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Metabolic control of quorum sensing signal molecule generation and response

Reference	BB/D007038/1
Principal Investigator / Supervisor	Professor Paul Williams
Co-Investigators / Co-Supervisors	Professor David Barrett, Professor Miguel Camara, Professor Kim Hardie, Dr Klaus Winzer
Institution	University of Nottingham
Department	Inst of Infections and Immunity
Funding type	Research
Value (£)	289,383
Status	Completed
Type	Research Grant
Start date	01/11/2005
End date	30/04/2009
Duration	42 months

Abstract

Cell-to-cell communication (quorum sensing [QS]) promotes multicellular behaviour in unicellular organisms by co-ordinating changes in gene expression with cell population density through the deployment of diffusible signal molecules (QSSMs). QS does not function in isolation but is one of multiple environmental parameters which a cell must integrate to adapt and survive within a given ecological niche. Thus adaptation to diverse environmental stresses will influence the activity of metabolic pathways which in turn will modulate QSSM synthesis and impact on the ability of an organism to exhibit the multicellular adaptive behaviour characteristic of QS. There have been few studies of the impact of metabolism on QS. Furthermore, the metabolic burden imposed upon the cell by the production of QSSMs has not been investigated. Despite their chemical diversity, QSSMs e.g. N-acylhomoserine lactones (AHLs), autoinducer-2 (AI-2) and the 4-quinolones (4Qs) draw upon a small subset of metabolic pathways, including the Activated Methyl Cycle (AMC), fatty acid and aromatic amino acid biosynthesis. Given this inter-dependence, metabolic activity will clearly impact on QSSM production with significant implications for both bacterial behaviour. To evaluate the impact of metabolism on QS in E. coli and Pseudomonas aeruginosa, we will exploit quantitative LC-MS and GC-MS metabolite and QSSM profiling as well as metabolite flux analysis and the NMR-MS and GC-MS metabolite fingerprinting and data handling facilities of MeT-RO. E. coli and P. aeruginosa were chosen for their contrasting lifestyles and because they use alternative AMC pathways to generate different QSSMs. E. coli produces AI-2 via an AMC incorporating LuxS (S-ribosylhomocysteine cleavage enzyme) and Pfs (5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase) whereas P. aeruginosa synthesizes AHLs using SAM generated via an AMC which incorporates an S-adenosylhomocysteine (SAH) hydolase rather than LuxS and Pfs. ForP. aeruginosa, 4Q biosynthesis is regulated by AHL-dependent QS and both depend on fatty acid biosynthesis for their acyl side chains. 4Q biosynthesis also draws from aromatic amino acid biosynthesis. Thus AHL and 4Q biosynthesis are intimately linked and we have also discovered that mutation of the mexGHI-opmD efflux pump results in the loss of QSSM synthesis in P. aeruginosa as a consequence of the altered metabolism of 4Q precursors. The primary aims of the project are to determine: (i) How growth phase, growth rate, nutrient limitation and population density influence QSSM biosynthesis through the modulation of metabolism. (ii) How manipulation of the AMC by mutagenesis of key AMC pathway enzymes impact on QS, (iii) the nature of the metabolic burden imposed by QSSM biosynthesis and (iv) How mutations in an efflux pump result in the loss of QSSM production through altered metabolism. To achieve our objective of understanding how environmental modulation of metabolism impacts on QS, we will employ both batch culture (to determine the influence of growth stage) and continuous culture (to determine the effects of growth rate and population density) in media limited for specific nutrients (C, N, S, P or Fe). How manipulation of the AMC impacts on QSSM synthesis will be investigated using mutants defective in key AMC enzymes (LuxS/Pfs in E. coli or Sah hydrolase in P. aeruginosa). The nature of the metabolic burden imposed by QSSM biosynthesis will be assessed by introducing AHL synthases (LasI and RhlI) into E. coli and by analysing the metabolome of lasI rhlI negative mutants of P. aeruginosa. Since the MexGHI-OpmD efflux pump has a dramatic effect on AHL and 4Q synthesis in P. aeruginosa, we will seek to characterize the metabolic defect. These approaches offer a systematic, quantitative approach to a comprehensive understanding of the how environmental modulation of metabolism impacts on QS.

Summary

Bacteria talk with each other by producing and detecting small signal molecules. These co-ordinate the behaviour of both beneficial and disease-causing bacteria and enable a population of bacteria to act as a group rather than as individuals. This is called quorum sensing (QS) and the mechanism through which the quorum sensing signals act has mainly been studied at the level of gene regulation. Despite the knowledge that the production of the QS signals is directly linked to the availability of starting molecules generated through the assimilation of nutrients by the cell (a process referred to as metabolism), there have been no studies aimed at determining the burden that QS signal production has upon metabolism and consequently the well-being of the organism. Studies to determine how changes in metabolism affect QS signal production have similarly not been attempted. In this project, our ultimate aim is to fill this gap. To do this, we will monitor the amounts of the starting molecules required for chemical signal production and the speed by which they are produced and turned into other products within two different bacterial species (E. coli and Pseudomonas aeruginosa). We have chosen these representative microbes because they have contrasting lifestyles, produce different kinds of QS signals and so have different pathways to synthesize them. We will assess the affects upon metabolism and QS signal synthesis simultaneously using liquid chromatography high resolution mass spectrometry and relate it to the speed of bacterial growth, the number of cells in the bacterial population, and the state of the cell (is it adapting to a new environment, is it reproducing at the maximum rate in a given environment, or has it exhausted the potential of the environment and is implementing long term survival mechanisms. In addition we shall investigate the impact of growth in conditions where there is a shortage of one of the major metabolic building blocks as this will have knock on effects on the concentration of QS signal molecules available in the cells since resources must be divided between their generation and that of more essential cellular components. Finally we shall look to see what happens if a protein which serves to pump molecules out of the cell is inactivated. We already know that disrupting the action of this pump prevents QS signal production, but the basis of this blockage is unclear. By characterizing the starting molecules available and their quantities we hope to find out what molecule is exported by the pump when it is working and how the loss of this function is linked to QS signal generation. Unravelling the interplay between bacterial metabolism and the production of QS signals will have applications within fundamental research (to understand bacterial metabolism), medicine (as a means of diagnosis, or to monitor treatment), agriculture (control of plant pathogens and use of bacteria as biocontrol agents) and industry (in the analysis of products). Thus this research will be of benefit by providing useful scientific tools and contribute to the understanding of important biological systems.

Committee	Closed Committee - Plant & Microbial Sciences (PMS)
Research Topics	Microbiology
Research Priority	X – Research Priority information not available
Research Initiative	Initiative in Plant and Microbial Metabolomics (MET) [2003-2005]
Funding Scheme	X – not Funded via a specific Funding Scheme

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