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

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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.