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

RUI：TraM 蛋白（根癌农杆菌群体传感器的抑制成分）的遗传和生化分析

• 批准号: 9723837
• 负责人: William Fuqua
• 金额: $ 24.7万
• 依托单位: Trinity University
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-15 至 1999-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9723837&HistoricalAwards=false
• 关键词: RUI; Genetic; Biochemical; Analysis; TraM

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..

9723837细菌监测并迅速响应其环境的物理和化学性质的变化。现在很清楚，许多细菌也进化出了测量自身种群增长的机制，这种现象通常被称为群体感应。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测细胞密度，这种信息素是一种称为自诱导物（AI）的酰化高丝氨酸内酯。植物病原体根癌农杆菌调节其主要毒力因子Ti（肿瘤诱导）质粒的接合转移，以响应这样的酰化高丝氨酸内酯细胞密度信号。本项目旨在了解人工智能介导的群体感应机制。tumefaciens，其长期目标是将这些知识应用于破坏或操纵采用自诱导物和群体感应的细菌的多样性和快速扩展的集合中的细胞密度依赖性过程。 在高细胞密度下A.根癌农杆菌将Ti质粒接合转移到受体农杆菌中。Ti质粒接合的细胞密度依赖性由LuxI-LuxR型群体感应器和农杆菌自诱导物（AAI）N-3-氧代（辛酰基）-L-高丝氨酸内酯介导。助理根癌农杆菌群体传感器由AAI合酶TraI和Ti质粒接合转移（tra）基因的AAI应答转录激活因子TraR组成。然而，与V. fischeri系统相反，A.根癌农杆菌细胞密度响应需要另外的称为TraM的调节蛋白。有轨电车是A.根瘤菌群体传感器和在TRAM缀合物中携带无效突变的菌株，而不管供体密度。Tram抑制TraR依赖性靶基因的转录激活。初步遗传学证据表明，Tram可能会干扰TraR与AAI、tra基因靶启动子或两者相互作用的能力。 本研究的目的是了解TraM作为A. tumefaciens群体传感器-特异性TraM如何抑制TraR依赖性靶基因的激活。使用互补的遗传和生物化学方法，将确定（i）TraM是否封闭来自TraR的tra启动子，（ii）是否降解和/或隔离AAI，或（iii）是否与TraR物理缔合从而限制其活性。一个正在研究的相关领域是traM表达的调节，以及这种调节对于控制Ti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，并扩展到其他LuxR型蛋白质。了解TraM的抑制功能可以提供一般调节LuxR型蛋白活性的模型。从更广泛的意义上说，这项研究将有助于理解原核生物中的多细胞行为以及信号转导和转录调控领域。