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

许多种类的细菌使用信号机制来监控自己的种群密度，并通过改变生理和发育途径来做出反应。这一过程通常需要释放一种可溶的信息素，其局部浓度提供了细胞密度的衡量标准。信号分子的释放和感知被称为群体感应。许多革兰氏阴性细菌的群体感应是通过产生酰化高丝氨酸内酯(Acyl HSL)来促进的。根癌农杆菌是植物冠瘤的病原菌，它利用酰基HSL N-3-(氧辛酰基)高丝氨酸内酯来调节其主要毒力因子Ti(肿瘤诱导)质粒向根际其他细菌的共生扩散。目前的建议集中在研究根癌农杆菌酰基HSL调控途径的一个独特和必要的抑制成分。对于大多数酰基HSL类型的群体传感器，信息素合成酶和受体蛋白也可以作为转录激活剂，足以传递细胞密度依赖的基因表达。然而，根癌农杆菌需要第三种成分，一种称为TRAM的调节蛋白，以促进群体感应。假设TRAM与酰基HSL受体TRAR形成非共价复合体，从而阻止转录激活剂与DNA结合。本研究的总体目标是剖析有轨电车和TRAR之间的相互作用，并了解这种相互作用是如何调节TRAR的活动的。生化分析将被用来检测抑制复合体，并确定在酰基HSL存在的情况下抑制对TRAM和TRAR活性的影响。表现出调节表型改变的突变的TRAR和TRAM蛋白将被分离并进行生化分析，以关联体外和体内的活性。其他研究将评估调控有轨电车表达和池子大小的影响。这项研究不仅将阐明重要的模式病原菌根癌农杆菌中酰基HSL群体感应的独特方面，而且也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.