.

[30]  Jef Huisman,et al.  Time-series resolution of gradual nitrogen starvation and its impact on photosynthesis in the cyanobacterium Synechocystis PCC 6803. , 2012, Physiologia plantarum.

[31]  Tomoyoshi Soga,et al.  Capillary electrophoresis-mass spectrometry for metabolomics. , 2007, Methods in molecular biology.