Collaborative research: Short-circuiting in bacterial quorum sensing

细菌细胞间的交流，也被称为群体感应（QS），是一个广泛传播的过程，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、群体结构和环境粘度。在了解QS的分子机制方面的研究已经有了爆炸式的发展，但是关于QS调控网络的生态生理含义和新兴特性的信息却很缺乏。目前的项目通过结合遗传学，生理学和系统生物学来理解模型细菌铜绿假单胞菌中的QS来解决这一需求。这种细菌通过扩散酰基-高丝氨酸内酯信号进行交流，控制数百个基因的表达。特别关注的是铜绿假单胞菌QS网络的两个中心特性，抗活化和协同调节。抗活化最初在植物病原体农杆菌中发现，它通过直接的蛋白-蛋白相互作用抑制同源QS受体的活性。共同调节允许将其他环境信号整合到群体反应中。这里的一个关键特征是主要铜绿假单胞菌QS受体LasR的饥饿依赖性转录。由于一些由qs控制的产物是参与营养获取的昂贵的细胞外酶，因此饥饿的共同调节在生态上是值得的。将研究抗活化和激光辐射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中一个悬而未决的主要问题：为什么扩散群体信号没有立即与产生它们的同一细胞中的同源受体结合，并独立于细胞密度激活基因表达？基于最近的建模数据，pi假设抗激活和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由细胞密度触发的短路或饥饿触发的群体反应的关键。因此，该项目将实验和计算建模相结合，其具体目标是：(1)直接观察抗激活剂缺乏和lasR过表达细胞中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度依赖性，(3)建立las QS网络模型，除了抗激活和共调控外，结合并评估关键特性，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impactsResearch。在美国国家科学基金会（NSF）的部分资助下，pi在过去十年中进行的研究已经建立了P. aeruginosa QS作为全球监管网络，深入了解了中央QS监管机构LasR的功能，证明了QS是一种受社会冲突影响的合作行为，并产生了P. aeruginosa QS的第一个计算模型。目前的项目将整合并扩展这些发现，以了解QS网络的基本设计特征，包括抗活化和环境信号的整合。这项工作将广泛受益，并将在合成生物学和生物技术中找到新的遗传反应电路设计的应用。教育。所描述的项目为学生提供了良好的教育机会。这些学院已经并将继续培养研究生和本科生。Schuster博士还将通过俄勒冈州立大学（Oregon State University）建立的暑期实习项目——科学与工程学徒计划（Apprenticeship for Science and Engineering）为高中生提供教育机会。许多拟议的实验在概念上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.