Inhibition of the Agrobacterium tumefaciens Quorum Sensor by the TraM Anti-Activator

许多种类的细菌利用信号机制来监测自身的种群密度，并通过改变生理和发育途径作出反应。这个过程通常需要可溶性信息素的释放，其局部浓度提供了细胞密度的量度。信号分子的释放和感知被称为群体感应。一些革兰氏阴性菌的群体感应是由酰基化的同丝氨酸内酯（酰基HSL）的产生促进的。农杆菌是植物冠瘿瘤的病原体，它利用酰基HSL N-3-(oxoocinoyl)同丝氨酸内酯调节其主要毒力因子Ti（肿瘤诱导）质粒向根际其他细菌的偶联传播。目前的建议侧重于检查a . tummefaciens酰基HSL调控途径的独特和必要的抑制成分。对于大多数酰基hsl型群体传感器，信息素合成酶和受体蛋白也可作为转录激活剂，足以传递细胞密度依赖性基因表达。然而，大肠杆菌需要第三种成分，一种称为TraM的调节蛋白来促进群体感应。假设是TraM与酰基HSL受体TraR形成非共价复合物，从而阻止转录激活剂结合DNA。本研究的总体目标是剖析TraM和TraR之间的相互作用，并了解这种相互作用如何调节TraR的活性。生化分析将用于检查抑制复合物，并确定在酰基HSL存在下抑制对TraM和TraR活性的影响。表现出改变的调节表型的TraR和TraM突变蛋白将被分离出来，并进行生化分析，以确定其体内和体外活性的相关性。进一步的研究将评估调节traM表达和池大小的影响。这项研究不仅将阐明重要的模型病原菌a. tummefaciens中酰基HSL群体感应的一个独特方面，而且将有助于理解其他细菌物种的细胞间通讯。

Many species of bacteria employ signaling mechanisms to monitor their own population density and respond by altering physiological and developmental pathways. This process usually entails the release of a soluble pheromone, the local concentration of which provides a measure of cell density. Release and perception of the signaling molecule has been termed quorum sensing. Quorum sensing in a number of gram-negative bacteria is facilitated by production of acylated homoserine lactones (acyl HSL). Agrobacterium tumefaciens, the causative agent of plant crown gall neoplasia, utilizes the acyl HSL N-3-(oxooctanoyl) homoserine lactone to regulate the conjugal dissemination of its primary virulence factor, the Ti (tumor-inducing) plasmid, to other bacteria in the rhizosphere. The current proposal focuses on examination of a unique and essential inhibitory component of the A. tumefaciens acyl HSL regulatory pathway. For most acyl HSL-type quorum sensors, a pheromone synthase and a receptor protein that also serves as a transcriptional activator are sufficient to impart cell-density-dependent gene expression. However, A. tumefaciens requires a third component, a regulator protein called TraM, to facilitate quorum sensing. The hypothesis is that TraM forms a noncovalent complex with the acyl HSL receptor, TraR, which prevents the transcriptional activator from binding DNA. The overall goal of this research is to dissect the interaction between TraM and TraR and to understand how this interaction modulates the activity of TraR. Biochemical analysis will be employed to examine the inhibitory complex and to determine the effect of inhibition on the activity of TraM and TraR in the presence of acyl HSL. Mutant TraR and TraM proteins that exhibit altered regulatory phenotypes will beisolated and biochemically analyzed to correlate in vitro and in vivo activities. Additional studies will assess the effects of regulation of traM expression and pool size. This research will not only elucidate a unique aspect of acyl HSL quorum sensing in the important model pathogen A. tumefaciens, but will also contribute to the understanding of cell-cell communication in other species of bacteria.