Collaborative research: Short-circuiting in bacterial quorum sensing

智能优势细菌细胞-细胞通讯，也称为群体感应(QS)，是一种广泛存在的过程，它协调多细胞行为，如毒力、生物膜形成和营养获取，以响应细胞密度、种群结构和环境粘度。针对QS的分子机制的研究已经出现了爆炸性增长，但关于QS的生态生理意义和QS调节网络的新特性的信息很少。目前的项目通过结合遗传学、生理学和系统生物学来解决这一需求，以了解模式细菌铜绿假单胞菌中的QS。这种细菌通过可扩散的酰基高丝氨酸内酯信号进行交流，以控制数百个基因的表达。特别关注铜绿假单胞菌QS网络的两个核心特性，即抗激活和协同调节。植物病原菌根癌农杆菌通过直接的蛋白质-蛋白质相互作用抑制同源QS受体的活性。共同监管允许将其他环境信号整合到法定反应中。这里的一个关键特征是主要的铜绿假单胞菌QS受体LasR的依赖于饥饿的转录。由于几种QS控制的产品是与营养获取有关的昂贵的胞外酶，饥饿的共同调节似乎在生态上是值得的。我们将研究抗激活和LasR调节在调节群体反应和防止“短路”中的作用。短路或自我诱导是细菌QS中一个主要的悬而未决的问题：为什么可扩散的群体信号不会立即与产生它们的同一细胞中的同源受体结合，并激活与细胞密度无关的基因表达？根据最近的建模数据，PI假设抗活化和LasR调节有助于防止短路，并且对LasR表达的严格环境控制是调节Quorum反应的关键，这些反应要么是短路的，要么是由细胞密度触发的，要么是由饥饿触发的。因此，该项目将实验和计算建模相结合，其具体目标是(1)直接观察在抗激活剂缺乏和LasR过表达的细胞中QS靶基因表达的短路，(2)研究野生型细胞中QS基因诱导和短路的生长速率依赖性，以及(3)开发一个LAS QS网络的模型，该模型除了抑制和共同调节外，还包括和评估关键特性，如受体-QS信号相互作用、受体二聚化、自动调节和主动外排。更广泛的影响研究。PIS在过去十年中进行的研究，部分由NSF资助，建立了铜绿假单胞菌QS作为全球监管网络，提供了对中央QS调节器LasR的功能的洞察，证明QS是一种受社会冲突影响的合作行为，并导致了铜绿假单胞菌QS的第一个计算模型。目前的项目将结合和扩展这些发现，以了解QS网络的基本设计特征，包括抗激活和环境线索的整合。这项工作将广泛受益，并将在合成生物学和生物技术中应用于设计新的遗传反应电路。教育。所描述的项目为学生提供了极好的教育机会。私人投资机构已经并将继续培训研究生和本科生。舒斯特博士还将通过俄勒冈州立大学已有的暑期实习项目--理工科学徒计划，为高中生提供教育机会。许多拟议的实验在概念和技术上都是直截了当的，特别适合高中生和本科生参与科学过程。

Intellectual meritBacterial cell-cell communication, also termed quorum sensing (QS) is a wide-spread process that coordinates multicellular behaviors such as virulence, biofilm formation, and nutrient acquisition in response to cell density, population structure and environmental viscosity. There has been an explosion in research directed at understanding the molecular mechanisms of QS, but there is a paucity of information on the ecophysiological implications and on the emergent properties of QS regulatory networks. The current project addresses this need by combining genetics, physiology, and systems biology in understanding QS in the model bacterium Pseudomonas aeruginosa. This bacterium communicates via diffusible acyl-homoserine lactone signals to control the expression of hundreds of genes. The particular focus is on two central properties of the P. aeruginosa QS network, antiactivation and co-regulation. Antiactivation, initially characterized in the plant pathogen Agrobacterium tumefaciens, inhibits the activity of cognate QS receptors through direct protein-protein interaction. Co-regulation permits the integration of other environmental signals into the quorum response. A key feature here is the starvation-dependent transcription of the main P. aeruginosa QS receptor, LasR. Because several QS-controlled products are costly extracellular enzymes involved in nutrient acquisition, co-regulation by starvation appears ecologically worthwhile. The roles of antiactivation and lasR regulation in modulating the quorum response and in preventing "short-circuiting" will be investigated. Short-circuiting, or self-induction, is a major unanswered question in bacterial QS: How is it that diffusible quorum-signals do not immediately bind to their cognate receptors in the same cell in which they are produced and activate gene expression independent of cell density? Based on recent modeling data, the PIs hypothesize that antiactivation and lasR regulation help prevent short-circuiting, and that the tight environmental control of lasR expression is key in modulating quorum responses that are either short-circuited, triggered by cell density, or triggered by starvation. The specific aims of the project, which integrate experimentation and computational modeling, are therefore to (1) directly observe short-circuiting of QS target gene expression in antiactivator-deficient and lasR overexpressing cells, (2) investigate the growth-rate dependence of QS gene induction and short-circuiting in wild-type cells, and (3) develop a model of the las QS network that, in addition to antiactivation and co-regulation, incorporates and evaluates key properties such as receptor-QS signal interaction, receptor dimerization, autoregulation, and active efflux. Broader impactsResearch. The research conducted by the PIs over the last decade, funded in part by NSF, has established P. aeruginosa QS as a global regulatory network, has provided insight into the function of the central QS regulator LasR, has demonstrated that QS is a cooperative behavior subject to social conflict, and has resulted in the first computational model of P. aeruginosa QS. The current project will incorporate and extend these findings to understand the basic design features of a QS network, including antiactivation and the integration of environmental cues. The work will broadly benefit and will find application in synthetic biology and biotechnology for the design of novel genetic response circuits. Education. The described project provides excellent educational opportunities for students. The PIs have and will continue to train graduate and undergraduate students. Dr. Schuster will also provide educational opportunities for high-school students through the Apprenticeship for Science and Engineering, an established summer internship program at Oregon State University. Many of the proposed experiments are conceptually and technically straight-forward and are particularly well suited for the engagement of high-school and undergraduate students in the scientific process.