Analysis of Toxin A synthesis in P. aeruginosa

铜绿假单胞菌中毒素 A 合成的分析

• 批准号: 6621010
• 负责人: Abdul N Hamood
• 金额: $ 22.2万
• 依托单位: TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-15 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6621010
• 关键词: Pseudomonas aeruginosa; bacterial genetics; exotoxins; functional /structural genomics; gel mobility shift assay; genetic regulation; genetic transcription; microorganism metabolism; mutant; protein biosynthesis; protein purification; recombinant DNA; toxin metabolism; transcription factor; virulence

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.

性状（由申请方提供）：外毒素A和铁载体（绿脓菌螯铁蛋白 和绿脓菌荧光素）在铜绿假单胞菌中通过复杂的过程产生， 涉及多个基因。该提案的长期目标是确定 调控外毒素A和绿脓菌荧光素合成的机制。 铜绿假单胞菌，并确定参与这一过程的不同因素， 调控我们已经确定了一个基因ptxR，它可以正向调节 toxA的转录，toxA调节因子regA，以及编码 绿脓菌荧光素系统的一些组分。现有证据表明， ptxR对这些基因的调节是间接的（通过独立的基因）。在 此外，ptxR表达本身的调节似乎涉及几个 调节蛋白，已鉴定和尚未鉴定的。一个所识别的 另一种sigma因子PvdS也调节toxA和 绿脓菌荧光素基因。未鉴定的蛋白质特异性结合到ptxR 上游地区。该提案的具体目标是：1）确定 toxA和绿脓菌荧光素基因通过ptxR调节的机制。 这包括鉴定ptxR靶序列和 携带它，并表征受调控的基因（直接或 2）研究ptxR在P. 铜绿。这包括广泛的体内和体外转录 分析ptxR上游区域并检查不同组织中ptxR的表达。 特异性铜绿假单胞菌突变体;以及3）分离和表征推定的铜绿假单胞菌突变体。 调节蛋白，并鉴定它们与之结合的核苷酸序列。 这家公司进退两难.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，二维 凝胶分析，体内和体外转录，以及不同的重组DNA 技术.