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

细菌能够用化学术语相互“交谈”。细菌之间交换的一组化学信号涉及N-酰基高丝氨酸内酯(N-AHL)。这些化学物质可以在细菌之间扩散，并可以在细胞内感受到。当这些化学信号从一个细菌细胞转移到一个新的细菌时，它们可以与特定的蛋白质结合，这些蛋白质负责打开或关闭接收细菌中不同的基因集。细菌使用这个系统将多基因表达的控制与可用化学信号的浓度联系起来。由于可用化学信号的浓度取决于产生该信号的细菌细胞的数量，因此该系统允许接收细菌间接确定种群中的细菌细胞的数量。因此，细菌可以使用这种化学信号方法将种群密度与多基因表达的控制联系起来。出于这个原因(细胞数量依赖)，这个过程被称为群体感知(QS)。受QS调控的基因包括某些动植物病原菌的毒力基因和次生代谢物(如抗生素)生物合成基因。基于N-乙酰高丝氨酸内酯(N-AHL)的QS调控系统的主要成员是Luxi-like蛋白和LuxR-like蛋白。Luxi-like蛋白是制造N-AHL的酶，而LuxR-like蛋白与N-AHL结合，并结合相应靶基因上游的DNA序列。这导致通过化学信号来控制相应的靶基因。一些LuxIR类型的基因是可移动的，是从其他细菌菌株获得的，它们可能是由一个类似于分子乐高(Lego)试剂盒的模块化系统进化而来的，在这个系统中，有各自蛋白质的功能部分的“混合和匹配”。我们将通过人工强制系统组成部分(Luxi和LuxR蛋白质)的进化来测试这种模块化概念，以便工程菌将制造与它们正常制造的分子不同的分子，并对与它们正常反应的分子不同的分子做出反应。这种以实验室为基础的进化过程的加速将使我们更好地了解QS系统的一些特征。我们将在两种细菌病原体中进行测试；一种对植物致病，另一种对动物致病。实际上，我们将在这些细菌中人工进化QS控制系统，使它们对一种新的化学语言做出反应。我们预计进化的版本不太“适合”，因为我们认为本地系统经过了很长一段时间的进化，以增强细菌的生存和传播。我们将在实验室测试这一想法。

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.