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Cyanobacteria are important primary producers of organic matter in diverse environments on a global scale. While mechanisms of CO 2 fixation are well understood, the distribution of the flow of fixed organic carbon within individual cells and complex microbial communities is less well characterized. To obtain a general overview of metabolism, we describe the use of deuterium oxide (D 2 O) to measure deuterium incorporation into the intracellular metabolites of two physiologically diverse cyanobacteria: a terrestrial filamentous strain (Microcoleus vaginatus PCC 9802) and a euryhaline unicellular strain (Synechococcus sp. PCC 7002). D 2 O was added to the growth medium during different phases of the diel cycle. Incorporation of deuterium into metabolites at nonlabile positions, an indicator of metabolite turnover, was assessed using liquid chromatography mass spectrometry. Expectedly, large differences in turnover among metabolites were observed. Some metabolites, such as fatty acids, did not show significant turnover over 12-24 h time periods but did turn over during longer time periods. Unexpectedly, metabolites commonly regarded to act as compatible solutes, including glutamate, glucosylglycerol, and a dihexose, showed extensive turnover compared to most other metabolites already after 12 h, but only during the light phase in the cycle. The observed extensive turnover is surprising considering the conventional view on compatible solutes as biosynthetic end points given the relatively slow growth and constant osmotic conditions. This suggests the possibility of a metabolic sink for some compatible solutes (e.g., into glycogen) that allows for rapid modulation of intracellular osmolarity. To investigate this, uniformly 13 C-labeled Synechococcus sp. PCC 7002 were exposed to 12 C glucosylglycerol. Following metabolite extraction, amylase treatment of methanol-insoluble polymers revealed 12 C labeling of glycogen. Overall, our work shows that D 2 O probing is a powerful method for analysis of cyanobacterial metabolism including discovery of novel metabolic processes.

Extensive Turnover of Compatible Solutes in Cyanobacteria Revealed by Deuterium Oxide (D2O) Stable Isotope Probing

Extensive Turnover of Compatible Solutes in Cyanobacteria Revealed by Deuterium Oxide (D₂O) Stable Isotope Probing