Global Molecular Analyses of Methane Metabolism in Methanotrophic Alphaproteobacterium, Methylosinus trichosporium OB3b. Part II. Metabolomics and 13C-Labeling Study

Song Yang,Martin Sadilek,Justin Clubb,Victoria J Orphan,David Beck,Abigail Green-Saxena,Janet B Matsen,Marina G Kalyuzhnaya,Michael Konopka

doi:10.3389/fmicb.2013.00070

Song Yang, Martin Sadilek + Show 7 more

Open Access

https://doi.org/10.3389/fmicb.2013.00070

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Abstract

In this work we use metabolomics and 13C-labeling data to refine central metabolic pathways for methane utilization in Methylosinus trichosporium OB3b, a model alphaproteobacterial methanotrophic bacterium. We demonstrate here that similar to non-methane utilizing methylotrophic alphaproteobacteria the core metabolism of the microbe is represented by several tightly connected metabolic cycles, such as the serine pathway, the ethylmalonyl-CoA (EMC) pathway, and the citric acid (TCA) cycle. Both in silico estimations and stable isotope labeling experiments combined with single cell (NanoSIMS) and bulk biomass analyses indicate that a significantly larger portion of the cell carbon (over 60%) is derived from CO2 in this methanotroph. Our13 C-labeling studies revealed an unusual topology of the assimilatory network in which phosph(enol) pyruvate/pyruvate interconversions are key metabolic switches. A set of additional pathways for carbon fixation are identified and discussed.

Highlights

Microbial oxidation of methane is one of the key elements of the global carbon cycle
The ability to oxidize methane has been demonstrated in two classes of Proteobacteria,Alpha, and Gamma
While the key metabolic pathways for carbon assimilation in M. trichosporium OB3b have been predicted (Strom et al, 1974), several fundamental questions have never been answered, such as how cells regenerate glyoxylate (Anthony, 1982), what is the role of the the citric acid (TCA) cycle in methanotrophic metabolism is (Patel et al, 1979; Shishkina and Trotsenko, 1982), and why CO2 supplementation has a significant positive effect on cell growth (Park et al, 1991, 1992)

Summary

INTRODUCTION

Microbial oxidation of methane is one of the key elements of the global carbon cycle. While the key metabolic pathways for carbon assimilation in M. trichosporium OB3b have been predicted (Strom et al, 1974), several fundamental questions have never been answered, such as how cells regenerate glyoxylate (Anthony, 1982), what is the role of the TCA cycle in methanotrophic metabolism is (Patel et al, 1979; Shishkina and Trotsenko, 1982), and why CO2 supplementation has a significant positive effect on cell growth (Park et al, 1991, 1992). Models of carbon assimilation pathways in M. trichosporium OB3b predicted by biochemical characterization (Strom et al, 1974; Trotsenko and Murrell, 2008) and global gene expression analysis (Matsen et al, 2013) are further tested by metabolomic and 13C-labeled studies

RESULTS AND DISCUSSION

CONCLUSION

EXPERIMENTAL PROCEDURES