Polyhydroxybutyrate (PHB) is a common carbon storage polymer among heterotrophic bacteria. It is also accumulated in some photoautotrophic cyanobacteria; however, the knowledge of how PHB accumulation is regulated in this group is limited. PHB synthesis in Synechocystis sp. PCC 6803 is initiated once macronutrients like phosphorus or nitrogen are limiting. We have previously reported a mutation in the gene sll0783 that impairs PHB accumulation in this cyanobacterium upon nitrogen starvation. In this study we present data which explain the observed phenotype. We investigated differences in intracellular localization of PHB synthase, metabolism, and the NADPH pool between wild type and mutant. Localization of PHB synthase was not impaired in the sll0783 mutant; however, metabolome analysis revealed a difference in sorbitol levels, indicating a more oxidizing intracellular environment than in the wild type. We confirmed this by directly measuring the NADPH/NADP ratio and by altering the intracellular redox state of wild type and sll0783 mutant. We were able to physiologically complement the mutant phenotype of diminished PHB synthase activity by making the intracellular environment more reducing. Our data illustrate that the NADPH pool is an important factor for regulation of PHB biosynthesis and metabolism, which is also of interest for potential biotechnological applications.
References
[1]
Stanier, R.Y.; Cohen-Bazire, G. Phototrophic prokaryotes: the cyanobacteria. Annu. Rev. Microbiol. 1977, 31, 225–274, doi:10.1146/annurev.mi.31.100177.001301.
[2]
Harder, W.; Dijkhuizen, L. Physiological responses to nutrient limitation. Annu. Rev. Microbiol. 1983, 37, 1–23, doi:10.1146/annurev.mi.37.100183.000245.
[3]
Gorl, M.; Sauer, J.; Baier, T.; Forchhammer, K. Nitrogen-starvation-induced chlorosis in Synechococcus PCC 7942: Adaptation to long-term survival. Microbiology 1998, 144, 2449–2458, doi:10.1099/00221287-144-9-2449.
[4]
Schwarz, R.; Forchhammer, K. Acclimation of unicellular cyanobacteria to macronutrient deficiency: Emergence of a complex network of cellular responses. Microbiology 2005, 151, 2503–2514, doi:10.1099/mic.0.27883-0.
[5]
Herrero, A.; Muro-Pastor, A.M.; Flores, E. Nitrogen control in cyanobacteria. J. Bacteriol. 2001, 183, 411–425.
[6]
Fadi Aldehni, M.; Sauer, J.; Spielhaupter, C.; Schmid, R.; Forchhammer, K. Signal transduction protein P(II) is required for NtcA-regulated gene expression during nitrogen deprivation in the cyanobacterium Synechococcus elongatus strain PCC 7942. J. Bacteriol. 2003, 185, 2582–2591, doi:10.1128/JB.185.8.2582-2591.2003.
[7]
Llacer, J.L.; Espinosa, J.; Castells, M.A.; Contreras, A.; Forchhammer, K.; Rubio, V. Structural basis for the regulation of NtcA-dependent transcription by proteins PipX and PII. Proc. Natl. Acad. Sci. USA 2010, 107, 15397–15402.
[8]
Panda, B.; Jain, P.; Sharma, L.; Mallick, N. Optimization of cultural and nutritional conditions for accumulation of poly-beta-hydroxybutyrate in Synechocystis sp. PCC 6803. Bioresource Technol. 2006, 97, 1296–1301, doi:10.1016/j.biortech.2005.05.013.
[9]
Taroncher-Oldenburg, G.; Nishina, K.; Stephanopoulos, G. Identification and analysis of the polyhydroxyalkanoate-specific beta-ketothiolase and acetoacetyl coenzyme A reductase genes in the cyanobacterium Synechocystis sp. strain PCC6803. Appl. Environ. Microbiol. 2000, 66, 4440–4448, doi:10.1128/AEM.66.10.4440-4448.2000.
