Global Proteomics Reveal an Atypical Strategy for Carbon/Nitrogen Assimilation by a Cyanobacterium Under Diverse Environmental Perturbations

Abstract

Cyanobacteria, the only prokaryotes capable of oxygenic photosynthesis, are present in diverse ecological niches and play crucial roles in global carbon and nitrogen cycles. To proliferate in nature, cyanobacteria utilize a host of stress responses to accommodate periodic changes in environmental conditions. A detailed knowledge of the composition of, as well as the dynamic changes in, the proteome is necessary to gain fundamental insights into such stress responses. Toward this goal, we have performed a large-scale proteomic analysis of the widely studied model cyanobacterium Synechocystis sp. PCC 6803 under 33 different environmental conditions. The resulting high-quality dataset consists of 22,318 unique peptides corresponding to 1955 proteins, a coverage of 53% of the predicted proteome. Quantitative determination of protein abundances has led to the identification of 1198 differentially regulated proteins. Notably, our analysis revealed that a common stress response under various environmental perturbations, irrespective of amplitude and duration, is the activation of atypical pathways for the acquisition of carbon and nitrogen from urea and arginine. In particular, arginine is catabolized via putrescine to produce succinate and glutamate, sources of carbon and nitrogen, respectively. This study provides the most comprehensive functional and quantitative analysis of the Synechocystis proteome to date, and shows that a significant stress response of cyanobacteria involves an uncommon mode of acquisition of carbon and nitrogen. Cyanobacteria, the only prokaryotes capable of oxygenic photosynthesis, are present in diverse ecological niches and play crucial roles in global carbon and nitrogen cycles. To proliferate in nature, cyanobacteria utilize a host of stress responses to accommodate periodic changes in environmental conditions. A detailed knowledge of the composition of, as well as the dynamic changes in, the proteome is necessary to gain fundamental insights into such stress responses. Toward this goal, we have performed a large-scale proteomic analysis of the widely studied model cyanobacterium Synechocystis sp. PCC 6803 under 33 different environmental conditions. The resulting high-quality dataset consists of 22,318 unique peptides corresponding to 1955 proteins, a coverage of 53% of the predicted proteome. Quantitative determination of protein abundances has led to the identification of 1198 differentially regulated proteins. Notably, our analysis revealed that a common stress response under various environmental perturbations, irrespective of amplitude and duration, is the activation of atypical pathways for the acquisition of carbon and nitrogen from urea and arginine. In particular, arginine is catabolized via putrescine to produce succinate and glutamate, sources of carbon and nitrogen, respectively. This study provides the most comprehensive functional and quantitative analysis of the Synechocystis proteome to date, and shows that a significant stress response of cyanobacteria involves an uncommon mode of acquisition of carbon and nitrogen. Cyanobacteria are a morphologically diverse group of Gram-negative bacteria capable of oxygenic photosynthesis. These organisms occur in almost every habitat and are credited with the transition of the Earth's atmosphere from an anaerobic state to the aerobic condition, the evolution of planetary primary production, and as being the progenitor of chloroplasts in higher plants (1.Kasting J.F. The rise of atmospheric oxygen.Science. 2001; 293: 819-820Crossref PubMed Scopus (171) Google Scholar, 2.Cavalier-Smith T. Chloroplast evolution: secondary symbiogenesis and multiple losses.Curr. Biol. 2002; 12: R62-R64Abstract Full Text Full Text PDF Scopus (227) Google Scholar, 3.Kasting J.F. Siefert J.L. Life and the evolution of Earth's atmosphere.Science. 2002; 296: 1066-1068Crossref PubMed Scopus (314) Google Scholar). Cyanobacteria contribute significantly to global photosynthetic productivity. It is estimated that more than half the total primary production essential for sustaining life on Earth is produced by cyanobacteria (4.Johnson Z.I. Zinser E.R. Coe A. McNulty N.P. Woodward E.M. Chisholm S.W. Niche partitioning among Prochlorococcus ecotypes along ocean-scale environmental gradients.Science. 2006; 311: 1737-1740Crossref PubMed Scopus (694) Google Scholar). Additionally, cyanobacteria have recently attracted significant interest because of their crucial role in the biogeochemical cycle (5.Falkowski P.G. Godfrey L.V. Electrons, life and the evolution of Earth's oxygen cycle.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2008; 363: 2705-2716Crossref PubMed Scopus (109) Google Scholar), and their ability to produce renewable carbon-neutral biofuels. Despite these important evolutionary, ecological, environmental, and biotechnological factors, many aspects of cyanobacterial physiology remain poorly understood. The physiological activities of cyanobacteria are intricately linked with the daily changes in environmental conditions. This is because the energy requirements for cyanobacterial cellular metabolism including fixation of carbon dioxide and assimilation of nutrients are derived from sunlight. Thus cyanobacteria must constantly modify the cellular machinery for efficient capture of light energy under continually changing natural conditions. Typically, they tightly coordinate growth with cellular energy levels to survive unfavorable conditions. Integration of nutrient specific pathways with photosynthetic processes is a key survival mechanism employed by cyanobacteria under changing environmental conditions (6.Lindahl M. Florencio F.J. Thioredoxin-linked processes in cyanobacteria are as numerous as in chloroplasts, but targets are different.