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Title: Characterisation of 'degenerate' cyclic di-GMP signalling proteins of Escherichia coli
Author: Whiting, Nicola
ISNI:       0000 0004 5350 5475
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2014
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Since its discovery 25 years ago, the cyclic di-GMP signalling pathway has been an everexpanding area of scientific research. This pathway, ubiquitous in Bacteria, regulates a range of bacterial phenotypes in response to various environmental cues. Two groups of enzymes make and break the secondary messenger c-di-GMP, these being the diguanylate cyclase (DGC) and phoshodiesterase (PDE) enzymes respectively. Both classes of enzymes contain a consensus motif of amino acid residues, necessary for the catalytic activity of the proteins; specifically a GGDEF motif for DGC proteins and an EAL or HD-GYP motif for PDE enzymes. However proteins exist which contain non-conserved sequences at these sites and are therefore deemed likely to be catalytically inactive. The roles of these so called 'degenerate' proteins are gradually being elucidated and have been found to be regulatory proteins, initiating a variety of cellular effects. This thesis focuses on the degenerate DGC and PDE enzymes in E. coli K-12, of which there are four. At the start of this project, two of these (YcgF and YdhA) had been characterised, whilst YdiV and YeaI were largely uncharacterised. Analysis of the YeaI protein confirmed the protein to be a catalytically inactive diguanylate cyclase, however the poor solubility of the protein vastly restricted characterisation of the protein. YdiV was shown to be a catalytically inactive phosphodiesterase. Chacterisation of the protein showed YdiV to bind to the E. coli transcriptional factor FlhDC, where it acted as an anti-FlhDC factor. The FlhDC factor is the master regulator of flagellar gene transcription, shown to bind to DNA and cause gene transcription. However, upon YdiV binding to FlhDC, DNA was shown to be dissociated thereby halting gene expression. Kinetic and thermodynamic analysis of the YdiV:FlhDC binding interaction revealed the proteins to have a nM binding affinity. In vitro transcription and EMSA analysis revealed the existence of a transitional or intermediate YdiV:FlhDC:DNA species which retains the FlhDC transcriptional activation ability. Phenotypically, overproduction of the ydiV gene caused a vast decrease in flagella production and FliC detection. The regulation of this YdiV:FlhDC interaction is currently unknown. Nucleotide binding assays have revealed a possible interaction between YdiV and c-di-GMP, which potentially could regulate the activity of YdiV and thereby the YdiV:FlhDC interaction. Furthermore, in vitro analysis has identified a YdiV:FlhDC:FliT species, which if produced in vivo, would add another level of regulation and complexity to the flagellar gene expression. Thus, YdiV has a gene regulatory role in E. coli, repressing flagella biosynthesis via its interactions with FlhDC.
Supervisor: Green, Jeff ; Artymiuk, Pete Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available