Allosteric control of type III secretion systems by the second message cyclic-di-GMP

Trampari, Eleftheria (2016) Allosteric control of type III secretion systems by the second message cyclic-di-GMP. Doctoral thesis, University of East Anglia.

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Cyclic di-GMP (cdG) is a ubiquitous second messenger in bacteria, regulating transcriptional and post-transcriptional processes and allosterically controlling protein function. While the mechanisms of cdG metabolism are well understood, the downstream targets of this molecule are poorly characterised.
To understand the role of cdG signalling in plant-associated species, cdG-capture compound pull-down experiments were performed to identify potential binding proteins in Pseudomonas fluorescens. One of the top targets identified was the flagella export AAA+ ATPase FliI, which was shown to bind specifically and tightly to cdG. FliI-cdG interaction was demonstrated for diverse bacterial FliI homologs. Excitingly, high-affinity binding was observed for the type-III secretion system (T3SS) homolog, HrcN and the type-VI ATPase, ClpB2. A combination of techniques was used to predict the FliI cdG binding site at the interface between two FliI subunits.
Although the addition of cdG inhibits the ATPase activity of both FliI and HrcN in vitro, this occurs at a non-physiological cdG concentration suggesting that this does not represent the in vivo role of binding. However, when cdG concentrations are artificially increased, the export of flagellin subunits is significantly reduced, suggesting a link between cdG binding and protein export. Changes in the in vitro multimerization state of the protein were also observed upon the addition of cdG.
As part of this study, novel and highly specific tools for nucleotide-protein interactions were developed and existing biochemistry techniques were optimised. These assays were
employed to characterise cdG binding to four more proteins, which were identified as cdG binders.
The results generated in this study broaden the existing knowledge about cdG binding protein diversity. The identification of FliI as a cdG binder suggests a novel cdG-dependent control mechanism for the function of bacterial export pathways including the flagellum and the T3SS, through allosteric interaction with export ATPase proteins.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Jackie Webb
Date Deposited: 30 Nov 2016 14:28
Last Modified: 24 Nov 2019 01:38


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