Dissertation
The Genetic Regulation of Biological Nitrogen Fixation
Washington State University
Doctor of Philosophy (PhD), Washington State University
2023
DOI:
https://doi.org/10.7273/000005381
Abstract
Access to available nitrogen commonly limits plant growth and the agricultural demand for nitrogen is largely supplemented with synthetic nitrogen fertilizers, which cause deleterious environmental effects including soil acidification and greenhouse gas emissions. To reduce our dependence on synthetic fertilizers, there is considerable interest in leveraging Biological Nitrogen Fixation (BNF) to supplement agricultural nitrogen demands, thereby improving land stewardship and agricultural sustainability. In the model diazotroph Azotobacter vinelandii, transcriptional control of components required for BNF is mediated by the NifL-NifA two-component system. NifA is a member of the enhancer-binding protein family that catalyzes the σ54-dependant transcription of genes involved in BNF. NifA activity is stringently controlled through interactions with the inhibitor protein NifL, which undergoes signal-dependent conformational changes, binding to NifA to form the inhibitory NifL-NifA complex in response to intracellular oxygen, low cellular energy, or interaction with the PII nitrogen signal transduction protein GlnK. In work described here, we take a physiological and structural approach to understand how the NifL-NifA system transduces redox conditions and small molecule binding to mediate the transcriptional regulation of BNF. Using a combination of protein structure prediction, small angle x-ray scattering, and a suite of mass spectrometry coupled techniques, we generate and empirically validate structural models NifL and GlnK poised in specific regulatory conformations. Results from both surface labelling coupled mass spectrometry and electron density maps calculated from small angle x-ray scattering data reveal conformational differences throughout the structure of NifL under reduced and oxidized conditions and an extended T-loop conformation in GlnK in the presence of ATP and 2-oxoglutarate that provide the basis for a model for modulating NifL-NifA and GlnK-NifL-NifA complex formation in the regulation of nitrogen fixation in response to oxygen and cellular fixed nitrogen status in the model diazotroph, Azotobacter vinelandii. Importantly, this work validates a strategy for structural investigation of conformationally dynamic and flexible proteins that can be applied to other interesting and important proteins.
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Details
- Title
- The Genetic Regulation of Biological Nitrogen Fixation
- Creators
- Nathaniel R. Boyer
- Contributors
- John W. Peters (Advisor)B. Markus Lange (Advisor)Mechthild Tegeder (Committee Member)Michael Kahn (Committee Member)Ray Dixon (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
- 152
- Identifiers
- 99901031240401842
- Language
- English
- Resource Type
- Dissertation