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Bacterial metabolic engineering: forced adaptive evolution of quorum sensing control of virulence and secondary metabolism by chemical selections

Reference	BB/E015581/1
Principal Investigator / Supervisor	Professor George Salmond
Co-Investigators / Co-Supervisors	Professor David Spring
Institution	University of Cambridge
Department	Biochemistry
Funding type	Research
Value (£)	465,021
Status	Completed
Type	Research Grant
Start date	01/09/2007
End date	31/08/2010
Duration	36 months

Abstract

Quorum sensing (QS) is a physiological mechanism based on small chemical signals through which bacteria regulate expression of multi-gene sets in response to cell population density. Genes under QS control include those for virulence and secondary metabolite biosynthesis in some pathogens of animals and plants. The main players in the N-acyl homoserine lactone (N-AHL)-based QS regulatory systems are LuxI-like proteins and LuxR-like proteins. LuxI-like proteins are enzymes that make the specific N-AHL whereas the LuxR-like proteins bind the ligand and bind DNA sequences upstream of the corresponding target genes. This leads to activation, repression or de-repression of the corresponding target genes. There have been recent suggestions that some luxIR-type genes are, or may have been, mobile and have been acquired by horizontal gene transfer in some bacterial strains, consistent with the view that these systems are essentially modular and evolutionarily plastic. We will test this modularity concept using a forced evolution approach in bacterial pathogens that are physiologically native except for the evolved QS component(s), avoiding multicopy / concentration artefacts. Using the phytopathogen, Erwinia, and the animal pathogen, Serratia, as simple test organisms, we will investigate artificial evolution of QS regulatory systems. We will mutagenise the LuxI homologues and re-engineer the bacteria to produce non-native QS signals then assess the phenotypic impacts. LuxR homologues controlling virulence and antibiotic biosynthesis will be evolved by localised chemical mutagenesis, error-prone PCR and DNA shuffling. Evolved variants responding to non-native N-AHLs of varying side chain length and oxidation state will be identified and characterised genetically, biochemically and physiologically. Variants responding to N-AHL-like xenobiotics will also be evolved and characterised. Fitness impacts of QS component evolution in the two pathogens will be assessed.

Summary

Bacteria are capable of 'talking' to each other in chemical terms. One set of chemical signals that is exchanged between bacteria involves N-acyl homoserine lactones (N-AHLs). These chemicals can diffuse between bacteria and can be sensed intracellularly. When these chemical signals move from one bacterial cell into a new bacterium they can bind to specific proteins that are responsible for switching on - or switching off - diverse sets of genes in the receiving bacterium. Bacteria use this system to link the control of multigene expression to the concentration of the available chemical signal. As the concentration of the chemical signal available is dependent on the number of bacterial cells producing the signal, then this system allows the receiving bacteria to determine indirectly the number of bacterial cells in the population. Therefore, bacteria can use this chemical signalling method to link the population density to the control of multigene expression. For that reason (cell number-dependency), this process is called quorum sensing (QS). Genes under QS control include those for virulence and secondary metabolite (such as antibiotic) biosynthesis in some pathogens of animals and plants. The main players in the N-acyl homoserine lactone (N-AHL)-based QS regulatory systems are LuxI-like proteins and LuxR-like proteins. LuxI-like proteins are enzymes that make the specific N-AHL whereas the LuxR-like proteins bind the N-AHL and bind DNA sequences upstream of the corresponding target genes. This leads to control of the corresponding target genes through chemical signalling. Some luxIR-type genes are mobile and have been acquired from other bacterial strains and they may have evolved by a modular system akin to a molecular 'lego' kit in which there is a 'mix-and-match' of functional parts of the respective proteins. We will test this modularity concept by artificailly forcing the evolution of the component parts of the system (LuxI and LuxR proteins) so that engineered bacteria will make different molecules to those they normally make and respond to different molecules from those to which they normally respond. This lab-based 'speeding up' of the evolutionary process will allow us to understand better some of the characteristics of the QS system. We will test this in two bacterial pathogens; one pathogenic to plants and another pathogenic to animals. In effect we will be artificially evolving the QS control systems in these bacteria such that they respond to a new chemical language. We expect the evolved versions to be less 'fit' because we think that the native system has evolved over a very long time period to enhance the survival and dissemination of the bacteria. We will test this idea in the lab.

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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