Characterisation of the fixABCX operon in symbiotic nitrogen fixation

Webb, Isabel (2016) Characterisation of the fixABCX operon in symbiotic nitrogen fixation. Doctoral thesis, University of East Anglia.

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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.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Users 4971 not found.
Date Deposited: 24 Apr 2017 13:45
Last Modified: 24 Apr 2017 13:45


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