cb6b00890_si_003.xlsx (10.35 kB)
Extensive Turnover of Compatible Solutes in Cyanobacteria Revealed by Deuterium Oxide (D2O) Stable Isotope Probing
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posted on 2017-01-09, 00:00 authored by Richard Baran, Rebecca Lau, Benjamin P. Bowen, Spencer Diamond, Nick Jose, Ferran Garcia-Pichel, Trent R. NorthenCyanobacteria
are important primary producers of organic matter
in diverse environments on a global scale. While mechanisms of CO2 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 (D2O) 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).
D2O 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 13C-labeled Synechococcus sp. PCC 7002 were exposed
to 12C glucosylglycerol. Following metabolite extraction,
amylase treatment of methanol-insoluble polymers revealed 12C labeling of glycogen. Overall, our work shows that D2O probing is a powerful method for analysis of cyanobacterial metabolism
including discovery of novel metabolic processes.
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euryhaline unicellular strainCO 2 fixationExtensive Turnoverdiel cyclecyanobacterial metabolismfilamentous strainCyanobacteria Revealedintracellular metabolitesnonlabile positionsDeuterium OxideSynechococcus sp12 C glucosylglycerolStable IsotopesolutePCC 7002D 2 Ochromatography mass spectrometrydeuterium oxideCompatible Solutesmethanol-insoluble polymerstime periodsintracellular osmolaritymetabolite turnoverbiosynthetic end pointslight phase12 Camylase treatmentgrowth mediummeasure deuterium incorporation12 h13 C-labeled Synechococcus spmetabolite extraction
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