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Title: Fluxomic Analysis Reveals Central Carbon Metabolism Adaptation for Diazotroph Azotobacter vinelandii Ammonium Excretion

Abstract

Diazotrophic bacteria are an attractive biological alternative to synthetic nitrogen fertilizers due to their remarkable capacity to fix atmospheric nitrogen gas to ammonium via nitrogenase enzymes. However, how diazotrophic bacteria tailor central carbon catabolism to accommodate the energy requirement for nitrogenase activity is largely unknown. In this study, we used Azotobacter vinelandii DJ and an ammonium excreting mutant, AV3 (ΔNifL), to investigate central carbon metabolism fluxes and central cell bioenergetics in response to ammonium availability and nitrogenase activity. Enabled by the powerful and reliable methodology of 13C-metabolic flux analysis, we show that the respiratory TCA cycle is upregulated in association with increased nitrogenase activity and causes a monotonic decrease in specific growth rate. Whereas the activity of the glycolytic Entner-Doudoroff pathway is positively correlated with the cell growth rate. These new observations are formulated into a 13C-metabolic flux model which further improves the understanding and interpretation of intracellular bioenergetics. This analysis leads to the conclusion that, under aerobic conditions, respiratory TCA metabolism is responsible for the supply of additional ATP and reducing equivalents required for elevated nitrogenase activity. This study provides a quantitative relationship between central carbon and nitrogen metabolism in an aerobic diazotroph for the first time.

Authors:
 [1];  [1]; ORCiD logo [1];  [1];  [1]; ORCiD logo [1]
  1. National Renewable Energy Lab. (NREL), Golden, CO (United States)
Publication Date:
Research Org.:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Org.:
USDOE National Renewable Energy Laboratory (NREL), Laboratory Directed Research and Development (LDRD) Program
OSTI Identifier:
1568045
Report Number(s):
NREL/JA-5100-74439
Journal ID: ISSN 2045-2322
Grant/Contract Number:  
AC36-08GO28308
Resource Type:
Accepted Manuscript
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Volume: 9; Journal Issue: 1; Journal ID: ISSN 2045-2322
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; fluxomic analysis; algae; azotobacter; carbon; Azotobacter vinelandii

Citation Formats

Wu, Chao, Herold, Ryan A., Knoshaug, Eric P., Wang, Bo, Xiong, Wei, and Laurens, Lieve M. L. Fluxomic Analysis Reveals Central Carbon Metabolism Adaptation for Diazotroph Azotobacter vinelandii Ammonium Excretion. United States: N. p., 2019. Web. doi:10.1038/s41598-019-49717-6.
Wu, Chao, Herold, Ryan A., Knoshaug, Eric P., Wang, Bo, Xiong, Wei, & Laurens, Lieve M. L. Fluxomic Analysis Reveals Central Carbon Metabolism Adaptation for Diazotroph Azotobacter vinelandii Ammonium Excretion. United States. doi:10.1038/s41598-019-49717-6.
Wu, Chao, Herold, Ryan A., Knoshaug, Eric P., Wang, Bo, Xiong, Wei, and Laurens, Lieve M. L. Fri . "Fluxomic Analysis Reveals Central Carbon Metabolism Adaptation for Diazotroph Azotobacter vinelandii Ammonium Excretion". United States. doi:10.1038/s41598-019-49717-6. https://www.osti.gov/servlets/purl/1568045.
@article{osti_1568045,
title = {Fluxomic Analysis Reveals Central Carbon Metabolism Adaptation for Diazotroph Azotobacter vinelandii Ammonium Excretion},
author = {Wu, Chao and Herold, Ryan A. and Knoshaug, Eric P. and Wang, Bo and Xiong, Wei and Laurens, Lieve M. L.},
abstractNote = {Diazotrophic bacteria are an attractive biological alternative to synthetic nitrogen fertilizers due to their remarkable capacity to fix atmospheric nitrogen gas to ammonium via nitrogenase enzymes. However, how diazotrophic bacteria tailor central carbon catabolism to accommodate the energy requirement for nitrogenase activity is largely unknown. In this study, we used Azotobacter vinelandii DJ and an ammonium excreting mutant, AV3 (ΔNifL), to investigate central carbon metabolism fluxes and central cell bioenergetics in response to ammonium availability and nitrogenase activity. Enabled by the powerful and reliable methodology of 13C-metabolic flux analysis, we show that the respiratory TCA cycle is upregulated in association with increased nitrogenase activity and causes a monotonic decrease in specific growth rate. Whereas the activity of the glycolytic Entner-Doudoroff pathway is positively correlated with the cell growth rate. These new observations are formulated into a 13C-metabolic flux model which further improves the understanding and interpretation of intracellular bioenergetics. This analysis leads to the conclusion that, under aerobic conditions, respiratory TCA metabolism is responsible for the supply of additional ATP and reducing equivalents required for elevated nitrogenase activity. This study provides a quantitative relationship between central carbon and nitrogen metabolism in an aerobic diazotroph for the first time.},
doi = {10.1038/s41598-019-49717-6},
journal = {Scientific Reports},
number = 1,
volume = 9,
place = {United States},
year = {2019},
month = {9}
}

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Figures / Tables:

Figure 1 Figure 1: Growth curves of A. vinelandii wt and AV3 (A,B) without and (C,D) with 50 mM ammonium. OD (green), glucose (blue), and ammonium (red) were determined during cultivation of A. vinelandii wt (solid line with triangles) and AV3 (dashed line with crosses). Data points with error bars represent meanmore » ± standard deviation of three replicates.« less

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    Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.