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Dissertation
INSIGHTS INTO MECHANISMS OF REDOX HOMEOSTASIS IN NITROGEN-FIXING AZOTOBACTER VINELANDII
Washington State University
Doctor of Philosophy (PhD), Washington State University
01/2021
DOI:
https://doi.org/10.7273/000003142
Handle:
https://hdl.handle.net/2376/122353
Abstract
Overuse of industrial nitrogen fertilizers causes environmental degradation through runoff, lowers soil pH, and generates greenhouse gas emissions. There is considerable interest in promoting biological nitrogen fixation (BNF) as a mechanism to reduce the inputs of nitrogen fertilizers in agriculture. However, considerable fundamental knowledge gaps need to be addressed to realize the potential impacts of BNF in agriculture. BNF is catalyzed by the enzyme nitrogenase, which requires a large amount of energy in the form of ATP and low potential electrons. Nitrogen-fixing organisms that respire aerobically have an advantage in meeting the energy demands of BNF but face challenges of protecting nitrogenase from inactivation by oxygen. The model aerobic nitrogen-fixing bacteria Azotobacter vinelandii has a unique electron transport system that accommodates excess oxygen while producing enough energy for nitrogenase. The mechanism, termed respiratory protection, consumes excess oxygen at the membrane and leads to lower oxygen accumulation in the cytosol. A. vinelandii also contains two enzymes, Rnf and Fix, that deliver reductant to nitrogenase in the form of low potential electrons. A physiological and systems-based approach was taken to understand how the dynamics of the electron transport systems are integrated to protect nitrogenase from oxygen while simultaneously creating enough energy for nitrogen fixation. A metabolic model of A. vinelandii was constructed that accurately determines growth rate under high oxygen and substrate concentrations, demonstrating the large flux directed to the respiratory protection mechanism. Interestingly, the respiratory protection mechanism is also required for accurate predictions even when ammonia is supplemented during growth, suggesting that respiratory protection might be an essential part of normal growth rather than strictly reserved for nitrogenase protection. The model has also shown how A. vinelandii can adapt under different oxygen concentrations, metal availability, and ammonia excreting phenotype by rearranging flux through the electron transport system. Through mutational studies of electron transport enzymes Rnf and Fix, Fix was shown to be required under low oxygen concentrations, and Rnf contributes to the regulation of redox homeostasis under high oxygen. Understanding the energy dynamics in aerobic nitrogen fixation will further allow the development of biotechnologies that can help supplement industrial nitrogenous fertilizer
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Details
- Title
- INSIGHTS INTO MECHANISMS OF REDOX HOMEOSTASIS IN NITROGEN-FIXING AZOTOBACTER VINELANDII
- Creators
- Alexander Alleman
- Contributors
- John W Peters (Advisor)Michael L Kahn (Committee Member)Helmut Kirchhoff (Committee Member)B. Markus Lange (Committee Member)
- Awarding Institution
- Washington State University
- Academic Unit
- Program in Molecular Plant Sciences
- Theses and Dissertations
- Doctor of Philosophy (PhD), Washington State University
- Publisher
- Washington State University
- Number of pages
- 162
- Identifiers
- 99900651901001842
- Language
- English
- Resource Type
- Dissertation