TOXIN A SYNTHESIS IN P AERUGINOSA

性状（改编自申请人摘要）：假单胞菌 铜绿假单胞菌是一种革兰氏阴性条件致病菌， 严重烧伤患者的严重感染，免疫功能低下 宿主和囊性纤维化患者。 毒素A是最具毒性的 铜绿假单胞菌产生的毒力因子。 毒素A的合成 在铜绿假单胞菌中的表达受不同环境因素的高度调节 尤其是铁。 尽管有几项研究，但其确切机制 规则并不完全清楚。 长期目标是 一项旨在确定调节毒素A合成的机制的提案 铜绿假单胞菌和参与这种调节的因素。 最近， 分离到一个新的toxA阳性调控基因ptxR。 分析 ptxR表明它能增强toxA和toxB的转录， 先前描述的toxA调节基因regAB。 核苷酸序列 分析显示存在一个开放的阅读框架， 一个34 kDa的预测蛋白。 计算机分析显示， 干扰ptxR功能的相邻基因。 开放式阅读 框架（ORF 2），其从ptxR（从负的 链）并编码30 kDa蛋白质。 该预测 蛋白与GalR的几种蛋白具有显著的同源性 家庭的压抑。 这项建议的具体目标是：1） 研究ptxR功能的机制; 2）确定 ptxR的转录调控;和3）检查ORF 2的作用 ptxR功能。 检查ptxR函数的机制将包括 ptxR产物纯化、DNA/蛋白结合实验 (gel阻滞实验），磷酸化实验，两个 三维凝胶实验，和PAO 1等基因的构建 ptxR的突变体。 监管研究将包括转录分析 ptxR，regA，和toxA ORF 2的功能分析将涉及到 体内和体外转录实验，免疫共沉淀 实验，和ORF 2同基因突变体的分离。

DESCRIPTION (adapted from the applicant's abstract): Pseudomonas aeruginosa is a gram negative opportunistic pathogen which causes serious infections in severely burned patients, immuno-compromised hosts, and Cystic Fibrosis patients. Toxin A is one of the most toxic virulence factors produced by P. aeruginosa. The synthesis of toxin A in P. aeruginosa is highly regulated by different environmental factors especially iron. Despite several studies, the exact mechanism of this regulation is not completely understood. The long term goal of this proposal is to determine the mechanisms of regulating toxin A synthesis in P. aeruginosa and the factors involved in this regulation. Recently, a new toxA positive regulatory gene, ptxR, was isolated. Analysis of ptxR showed that it enhances transcription of both toxA and the previously described toxA regulatory gene, regAB. Nucleotide sequence analysis revealed the presence of an open reading frame which codes for a 34 kDa predicted protein. Computer analysis revealed the presence of an adjacent gene which interferes with ptxR function. An open reading frame (ORF2) which is divergently transcribed from ptxR (from the minus strand) and codes for a 30 kDa protein was identified. This predicted protein has a significant homology to several proteins of the GalR family of repressors. The specific aims of this proposal are: 1) to examine the mechanism of ptxR function; 2) to determine the transcriptional regulation of ptxR; and 3) to examine the effect of ORF2 on ptxR function. Examining the mechanism of ptxR function will include the purification of ptxR production, DNA/protein binding experiments (gel retardation experiments), phosphorylation experiments, two dimensional gel experiments, and the construction of a PAO1 isogenic mutant in ptxR. Regulatory studies will include transcriptional analysis of ptxR, regA, and toxA. Analysis of ORF2 function will involved in vivo and in vitro transcription experiments, co-immunoprecipitation experiments, and the isolation of an ORF2 isogenic mutant.