Analysis of Toxin A synthesis in P. aeruginosa

描述(申请人提供)：外毒素A和铁载体(绿球蛋白 和吡夫定)在铜绿假单胞菌中通过一个复杂的过程产生 涉及多个基因。这项提案的长期目标是定义 外毒素A和吡哆醇合成的调控机制。 铜绿假单胞菌并确定参与其中的不同因素 监管。我们已经确定了一个基因，ptxR，它可以积极地调节 Toxa的转录，Toxa调节因子RegA，以及编码 吡哆醇系统的一些组件。现有证据表明， PtxR对这些基因的调节是间接的(通过一个独立的基因)。在……里面 此外，ptxR表达的调节本身似乎涉及几个 调控蛋白，既有已鉴定的，也有尚未鉴定的。一名已确定的 蛋白质，另一种sigma因子PvdS，也调节弓形虫和 吡咯烷酮基因。未知蛋白与ptxR特异结合 上游地区。这项建议的具体目的是：1)确定 PtxR调控TOXA和PYOVDINE基因的机制。 这包括鉴定ptxR靶序列和 携带它和受调控的基因的特征(直接或 2)通过ptxR间接调节ptxR的表达。 铜绿假单胞菌。这包括广泛的体内和体外转录 PtxR上游区分析及不同组织中ptxR表达的检测 特异性铜绿假单胞菌突变体；以及3)分离和鉴定推定的 调节蛋白，并鉴定它们与之结合的核苷酸序列 捆绑。这些目标将通过蛋白质纯化实验来实现， 简单和改进的DNA/蛋白质结合实验、功能基因组学、2-D 凝胶分析，体内和体外转录，以及不同的重组DNA 技巧。

DESCRIPTION (provided by applicant): Exotoxin A and the siderophores (pyochelin and pyoverdine) are produced in P. aeruginosa by a complicated process that involves several genes. The long-term goal of this proposal is to define the mechanism that regulates the synthesis of exotoxin A and pyoverdine in P. aeruginosa and to identify the different factors that participate in this regulation. We have identified a gene, ptxR, which positively regulates the transcription of toxA, the toxA regulator regA, and the genes that code for some components of the pyoverdine system. Available evidence suggests that the regulation of these genes by ptxR is indirect (through an independent gene). In addition, the regulation of ptxR expression itself appears to involve several regulatory proteins, both identified and as yet unidentified. One identified protein, the alternative sigma factor PvdS, also regulates toxA and the pyoverdine genes. The unidentified proteins bind specifically to the ptxR upstream region. The specific aims of this proposal are: 1) to determine the mechanism through which toxA and the pyoverdine genes are regulated by ptxR. This includes identification of the ptxR target sequence and the gene that carries it and characterization of the genes that are regulated (directly or indirectly) by ptxR; 2) to examine the regulation of ptxR expression in P. aeruginosa. This includes extensive in vivo and in vitro transcriptional analysis of the ptxR upstream region and examining ptxR expression in different specific P. aeruginosa mutants; and 3) to isolate and characterize the putative regulatory proteins and to identify the nucleotide sequences to which they bind. These aims will be accomplished using protein purification experiments, simple and modified DNA/protein binding experiments, functional genomics, 2-D gel analysis, in vivo and in vitro transcription, and different recombinant DNA techniques.