RUI: Genetic and Biochemical Analysis of the TraM Protein, an Inhibitory Component of the Agrobacterium tumefaciens Quorum-Sensor

福库9723837细菌监测环境的物理和化学性质的变化，并迅速做出反应。现在很明显，许多细菌也进化了测量自己种群增长的机制，这种现象通常被描述为群体感应。广泛的细菌，通常是动物或植物的病原体或共生体，通过产生和感知一种可扩散的细菌信息素来监控细胞密度，这种信息素是一种被称为自身诱导剂(AI)的酰化高丝氨酸内酯。植物病原菌根癌农杆菌调节其主要毒力因子(肿瘤诱导)质粒的结合转移，以响应这种酰化的高丝氨酸内酯细胞密度信号。这个项目旨在了解AI介导的根癌农杆菌群体感应的机制，长期目标是应用这一知识来扰乱或操纵使用自动诱导剂和群体感应的多样化和快速扩大的细菌集合中依赖细胞密度的过程。在细胞密度较高的情况下，根癌农杆菌将钛质粒接合转移到受体农杆菌。利用Luxi-LuxR型群体传感器和农杆菌自身诱导剂N-3-氧代(辛酰基)-L-高丝氨酸内酯介导了钛质粒接合的细胞密度依赖性。根癌农杆菌群体感受器由AAI合成酶TraI和钛质粒共轭转移(Tra)基因的AAI反应转录激活子TraR组成。然而，与费氏弧菌系统不同的是，根癌农杆菌的细胞密度反应需要一种名为TRAM的额外调节蛋白。TRAM是根癌农杆菌群体感应器的一个组成部分，无论供体密度如何，TRAM结合物中携带零突变的菌株。TRAM抑制TRAR依赖的靶基因的转录激活。初步的遗传学证据表明，TRAM可能干扰TRAR与AAI、trA基因靶标启动子或两者的相互作用。这项研究的目的是了解TRAM作为根癌农杆菌群体传感器的一个组成部分的作用--特别是TRAM如何抑制依赖TRAR的靶基因的激活。使用互补的遗传和生化方法，将确定(I)TRAM是否封闭了TRAR的反式启动子，(Ii)降解和/或隔离AAI，或(Iii)物理上与TRAR结合从而限制其活性。正在研究的一个相关领域是TRAM表达的调节，以及这种调节对控制钛质粒接合转移的重要性。在剖析TRAM的作用机制时，将深入了解TRAR本身的功能，并由此延伸到其他LuxR类型的蛋白质。了解TRAM的抑制功能可能为调节LuxR型蛋白的活性提供一般的模型。从更广泛的意义上讲，这项研究将有助于理解原核生物的多细胞行为以及信号转导和转导调控领域。

Fuqua 9723837 Bacteria monitor and respond rapidly to changes in the physical and chemical character of their environment. It is now clear that many bacteria also have evolved mechanisms by which they measure their own population growth, a phenomenon generally described as quorum sensing. A wide range of bacteria, often pathogens or symbionts of animals or plants, monitor cell density by production and perception of a diffusible bacterial pheromone, an acylated homoserine lactone called an autoinducer (AI). The plant pathogen Agrobacterium tumefaciens regulates conjugal transfer of its primary virulence factor, the Ti (tumor-inducing) plasmid, in response to such an acylated homoserine lactone cell density signal. This project is directed towards understanding the mechanism of AI-mediated quorum sensing in A. tumefaciens, with the long term aim of applying this knowledge towards disrupting or manipulating cell density-dependent processes in the diverse and rapidly expanding collection of bacteria that employ autoinducers and quorum sensing. At high cell densities A. tumefaciens conjugally transfer the Ti plasmid to recipient agrobacteria. Cell density-dependence of Ti plasmid conjugation is mediated by a LuxI-LuxR type quorum-sensor and the Agrobacterium autoinducer (AAI), N-3-oxo(octanoyl)-L-homoserine lactone. The A. tumefaciens quorum-sensor consists of the AAI synthase TraI and the AAI-responsive transcriptional activator of Ti plasmid conjugal transfer (tra) genes, TraR. However, in contrast to the V. fischeri system, A. tumefaciens cell density responsiveness requires an additional regulatory protein called TraM. Tram is an integral component of the A. tumefaciens quorum sensor and strains carrying null mutations in tram conjugate irrespective of donor density. Tram inhibits transcriptional activation of TraR-dependent target genes. Preliminary genetic evidence suggests that Tram may interfere with the ability of TraR to interact with AAI, the tra gene target promoters, or both. The go al of this research is to understand the role of TraM as a component of the A. tumefaciens quorum sensor-specifically how TraM inhibits activation of TraR-dependent target genes. Using complementary genetic and biochemical approaches it will be determined whether (i) TraM occludes tra promoters from TraR, (ii) degrades and/or sequesters AAI, or (iii) physically associates with TraR thereby limiting its activity. A related area that is being investigated is the regulation of traM expression, and the importance of this regulation for control of Ti plasmid conjugal transfer. In dissecting the TraM mechanism of action, insights will be gained into the function of TraR itself, and by extension other LuxR-type proteins. Understanding the inhibitory function of TraM may provide models for modulating the activity of LuxR-type proteins in general. In a broader sense, this study will contribute to the understanding of multicellular behavior in prokaryotic organisms as well as the areas of signal transduction and transciptional regulation..