Regulation of Nitrate and Nitrite Assimilation in Paracoccus denitrificans at the Level of RNA

Pinchbeck, Benjamin (2016) Regulation of Nitrate and Nitrite Assimilation in Paracoccus denitrificans at the Level of RNA. Doctoral thesis, University of East Anglia.

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Paracoccus denitrificans, a model Alphaproteobacteria soil denitrifier, can grow solely on nitrate or nitrite as inorganic nitrogen sources using a specialised cytoplasmic assimilatory nitrate/nitrite reducing pathway; Nas. This growth capability is phylogenetically represented throughout heterotrophic and autotrophic bacteria, plants and fungi. Whilst this metabolism has been extensively studied in the latter two, the regulatory mechanisms by which organoheterotrophic bacteria govern this nitrate-dependant metabolism are less understood. The work conducted here primarily investigated genetic regulation of Nas expression in P. denitrificans.
In Gram-negative bacteria, transcription of proteins required to import and reduce nitrate/nitrite to ammonium, for nitrogen assimilation, are subject to dual control; promotion in the absence of ammonium by the general nitrogen regulatory system, NtrBC, and nitrate-induced transcriptional anti-termination by the two-component, NasT-NasS complex. Here, a hypothetical gene, nifR3, conserved with the ntr cluster throughout Alphaproteobacteria, was shown to regulate Nas biosynthesis.
We report nifR3 encodes a nitrogen-responsive, tRNA-dihydrouridine synthase required for nasABGHC translation. Genomic deletion of nifR3 from P. denitrificans resulted in the lethal loss of nitrate assimilation and severe deficiency of dihydrouridine in tRNA, restored by genetic complementation of nifR3 in trans. Pure NifR3 harboured an FMN cofactor and reversibly catalysed NADH-dependant reduction of uridine, a physiological important post-transcriptional modification. Native band-shift assays using an isolated tRNA fraction of P. denitrificans identified specific targets of NifR3: mature tRNA transcripts encoding PheGAA, LysUUU and TrpCCA. This novel regulatory role of bacterial NifR3 and tRNA-dihydrouridine formation concerning post-transcriptional fine-turning of protein expression will be discussed throughout this thesis, in addition to the function of several other nitrogen-responsive proteins explored here.
Separately, we demonstrated that NarJ, the molybdenum-chaperone for biogenesis of respiratory nitrate reductase, NarG, performs an unprecedented wide-spread maturation role of non-Nar nitrate reductases. Here, we found NarJ is solely responsible for fully assembling the functional assimilatory nitrate reductase, NasC, complete with cofactors, even under aerobic conditions.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Users 4971 not found.
Date Deposited: 21 Jul 2017 08:27
Last Modified: 30 Apr 2020 00:38


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