[10]
Hein, S.; Tran, H.; Steinbuchel, A. Synechocystis sp. PCC6803 possesses a two-component polyhydroxyalkanoic acid synthase similar to that of anoxygenic purple sulfur bacteria. Arch. Microbiol. 1998, 170, 162–170, doi:10.1007/s002030050629.
[11]
Schlebusch, M.; Forchhammer, K. Requirement of the nitrogen starvation-induced protein Sll0783 for polyhydroxybutyrate accumulation in Synechocystis sp. strain PCC 6803. Appl. Environ. Microbiol. 2010, 76, 6101–6107, doi:10.1128/AEM.00484-10.
[12]
Miyake, M.; Kataoka, K.; Shirai, M.; Asada, Y. Control of poly-beta-hydroxybutyrate synthase mediated by acetyl phosphate in cyanobacteria. J. Bacteriol. 1997, 179, 5009–5013.
[13]
Cho, M.; Brigham, C.J.; Sinskey, A.J.; Stubbe, J. Purification of polyhydroxybutyrate synthase from its native organism, Ralstonia eutropha: implications for the initiation and elongation of polymer formation in vivo. Biochemistry 2012, 51, 2276–2288, doi:10.1021/bi2013596.
[14]
Jendrossek, D. Polyhydroxyalkanoate granules are complex subcellular organelles (carbonosomes). J. Bacteriol. 2009, 191, 3195–3202.
[15]
Krasikov, V.; Aguirre von Wobeser, E.; Dekker, H.L.; Huisman, J.; Matthijs, H.C. Time-series resolution of gradual nitrogen starvation and its impact on photosynthesis in the cyanobacterium Synechocystis PCC 6803. Physiol. Plantarum 2012, 145, 426–439, doi:10.1111/j.1399-3054.2012.01585.x.
[16]
Muro-Pastor, A.M.; Olmedo-Verd, E.; Flores, E. All4312, an NtcA-regulated two-component response regulator in Anabaena sp. strain PCC 7120. FEMS Microbiol. Lett. 2006, 256, 171–177, doi:10.1111/j.1574-6968.2006.00136.x.
[17]
Zinchenko, V.V.; Piven, I.V.; Melnik, V.A.; Shestakov, S.V. Vectors for the complementation analysis of cyanobacterial mutants. Russ. J. Genet. 1999, 35, 228–232.
[18]
Wolk, C.P.; Vonshak, A.; Kehoe, P.; Elhai, J. Construction of shuttle vectors capable of conjugative transfer from Escherichia coli to nitrogen-fixing filamentous cyanobacteria. Proc. Natl. Acad. Sci. USA 1984, 81, 1561–1565, doi:10.1073/pnas.81.5.1561.
[19]
Eisenhut, M.; Huege, J.; Schwarz, D.; Bauwe, H.; Kopka, J.; Hagemann, M. Metabolome phenotyping of inorganic carbon limitation in cells of the wild type and photorespiratory mutants of the cyanobacterium Synechocystis sp. strain PCC 6803. Plant. Physiol. 2008, 148, 2109–2120, doi:10.1104/pp.108.129403.
[20]
Zhang, S.; Bryant, D.A. The tricarboxylic acid cycle in cyanobacteria. Science 2011, 334, 1551–1553, doi:10.1126/science.1210858.
[21]
Wolk, C.P.; Thomas, J.; Shaffer, P.W.; Austin, S.M.; Galonsky, A. Pathway of nitrogen metabolism after fixation of 13N-labeled nitrogen gas by the cyanobacterium, Anabaena cylindrica. J. Biol. Chem. 1976, 251, 5027–5034.
[22]
Collier, J.L.; Grossman, A.R. A small polypeptide triggers complete degradation of light-harvesting phycobiliproteins in nutrient-deprived cyanobacteria. EMBO J. 1994, 13, 1039–1047.
[23]
Sauer, J.; Gorl, M.; Forchhammer, K. Nitrogen starvation in synechococcus PCC 7942: involvement of glutamine synthetase and NtcA in phycobiliprotein degradation and survival. Arch. Microbiol. 1999, 172, 247–255, doi:10.1007/s002030050767.
