Quorum Sensing in Pseudomonas aeruginosa

描述（由申请人提供）：酰基-高丝氨酸内酯（HSL）群体感应发生在多种革兰氏阴性细菌中。信号、信号发生器和信号受体的细节在过去几年中得到了快速的研究。群体感应作为基因表达的全球调节者，研究人员刚刚开始探索信号传导的这一方面。铜绿假单胞菌是研究基因表达全局群体控制的一个特别有用的模型。基因组测序已经完成，商业化的基因芯片已经问世，插入突变体的基因库也正在创建中。信号和信号基因已经被识别，申请人已经在群体控制的“规则”中识别了300多个基因。铜绿假单胞菌群体感应是特别相关的，因为它是需要对这种新出现的人类病原体的毒力。本项目的目标如下：1 .精细绘制“早期”群体控制基因的诱导图谱。这一分析将是验证以下假设的一步：转录激活因子结合位点的强度决定了诱导的精确时间。2。利用研究者可控制的LasR和RhlR表达模块设计铜绿假单胞菌菌株，并利用这些构建体验证在高表达水平下，LasR或RhlR直接调控的所有基因的时间都应该早的假设。在低水平表达时，只有那些与转录因子亲和力最大的启动子才会响应信号添加。3。在体外研究信号受体（转录因子），以测试控制群体基因复杂调控模式的机制模型。研究一个特定的群体控制基因，该基因在固定期特异性表达。研究群体感应在生物膜发育中的作用。铜绿假单胞菌群体感应系统提供了一个机会，了解一个简单的调控元件如何影响细菌培养物中数百个基因的有序表达。本研究将有助于丰富对群体感应机制和生理意义的认识。

DESCRIPTION (provided by applicant): Acyl-homoserine lactone (HSL) quorum sensing occurs in a wide variety of Gram-negative bacterial species. Details of the signals, signal generators and signal receptors have been generated at a rapid pace in the past several years. Quorum sensing functions as a global regulator of gene expression and the investigators are just beginning to explore this aspect of the signaling. Pseudomonas aeruginosa is a particularly useful model to study global quorum control of gene expression. The genome has been sequenced, there is a commercial GeneChip, and libraries of mapped insertion mutants are being created. The signals and signal genes have been identified, and the applicants have identified over 300 genes in the quorum-controlled "regulon." P. aeruginosa quorum sensing is particularly relevant as it is required for virulence of this emerging human pathogen. The goals of this project are as follows: I. To fine map the induction of "early" quorum-controlled genes. This analysis will be a step in testing the hypothesis that the strength of the binding sites for the transcriptional activators defines the precise timing of induction. II. To engineer P. aeruginosa strains with investigator-controllable LasR and RhlR expression modules and use the constructs to test the hypothesis that at high levels of expression the timing of all genes directly regulated by LasR or RhlR should be early. At low levels of expression only those promoters with the greatest affinity for the transcription factors should respond to signal addition. III. To study the signal receptors (transcription factors) in vitro to test models for mechanisms, which control the complex quorum gene regulatory patterns. IV. To study one particular quorum-controlled gene that is specifically expressed in stationary phase. V. To examine the role of quorum sensing in biofilm development. The P. aeruginosa quorum sensing system provides an opportunity to understand how a simple regulatory element can affect an ordered expression of hundreds of genes in a bacterial culture. The proposed research will serve to enrich the understanding of the mechanisms and physiological significance of quorum sensing.