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Characterization of an anaerobic Escherichia coli K-12 cyclic-di-GMP phosphodiesterase

Reference	BB/G006024/1
Principal Investigator / Supervisor	Professor J Green
Co-Investigators / Co-Supervisors
Institution	University of Sheffield
Department	Molecular Biology and Biotechnology
Funding type	Research
Value (£)	326,364
Status	Completed
Type	Research Grant
Start date	01/07/2009
End date	03/03/2013
Duration	44 months

Abstract

Cyclic-di-GMP signalling pathways are widespread in bacteria and have important roles in controlling motility, biofilm formation and virulence. We have identified the Escherichia coli YfgF protein as a cyclic-di-GMP phosphodiesterase that is expressed under anaerobic conditions. The YfgF protein has three domains and we wish to learn more about each of these and how they contribute to the role played by YfgF in the bacterial cell. In the N-terminal region of YfgF is a MASE1 domain, a predicted membrane bound sensory region. This is followed by a GGDEF diguanylate cyclase domain that is predicted to be inactive. Finally the protein has an active EAL phosphodiesterase domain. We have shown that a yfgF mutant has altered surface properties and is sensitive to peroxide stress. By investigating the role of YfgF and its component domains we will learn more about the specific role of YfgF in the model bacterium E. coli and also discover more general features of the as yet poorly defined mechanisms underpinning cyclic-di-GMP signalling. Our preliminary work has led us to formulate an overarching hypothesis: YfgF influences E. coli cell surface properties by altering gene expression through modulation of intracellular cyclic-di-GMP concentrations in response to signals received through the MASE1 domain (peroxide?) and through the catalytically inactive GGDEF domain (nucleotide binding). To test our hypothesis we will address the following questions: 1. What is the role of the MASE1 domain in YfgF function? 2. What is the role of the GGDEF domain in YfgF function? 3. How does YfgF alter gene expression and surface protein profiles in E. coli? By answering these questions we hope to shed new light on how cyclic-di-GMP controls bacterial behaviour and how YfgF acts as a communication hub linking two important global regulatory systems, namely oxygen-sensing and cyclic-di-GMP signalling.

Summary

Bacteria are the most adaptable organisms on the planet. At the root of their ability to adapt to different conditions is the ability to select and express optimal combinations of genes for particular environments. This is generally achieved by proteins known as transcription factors which sense signal molecules and then act to turn specific genes on or off. Sometimes the transcription factor does not sense the signal directly but rather interacts with a small molecule that acts as a messenger. In these cases the signal has an effect on a metabolic process that changes the concentration of the messenger molecule in the bacterial cell, which is then sensed by the transcription factor. Recently a messenger molecule called cyclic-di-GMP has been identified in many bacteria. Cyclic-di-GMP signalling is associated with many important properties of bacteria, including the ability to move and the ability to form biofilms (communities of bacteria attached to surfaces). However, despite the widespread occurrence of cyclic-di-GMP signalling pathways little is known about the way they work. In this project we want to learn more about a protein called YfgF. Our previous work has shown us that YfgF is made by the bacterium Escherichia coli when it is starved of oxygen. We have also discovered that YfgF has the ability to breakdown cyclic-di-GMP and so should have an effect on processes controlled by this molecule, e.g. motility and biofilm formation. Because so little is known about cyclic-di-GMP signalling pathways we want to use YfgF, supported by the enormous amount of information that is known about E. coli as a model bacterium, to learn more about how cyclic-di-GMP alters patterns of gene expression. Our approach will be to isolate the YfgF protein and characterise its catalytic activities and how these change under different conditions. We will also discover which genes are influenced by the presence or absence of YfgF and then attempt to fill in the missing links betweenthe signal perceived, the change in YfgF activity and the genes that are regulated. In this way we will learn more about how YfgF works and also learn some general lessons on how other proteins that interact with cyclic-di-GMP work. This could have important implications because bacteria because many infections are associated with oxygen-starved biofilms.

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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