Abstract
BackgroundRecent studies using transcript and metabolite profiles of wild-type and gene deletion mutants revealed that photorespiratory pathways are essential for the growth of Synechocystis sp. PCC 6803 under atmospheric conditions. Pool size changes of primary metabolites, such as glycine and glycolate, indicated a link to photorespiration.Methodology/Principal FindingsThe 13C labelling kinetics of primary metabolites were analysed in photoautotrophically grown cultures of Synechocystis sp. PCC 6803 by gas chromatography-mass spectrometry (GC-MS) to demonstrate the link with photorespiration. Cells pre-acclimated to high CO2 (5%, HC) or limited CO2 (0.035%, LC) conditions were pulse-labelled under very high (2% w/w) 13C-NaHCO3 (VHC) conditions followed by treatment with ambient 12C at HC and LC conditions, respectively. The 13C enrichment, relative changes in pool size, and 13C flux of selected metabolites were evaluated. We demonstrate two major paths of CO2 assimilation via Rubisco in Synechocystis, i.e., from 3PGA via PEP to aspartate, malate and citrate or, to a lesser extent, from 3PGA via glucose-6-phosphate to sucrose. The results reveal evidence of carbon channelling from 3PGA to the PEP pool. Furthermore, 13C labelling of glycolate was observed under conditions thought to suppress photorespiration. Using the glycolate-accumulating ΔglcD1 mutant, we demonstrate enhanced 13C partitioning into the glycolate pool under conditions favouring photorespiration and enhanced 13C partitioning into the glycine pool of the glycine-accumulating ΔgcvT mutant. Under LC conditions, the photorespiratory mutants ΔglcD1 and ΔgcvT showed enhanced activity of the additional carbon-fixing PEP carboxylase pathway.Conclusions/SignificanceWith our approach of non-steady-state 13C labelling and analysis of metabolite pool sizes with respective 13C enrichments, we identify the use and modulation of major pathways of carbon assimilation in Synechocystis in the presence of high and low inorganic carbon supplies.
Highlights
Cyanobacteria are considered the first organisms to have evolved the capacity for oxygenic photosynthesis around three billion years ago [1]
Experimental design of metabolic flux analysis Pulse labelling of photoautotrophic Synechocystis cultures was performed by adding aliquots of a saturated solution of 13C labelled NaHCO3 to a final concentration of 2% (w/w)
We focused on the metabolite pools that reached high 13C enrichment and allowed optimal gas chromatography-mass spectrometry (GC-MS)-based analysis
Summary
Cyanobacteria are considered the first organisms to have evolved the capacity for oxygenic photosynthesis around three billion years ago [1]. Many of the initial cyanobacterial proteins are still detectable within the chloroplasts and nuclear genomes of current higher plants [2,3]. In both cyanobacteria and C3 plants, CO2 fixation is primarily catalysed by the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Recent studies using transcript and metabolite profiles of wild-type and gene deletion mutants revealed that photorespiratory pathways are essential for the growth of Synechocystis sp. Pool size changes of primary metabolites, such as glycine and glycolate, indicated a link to photorespiration
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