[24]
Senior, P.J.; Dawes, E.A. Poly-β-hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijerinckii. Biochem. J. 1971, 125, 55–66.
[25]
England, P.J.; Denton, R.M.; Randle, P.J. The influence of magnesium ions and other bivalent metal ions on the aconitase equilibrium and its bearing on the binding of magnesium ions by citrate in rat heart. Biochem. J. 1967, 105, 32C–33C.
[26]
Huege, J.; Goetze, J.; Schwarz, D.; Bauwe, H.; Hagemann, M.; Kopka, J. Modulation of the major paths of carbon in photorespiratory mutants of synechocystis. PloS One 2011, 6, e16278.
[27]
Grundel, M.; Scheunemann, R.; Lockau, W.; Zilliges, Y. Impaired glycogen synthesis causes metabolic overflow reactions and affects stress responses in the cyanobacterium Synechocystis sp. PCC 6803. Microbiology 2012, 158, 3032–3043, doi:10.1099/mic.0.062950-0.
[28]
Koksharova, O.; Schubert, M.; Shestakov, S.; Cerff, R. 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. Plant. Mol. Biol. 1998, 36, 183–194, doi:10.1023/A:1005925732743.
[29]
Metz, J.G.; Pakrasi, H.B.; Seibert, M.; Arntzen, C.J. Evidence for a Dual Function of the Herbicide-Binding D1-Protein in Photosystem-Ii. FEBS Lett. 1986, 205, 269–274, doi:10.1016/0014-5793(86)80911-5.
[30]
Zürrer, H.; Snozzi, M.; Bachofen, R. Specific binding of DCCD to reaction centers of the photosynthetic bacterium Rhodospirillum rubrum and its effect of certain photosynthetic reactions. FEBS Lett. 1983, 153, 151–155, doi:10.1016/0014-5793(83)80137-9.
[31]
Sauer, J.; Schreiber, U.; Schmid, R.; Volker, U.; Forchhammer, K. Nitrogen starvation-induced chlorosis in Synechococcus PCC 7942. Low-level photosynthesis as a mechanism of long-term survival. Plant. Physiol. 2001, 126, 233–243, doi:10.1104/pp.126.1.233.
[32]
Mallick, N.; Sharma, L.; Singh, A.K. Poly-beta-hydroxybutyrate accumulation in Nostoc muscorum: Effects of metabolic inhibitors. J. Plant. Physiol. 2007, 164, 312–317, doi:10.1016/j.jplph.2006.01.012.
[33]
Dephilippis, R.; Ena, A.; Guastini, M.; Sili, C.; Vincenzini, M. Factors Affecting Poly-Beta-Hydroxybutyrate Accumulation in Cyanobacteria and in Purple Nonsulfur Bacteria. FEMS Microbiol. Rev. 1992, 9, 187–194.
[34]
Rippka, R. Isolation and Purification of Cyanobacteria. Method Enzymol. 1988, 167, 3–27.
[35]
Valentin, H.; Steinbüchel, A. Application of enzymatically synthesized short-chain-length hydroxy fatty acid coenzyme A thioesters for assay of polyhydroxyalkanoic acid synthases. Appl. Microbiol. Biotechnol. 1994, 40, 699–709, doi:10.1007/BF00173332.
[36]
Marin, K.; Stirnberg, M.; Eisenhut, M.; Kramer, R.; Hagemann, M. Osmotic stress in Synechocystis sp. PCC 6803: Low tolerance towards nonionic osmotic stress results from lacking activation of glucosylglycerol accumulation. Microbiology 2006, 152, 2023–2030, doi:10.1099/mic.0.28771-0.
[37]
Estepa, J.; Luque-Almagro, V.M.; Manso, I.; Escribano, M.P.; Martinez-Luque, M.; Castillo, F.; Moreno-Vivian, C.; Roldan, M.D. The nit1C gene cluster of Pseudomonas pseudoalcaligenes CECT5344 involved in assimilation of nitriles is essential for growth on cyanide. Env. Microbiol. Rep. 2012, 4, 326–334, doi:10.1111/j.1758-2229.2012.00337.x.