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 16107-16112Crossref PubMed Scopus (140) Google Scholar, 7.Singh A.K. Elvitigala T. Bhattacharyya-Pakrasi M. Aurora R. Ghosh B. Pakrasi H.B. Integration of carbon and nitrogen metabolism with energy production is crucial to light acclimation in the cyanobacterium Synechocystis.Plant Physiol. 2008; 148: 467-478Crossref PubMed Scopus (73) Google Scholar, 8.Tsinoremas N.F. Castets A.M. Harrison M.A. Allen J.F. Tandeau de Marsac N. Photosynthetic electron transport controls nitrogen assimilation in cyanobacteria by means of posttranslational modification of the glnB gene product.Proc. Natl. Acad. Sci. U.S.A. 1991; 88: 4565-4569Crossref PubMed Scopus (69) Google Scholar). Such adaptation strategies allow cyanobacteria to balance the supply of electrons from photosynthetic processes with the demands of cellular metabolism, and prevent the generation of damaging reactive oxygen species by excess reducing power. Assimilation of carbon and nitrogen in photosynthetic organisms is one of the main sinks for the reducing power produced by the light reactions of photosynthesis. Accordingly, cyanobacteria have developed intricate mechanisms to control and coordinate several pathways involved in the acquisition of carbon and nitrogen. For example, PII, a regulatory protein, has been suggested to balance the acquisition of the two nutrients by sensing the carbon and nitrogen ratio (9.Forchhammer K. Global carbon/nitrogen control by PII signal transduction in cyanobacteria: from signals to targets.FEMS Microbiol. Rev. 2004; 28: 319-333Crossref PubMed Scopus (203) Google Scholar). Thioredoxins have also been shown to link the activity of the photosynthetic electron transport chain with carbon and nitrogen assimilation (6.Lindahl M. Florencio F.J. Thioredoxin-linked processes in cyanobacteria are as numerous as in chloroplasts, but targets are different.Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 16107-16112Crossref PubMed Scopus (140) Google Scholar). Despite the active participation of these proteins, cyanobacteria can assimilate carbon and nitrogen at disparate levels exceeding cellular demands. Excess carbon and nitrogen are stored in the forms of glycogen and cyanophycin granules, respectively, and are subsequently utilized under limiting conditions. Synechocystis sp. PCC 6803 (hereafter Synechocystis), a model cyanobacterium, utilizes the oxidative pentose phosphate and glycolytic pathways to obtain carbon from glycogen granules (10.Yang C. Hua Q. Shimizu K. Quantitative analysis of intracellular metabolic fluxes using GC-MS and two-dimensional NMR spectroscopy.J. Biosci. Bioeng. 2002; 93: 78-87Crossref PubMed Scopus (62) Google Scholar). However, the pathway for utilization of cyanophycin is well understood. is a of and must catabolized to the nitrogen for cellular are at pathways for the of arginine in a pathway and the urea cycle has been shown to active in Synechocystis A.M. A. in the cyanobacterium Synechocystis sp. PCC 6803 involves the urea cycle and PubMed Scopus Google Scholar). of gene linked to the assimilation of carbon and nitrogen, as well as cellular adaptation mechanisms under different environmental have significantly from global analysis of Synechocystis A.K. Elvitigala T. Ghosh Bhattacharyya-Pakrasi M. Pakrasi H.B. analysis of response and multiple cellular processes in a Biol. 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Elvitigala T. Bhattacharyya-Pakrasi M. Aurora R. Ghosh B. Pakrasi H.B. Integration of carbon and nitrogen metabolism with energy production is crucial to light acclimation in the cyanobacterium Synechocystis.Plant Physiol. 2008; 148: 467-478Crossref PubMed Scopus (73) Google Scholar, A.K. Bhattacharyya-Pakrasi M. Elvitigala T. Ghosh B. Aurora R. Pakrasi H.B. A analysis of the of light on the metabolism of a Physiol. PubMed Scopus Google Scholar). It has also been suggested that changes in environmental conditions to transport of the pathway involved in the transport of nitrogen including and arginine A.K. Bhattacharyya-Pakrasi M. Elvitigala T. Ghosh B. Aurora R. Pakrasi H.B. A analysis of the of light on the metabolism of a Physiol. PubMed Scopus Google Scholar). A mechanism under changing nutrient conditions is from the analysis of proteomic shows that the for nitrogen acquisition are urea and arginine. 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The of this is that by is the production of In the proteome analysis in this study has a unique and comprehensive of the proteomic of Synechocystis under various environmental conditions. that the knowledge of the functional of and with as well as are is to a for of dynamic changes in the proteome has insights into cellular under various environmental that a key cellular adaptation to the activation of pathways for the acquisition of carbon and nitrogen, are the two sinks for reducing by the photosynthetic light reactions in the of the Pakrasi and for N. for a dataset for Synechocystis under with