Evolution of bacterial communication systems

项目总结/摘要 群体感应（QS）是细菌通讯的一种重要形式，用于协调基因表达， 以细胞密度依赖的方式进行群体行为。许多细菌使用一种形式的QS，其中信号合酶 产生一种膜可渗透的小分子，成对的胞质受体对其作出反应。数百名 已经鉴定出具有不同性质的QS系统，其中许多对于细菌的毒力是重要的。 致病菌这种多样性是如何演变的，这对该领域来说是一个至关重要的问题。出头 在对铜绿假单胞菌的模型QS系统LasI-LasR的博士后工作中，我观察到， 受体LasR尚未进化到最大的信号敏感性，LasI和LasR的变体倾向于 选择性低于野生型。此外，QS系统通常受到严格监管，以确保活动仅在 比细胞密度基于这些观察，我假设QS系统进化到平衡信号 敏感性、选择性和适应性。本提案使用LasI-LasR模型系统来检验这一假设。要求1 将使用高灵敏度和低灵敏度LasR变体I研究信号灵敏度的成本和收益 以前识别的。我将测量这些突变体中QS活性的时间和水平， 突变体的信号干扰，并最终适应突变体在单一培养和竞争， 野生型铜绿假单胞菌目的2将评估铜绿假单胞菌如何响应压力对LasI-LasR信号的影响 选择性我将使用我最近发现的非选择性LasI和LasR变体作为体外研究的起点。 新信号选择性的演变。这一目标将阐明QS选择性的其他决定因素， 生成工具，用于未来研究QS信号选择性演变的轨迹。这些目标一起 将加深我们对细菌通讯系统进化的理解，有望促进 未来研究这些系统在多微生物群落中的作用，并将为 了解和治疗感染。这项研究计划将成为我申请一个 独立的教师职位和我获得的结果有望帮助我成功地竞争未来 经费这些实验将在K99阶段启动，并将包括以下培训： 细菌共培养、体外进化和生物统计学方法。此外，该提案还包括职业生涯 发展和培训计划，以补充我以前的经验。我召集了一个多学科的顾问团 团队帮助我实现我的目标，我的导师，格林伯格博士，是一个先驱和专家，在细菌 沟通与长期成功的指导记录。该组织提供的培训和支持 获奖将使我能够实现我的长期目标，建立一个独立的研究计划，重点是 在复杂环境中的细菌信号和生理学。

PROJECT SUMMARY/ ABSTRACT Quorum sensing (QS) is an important form of bacterial communication used to coordinate gene expression and group behaviors in a cell density-dependent manner. Many bacteria use a form of QS in which a signal synthase produces a membrane-permeable small molecule to which a paired cytosolic receptor responds. Hundreds of QS systems with diverse properties have been identified, many of which are important for the virulence of pathogenic bacteria. How this diversity evolved is a question of fundamental importance to the field. In my early postdoctoral work on the model QS system LasI-LasR from Pseudomonas aeruginosa, I observed that the receptor, LasR, has not evolved to maximal signal sensitivity and that variants of both LasI and LasR tend to be less selective than wildtype. Further, QS systems are often tightly regulated to ensure activity only occurs at a specific cell density. Based on these observations, I hypothesize that QS systems evolve to balance signal sensitivity, selectivity, and fitness. This proposal tests that hypothesis using the LasI-LasR model system. Aim 1 will investigate the costs and benefits of signal sensitivity using hyper- and hypo-sensitive LasR variants I identified previously. I will measure the timing and level of QS activity in these mutants, the susceptibility of the mutants to signal interference, and ultimately the fitness of the mutants in monoculture and in competition with wildtype P. aeruginosa. Aim 2 will evaluate how P. aeruginosa responds to pressure on LasI-LasR signal selectivity. I will use my recently identified non-selective LasI and LasR variants as a starting point for the in vitro evolution of new signal selectivity. This aim will illuminate additional determinants of QS selectivity and will generate tools for future research on the trajectory by which QS signal selectivity evolves. Together these aims will deepen our understanding of the evolution of bacterial communication systems, are expected to facilitate future research into the roles of these systems in polymicrobial communities, and will lead to new avenues for understanding and treating infections. This research proposal will form the foundation of my applications for an independent faculty position and the results I obtain are expected to help me successfully compete for future funding. These experiments will be initiated during the K99 phase of the award and will include training in bacterial co-culture, in vitro evolution, and biostatistical methods. Additionally, this proposal includes a career development and training plan to complement my prior experience. I have assembled a multidisciplinary advisory team to help me achieve my goals and my mentor, Dr. Greenberg, is a pioneer and expert in bacterial communication with a long track record of successful mentorship. The training and support provided by this award will enable me to achieve my long-term goal of establishing an independent research program focused on bacterial signaling and physiology in complex environments.