QUORUM SENSING IN PSEUDOMONAS AERUGINOSA

群体感应，即细菌物种监测自己的种群密度并在适当密度下激活特定基因的能力，是一种共同的特征，对于适应环境变化至关重要。这是铜绿假单胞菌中的两个群体感应系统，它们控制着一组定义不清的毒力基因。其中一个系统也是开发铜绿假单胞菌生物膜所必需的。这项拟议研究的目的是确定这两个系统各自控制的基因，并确定单个基因的控制是直接的还是间接的。我们还将确定我们确定的哪些目标基因是生物膜发育所必需的。将采取两种方法来识别目标基因。I.P.铜绿假单胞菌群体感应突变体将被用来识别在有或没有每个信号分子的情况下在较高水平表达的蛋白质。利用启动子探针转座子对铜绿假单胞菌群体感应缺失突变体进行诱变，将被用来识别需要表达的基因，以及是否存在群体感应信号。为了确定靶基因的群体感应控制是直接的还是间接的，我们将检测LacZ与靶基因启动子的融合在重组大肠杆菌中的表达。为了确定哪些群体感应靶点是正常生物膜发育所必需的，我们将测试被鉴定为群体感应调控的突变体，以确定它们形成典型的铜绿假单胞菌结构、抗杀菌剂生物膜的能力。很明显，群体感应在细胞的一般调节电路中的作用是复杂的，但这还没有得到系统的研究。这项拟议的研究将开辟细菌细胞间通讯的新领域。出于几个原因，铜绿假单胞菌是进行这些研究的合适模型。基因组正在测序(大部分基因组序列已经可以分析)。在矮化对虾中有两个特征良好的群体感应系统，并且已经构建了适当的零突变。虽然目前还没有系统的研究，但我们知道在铜绿假单胞菌中群体感应控制着许多非连锁基因的表达。在一些动物或植物模型系统中，群体感应已被证明是铜绿假单胞菌毒力的关键。

Quorum sensing, the ability of a bacterial species to monitor its own population density and activate specific genes at appropriate densities, is a common trait, critical for adaptation to environmental changes. These are two quorum sensing systems in Pseudomonas aeruginosa that control an ill-defined battery of virulence genes. One of the systems also is required for development of P. aeruginosa biofilms. The aims of the proposed research are to identify the genes controlled by each of the two systems and to determine whether control of individual genes is direct of indirect. We will also determine which of the target genes we identify are required for biofilm development. Two approaches will be taken to identify target genes. I.P. aeruginosa quorum sensing mutants will be employed to identify proteins expressed at higher levels with or without each of the signal molecules. II. Mutagenesis of a P. aeruginosa quorum sensing null mutant with promoter probe transposons will be used to identify genes that require for their expression, the presence or absence of either of the quorum sensing signals. To determine whether quorum sensing control of target genes is direct of indirect we will examine expression of lacZ fusions to target gene promoters in recombinant Escherichia coli. To determine which targets of quorum sensing are required for normal biofilm development we will test mutants identified as quorum sensing regulated for their ability to form typical P. aeruginosa structured, biocide resistant biofilms. It is clear that the role of quorum sensing in the general regulatory circuits of the cell is complex, yet this has not been investigated systematically. The proposed research will open a new area of study in bacterial cell-to-cell communication. P. aeruginosa is an appropriate model for these studies for several reasons. The genome is being sequenced (most of the genome sequence is already available for analysis). There are two well-characterized quorum sensing systems in P. aeurginosa and appropriate null mutations have been constructed. Although there have been no systematic surveys, we know that quorum sensing in P. aeruginosa controls expression of many unlinked genes. Quorum sensing has been shown to be critical for P. aeruginosa virulence in several animal or plant model systems.