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Title: Characterisation of the fixABCX operon in symbiotic nitrogen fixation
Author: Webb, Isabel
ISNI:       0000 0004 6059 9297
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2016
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The fixABCX genes are essential for nitrogen fixation in R. leguminosarum bv. viciae 3841. Comparison of FixABCX to homologous proteins across the Kingdoms of Life suggests a role in electron transport to nitrogenase, which requires eight electrons per molecule of dinitrogen fixed. Mutation of this operon leads to bacteroids unable to fix nitrogen in symbiosis with P. sativum (pea). Electron microscopy revealed a drastically altered bacteroid morphology in fixAB mutants, revealing insights into the developmental response of both plant and bacteria to a lack of nitrogen fixation. Observations from electron microscopy were coupled to data obtained using single-cell Raman microscopy in order to understand metabolite production in nitrogen fixing and non-fixing bacteroids. The promoter controlling fixA has been characterised to a minimal region consisting of binding sites for NifA, the general transcriptional activator of nitrogen fixation, and RpoN (σ54), its cognate sigma factor. Mutation analysis reveals that fixABCX is part of a larger operon including the nifA gene. Promoter analysis of the downstream genes has identified a set of basal promoters found within the fixCX region, which control expression of the nifA gene. Control of nitrogen fixation occurs at the post-transcriptional level, whereby NifA is able to activate nitrogen fixation genes, including the fixABCXnifA operon, autoregulating its own expression under nitrogen-fixing conditions. Pull-down assays have revealed protein-protein interactions between FixAB and nitrogenase, as well as an interaction with both pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase. FixAB may interact with these dehydrogenases and via electron bifurcation couple the exergonic reduction of the quinone pool to the endergonic reduction of ferredoxin and subsequently nitrogenase. Furthermore, FixAB, nitrogenase and pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase may form a supra-molecular complex within nitrogen fixing bacteroids.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available