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Structure and activity investigations of the cell fate determinant, SpoIIE, from Bacillus subtilis

Tunaley, J A (2013) Structure and activity investigations of the cell fate determinant, SpoIIE, from Bacillus subtilis. PhD thesis, University of York.

James Alan Tunaley - Structure and Activity Investigations of the Cell Fate Determinant, SpoIIE, from Bacillus subtilis - Submission2.pdf
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For many years the Gram positive bacterium Bacillus subtilis has been a model organism for prokaryotic cell and molecular biology. The asymmetric cell division which B. subtilis undergoes during sporulation is a simple system by which to study the process of cell differentiation. Sporulation is governed by a series of genetic temporal and spatial controls. Gene regulation brought about by a series of σ factors and transcriptional regulators is coupled to key morphological stages or checkpoints. σF initiates the first step in a cascade of complex genetic control which eventually produces a resilient endospore. The activation of σF, the first compartment-specific sigma factor, in the forespore and its regulation through interaction between three proteins; SpoIIAA, SpoIIAB and SpoIIE, is of particular interest. SpoIIE, a protein phosphatase which binds to the asymmetric division septum, is a crucial factor in the selective activation of σF in the forespore. Of three putative domains in SpoIIE only the C-terminal PP2C phosphatase domain has been structurally characterised. The central domain, domain II, of SpoIIE has been assigned a role in interaction with the cell division machinery; however mutational studies have shown that, in addition, this domain is also responsible for the regulation of phosphatase activity. This work describes the isolation and characterisation of three new fragments of SpoIIE containing elements of the central cytoplasmic domain of SpoIIE. These include a fragment found to accurately represent the N-terminal solubility limit of domain II which shows a high degree of oligomeric character. The fragments isolated show specific phosphatase activity against SpoIIAA~P, albeit at reduced rates compared to the free phosphatase domain, which indicates an inhibitory role for SpoIIE domain II against the PP2C domain. Three ultimately unsuccessful approaches were attempted to isolate a co-complex of SpoIIE and SpoIIAA~P for structural characterisation. A tendency for domain II containing SpoIIE fragments to precipitate in the presence of Mn(2+) is also identified. An in vivo investigation into the sporulation efficiencies of amino acid substitutions in a potential regulatory interface between domains II and III of SpoIIE indicated no strong sporulation defects.

Item Type: Thesis (PhD)
Keywords: Sporulation, Bacillus, SpoIIE, regulation, spore, endospore, sigma factor.
Academic Units: The University of York > Chemistry (York)
Identification Number/EthosID: uk.bl.ethos.595180
Depositing User: Mr J A Tunaley
Date Deposited: 25 Mar 2014 13:16
Last Modified: 08 Sep 2016 13:30
URI: http://etheses.whiterose.ac.uk/id/eprint/5430

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