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Burkholderia sigma factor systems with metal-responsive regulatory domains

Banyard, Christopher (2019) Burkholderia sigma factor systems with metal-responsive regulatory domains. PhD thesis, University of Sheffield.

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Burkholderia pseudomallei and Burkholderia cenocepacia are pathogenic bacteria that are the causative agents of the deadly diseases melioidosis and ‘cepacia syndrome’, respectively. Both species utilise extracytoplasmic function (ECF) sigma factors, alternative subunits of bacterial RNA polymerase, to transduce extracellular signals and drive the transcription of specific genes. MbaS, from Burkholderia pseudomallei, and OrbS, from Burkholderia cenocepacia, are two iron starvation (IS) sigma factors that regulate gene clusters associated with the iron-chelating siderophores mallebaction and ornibactin, respectively. Evidence has been presented in this study that these proteins contain on-board C-terminal regulatory extensions that transduce the presence of selected metal ions via cysteine thiol groups. Firstly, C-terminal trucnations of OrbS were assayed through colorimetric siderophore assays and promoter-reporter analyses to demonstrate that the C-terminal extension is required for sigma factor activity, and may be necessary for iron-dependent regulation. A series of gene deletions were also introduced into B. thailandensis, a safer surrogate species of B. pseudomallei, through allelic exchange to delete mbaS and the secondary siderophore pyochelin. Through use of these constructed mutants and promoter-reporter analyses, the target promoters of MbaS have been identified and Fur regulation of the sigma factor has been established. Using further promoter-reporter analyses and in vitro techniques to assay transcription activity and protein-protein interaction, it has been demonstrated that the sigma factor activity of OrbS is inhibited in the presence of Fe(II), Cu(II) and Zn(II) ions, and the sigma factor activity of MbaS is inhibited in the presence of Zn(II). This could represent a novel regulation mechanism of a class of bacterial sigma factors.

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Medicine, Dentistry and Health (Sheffield)
Depositing User: Christopher Banyard
Date Deposited: 08 Apr 2020 14:01
Last Modified: 08 Apr 2020 14:01
URI: http://etheses.whiterose.ac.uk/id/eprint/26355

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