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..

Fuqua 9723837细菌监测器并迅速响应其环境的物理和化学特征的变化。现在很明显，许多细菌也已经进化出了它们来衡量自己人口生长的机制，这种现象通常被描述为群体传感。多种细菌，通常是动物或植物的病原体或共生体，通过生产和感知可扩散的细菌信息素（一种称为自动诱导剂（AI））来监测细胞密度。植物病原体农杆菌tumefaciens调节其主要的毒力因子Ti（肿瘤诱导）质粒的结合转移，以响应于这种酰基化的同层holy-同碱性内酯细胞密度信号。该项目旨在理解A. tumefaciens中AI介导的群体传感的机制，其长期目的是将这些知识应用于破坏或操纵细胞密度依赖性过程中，并在多样化和快速扩展的细菌收集中采用自动诱导者和Quorum传感。 在高细胞密度下，A。tumefaciens共轭将Ti质粒转移到受体农杆菌。 Ti质粒偶联的细胞密度依赖性是由Luxi-Luxr型Quorum-Sensor和Anai引诱剂（AAI），N-3-OXO（Octanoyl）-L-HOMOSERINE LACTONE介导的。 A. tumefaciens法定人数传感器由AAI合酶TRAI和Ti质粒偶联物转移（TRA）基因的AAI响应转录激活剂，Trar。然而，与F. fischeri系统相比，曲霉细胞密度的响应性需要一种称为TRAM的调节蛋白。电车是A. tumefaciens法定传感器的组成部分，无论供体密度如何，在电车结合物中带有无效突变的菌株。电车抑制Trar依赖性靶基因的转录激活。初步的遗传证据表明，电车可能会干扰Trar与AAI相互作用的能力，TRA基因靶启动子或两者兼而有之。 这项研究的研究是了解Tram作为A. tumefaciens Quorum传感器特异性的组成部分的作用，特定于Tram如何抑制Trar依赖性靶基因的激活。使用互补的遗传和生化方法，将确定（i）电车是否会闭塞Trar的Tra启动子，（ii）降解和/或隔离器AAI，还是（III）与Trar的物理联系，从而限制其活性。正在研究的相关区域是电车表达的调节，以及该调节对控制Ti质粒结合转移的重要性。 在剖析动作的电车机理时，将获得洞察力，并通过扩展其他LuxR型蛋白质的功能。了解电车的抑制功能可能会提供模型，以调节LuxR型蛋白的活性。从广义上讲，这项研究将有助于理解原核生物中多细胞行为，以及信号转导和透射调节的领域。