The unicellular diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142 (Cyanothece 51142), is able to grow aerobically in nitrogen-fixing conditions under continuous illumination. This study investigated the impacts of carbon and nitrogen sources on Cyanothece 51142 metabolism via 13C-assisted metabolite analysis and biochemical measurements. Cyanothece 51142 was cultivated using 13C-labeled glycerol, glucose or pyruvate, and the resulting patterns of derivatized metabolites were determined by gas chromatography-mass spectrometry (GC-MS) to track the active pathways. The isotopomer data were then integrated with transcription and enzyme activity measurement to link Cyanothece 51142 physiology with functional genes in the central metabolism. Under continuous light and nitrogen-fixing conditions, we find glycerol addition promoted aerobic catabolism and nitrogenase-dependent hydrogen production (up to 25 µmol H2/mg chlorophyll/hr), but strongly reduced phototrophic CO2 utilization. Under nitrogen-sufficient conditions, Cyanothece 51142 was able to metabolize glycerol photoheterotrophically without measurable CO2 fixation, while its activity of light dependent reaction was not reduced. Isotopomer analysis also detected that Cyanothece 51142 was able to fix CO2 via anaplerotic pathways, and to uptake glucose and pyruvate for mixotrophic biomass synthesis. Based on the unique isoleucine labeling pattern, we discovered that Cyanothece 51142 employs an atypical citramalate pathway for isoleucine synthesis, with pyruvate and acetyl-CoA as precursors. This study demonstrates the merits of combining 13C-assisted metabolite analysis, enzyme assays, and metabolite detection not only to understand cellular metabolisms, but also to discover novel enzyme activities.
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