Quorum Sensing in Pseudomonas aeruginosa

描述(由申请人提供)：酰基高丝氨酸内酯(HSL)群体感应存在于各种革兰氏阴性细菌中。在过去的几年里，信号、信号发生器和信号接收器的细节都以快速的速度产生。群体感应作为基因表达的全球调节器发挥作用，研究人员才刚刚开始探索这方面的信号。铜绿假单胞菌是研究基因表达的全球群体控制的一个特别有用的模型。基因组已经测序，有了商业基因芯片，绘制的插入突变体的文库正在创建中。信号和信号基因已经被识别出来，申请者已经在群体控制的“调节子”中识别了300多个基因。铜绿假单胞菌群体感应特别相关，因为它是这种新出现的人类病原体毒力所必需的。该项目的目标如下：1.对“早期”群体控制基因的诱导进行精细定位。这一分析将是检验这一假设的一步，该假设认为转录激活剂的结合位点的强度决定了诱导的准确时间。Ii.利用研究者可控制的LasR和RhlR表达模块来设计铜绿假单胞菌菌株，并使用这些构建体来检验这样的假设，即在高表达水平时，所有由LasR或RhlR直接调控的基因的时机都应该较早。在低水平表达时，只有那些与转录因子具有最大亲和力的启动子才能对信号添加做出反应。在体外研究信号受体(转录因子)，以检验控制复杂法定基因调控模式的模型的机制。IV.研究一种特定的群体控制基因，该基因在稳定期特异表达。五、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为了解一个简单的调控元件如何影响细菌培养中数百个基因的有序表达提供了机会。提出的研究将有助于丰富对群体感应的机制和生理意义的理解。

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.