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

Lead Research Organisation: University of Sheffield
Department Name: Molecular Biology and Biotechnology

Abstract

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 between the 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.

Technical Summary

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.
 
Description Bacteria are remarkably versatile. They can adapt their behaviour to survive and thrive in very diverse environments. Many bacteria produce a small molecule known as cyclic di-GMP to control whether they are adopt a motile, free-swimming lifestyle or whether they become attached to surfaces as a biofilm. Generally, low concentrations of cyclic di-GMP result in motility, whereas high concentrations of cyclic di-GMP promote biofilm formation. The concentration of cyclic di-GMP in the bacterium is set by the action of opposing enzymes. Multiple diguanylate cyclases (DGC) synthesise cyclic di-GMP, which can be degraded by multiple phosphodiesterases (PDE). The relative activities of these enzymes are governed by environmental signals that are perceived by protein modules often fused to the DGC or PDE domains. In this research we have focussed on the YfgF proteins of the enteric bacteria Escherichia coli and Salmonella. YfgF is anchored to the bacterial membrane by a MASE1 domain; the function of this domain was unknown. Attached to the YfgF MASE1 domain is a DGC, which is followed by a PDE domain. We showed that the yfgF gene is maximally expressed when the bacteria are starved of oxygen. Measurements of DGC and PDE activities demonstrated that the DGC component of YfgF is inactive but the PDE region is active. Furthermore, the PDE activity of YfgF was modulated by the MASE1 and DGC domains suggesting that these latter regions of the protein were involved in signal perception/transduction. A mutant of E. coli lacking the yfgF gene was sensitive to oxidative shock, suggesting that YfgF played a role in counteracting the deleterious effects that occur when anaerobic bacteria encounter oxygen. To investigate this possibility we created a strain of Salmonella that was able to express YfgF in the presence and absence of oxygen. This sustained expression of YfgF was shown to inhibition the ability of Salmonella to move towards aspartate, an amino acid that can support growth of the bacterium; this phenomenon is known as aspartate chemotaxis. However, this inhibition only occurred in the presence of oxygen and was independent of the ability of YfgF to degrade cyclic di-GMP, because it occurred in a Salmonella strain that lacked the ability to synthesize cyclic di-GMP and also when PDE activity of YfgF was abolished by replacing an essential amino acid of the active site. The YfgF-mediated inhibition of aerobic aspartate chemotaxis was impaired in the presence of the reagent p-benzoquinone, which alters the redox environment of the bacterium. We then showed that the ability of YfgF to inhibit aspartate chemotaxis required a motif of charged amino acids located in the MASE1 domain of the protein. When anaerobic bacteria are exposed to oxygen some enzymes can transfer electrons to oxygen producing highly damaging reactive oxygen species. The enzymes that are the major sources of the reactive oxygen species are fuelled directly or indirectly by aspartate. Thus, the YfgF protein provides the bacteria with a fuse to cut-off the supply of aspartate when anaerobic bacteria are exposed to oxygen, thereby limiting the damage to cell components by reactive oxygen species. Furthermore, these studies demonstrated for the first time the function of a MASE1 domain as a redox-responsive regulator. This is significant because MASE1 domains are known to be present in ~500 bacterial species where they are often linked to other protein domains involved in signalling pathways, suggesting that these poorly characterized MASE1 domains could have widespread effects on bacterial behaviour.
Exploitation Route We have provided a better understanding of function of a previously uncharacterized transmembrane protein domain and its relationship to a downstream signalling unit. This might in the longer term find some utility as a biosensor.
Sectors Education,Other
URL https://www.sheffield.ac.uk/mbb/staff/green
 
Description Improved quality of life by increased understanding of a fundamental biological process.
First Year Of Impact 2013
Sector Education
Impact Types Cultural
 
Description From the earth to our plate: exploring current food issues 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact This project included a visit to a local community farm as well as classroom-based sessions with a local primary school. The presence of good and bad microbes at the farm was be discussed, with links to swine flu and other animal-borne diseases introduced, and a microbiology practical carried out on the farm.

no actual impacts realised to date
Year(s) Of Engagement Activity 2010
 
Description Science Badge Sheffield Guide Group 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact General microbiology, crystalography and general science (2 x 1.5 hour sessions). September 2009.

no actual impacts realised to date
Year(s) Of Engagement Activity 2009
 
Description Science badge with local brownie group (7-10 years) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact General microbiology, hand hygiene and general science (2 x 1 hour sessions). November 2011.

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
Description Workshops on general microbiology, antibiotic resistance, microbiology at the farm. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact National science and engineering week events with Primary school pupils (5-11 years). March 2010.

no actual impacts realised to date
Year(s) Of Engagement Activity 2010