ALGB AND P AERUGINOSA ALGINATE GENE EXPRESSION

ALGB 和绿脓杆菌藻酸盐基因表达

• 批准号: 2070637
• 负责人: Daniel J Wozniak
• 金额: $ 9.21万
• 依托单位: WAKE FOREST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-09-01 至 1999-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2070637
• 关键词: DNA binding protein; DNA footprinting; Escherichia coli; Pseudomonas aeruginosa; alginates; bacterial polysaccharides; carbohydrate biosynthesis; cystic fibrosis; gel mobility shift assay; gene deletion mutation; gene expression; gene interaction; genetic regulation; genetic strain; library; molecular cloning; molecular pathology; nucleic acid sequence; operon; plasmids; polymerase chain reaction; transcription factor

Most patients with the genetic disease cystic fibrosis (CF) become colonized and infected with mucoid strains of Pseudomonas aeruginosa. These strains are mucoid due to the overproduction of a polysaccharide called alginate, which plays a role in the pathogenesis of P. aeruginosa in the lungs of CF patients. This viscous polymer impairs lung function and makes it almost impossible to eradicate the organism from the CF respiratory tract, making P. aeruginosa infections the major cause of death in CF patients. Therefore, the long-term objective of this proposal is to understand the molecular basis for the overproduction of alginate by strains of P. aeruginosa which infect cystic fibrosis patients. The alginate biosynthetic genes are controlled by a complex, multigenic regulatory network. The research proposed here will focus on the role of a AlgB, a key alginate regulatory protein, in the control of alginate biosynthesis. In order to understand this, two critical questions which constitute the aims of this proposal will be addressed. The first goal is to identify the molecular targets of AlgB. This will be accomplished by DNA binding studies, a deletion analysis of algD, genetic characterization of algB suppressor strains, and the use of transcriptional fusions to isolate genes under AlgB control. Another aim of this proposal is to determine the mechanism of algB transcriptional activation. In particular, the roles of the alginate regulator AlgT and the DNA binding/bending protein integration host factor in algB expression will be investigated. The algB promoter(s) responsive to these proteins will be characterized by ribonuclease protection mapping and primerextension analysis. The combination of biochemical and genetic approaches utilized in these studies should provide insights into the complex regulation of this important virulence factor with the ultimate goal of an improved quality of lite for CF patients colonized with alginate-producing strains of P. aeruginosa.

大多数患有遗传性疾病囊性纤维化（CF）的患者 定植并感染铜绿假单胞菌的粘液型菌株。 这些菌株是粘液由于过度生产的多糖 称为藻酸盐，它在铜绿假单胞菌的发病机制中发挥作用， CF患者的肺部。 这种粘性聚合物损害肺功能， 几乎不可能从CF中根除微生物 呼吸道，使铜绿假单胞菌感染的主要死因 CF患者中。 因此，本建议的长期目标是 了解海藻酸盐生产过剩的分子基础， 感染囊性纤维化患者的铜绿假单胞菌菌株。 藻酸盐生物合成基因由一个复杂的多基因调控系统控制， 监管网络。 本文提出的研究将侧重于以下方面的作用： 一种关键的海藻酸调控蛋白AlgB，在海藻酸调控中， 生物合成 为了理解这一点，有两个关键问题， 本提案的目的将得到解决。 第一个目标是 以确定AlgB的分子靶点。 这将通过 DNA结合研究，algD缺失分析，遗传表征 的algB抑制菌株，以及使用转录融合， 在AlgB控制下分离基因。 这项建议的另一个目的是 确定algB转录激活的机制。 特别是， 藻酸盐调节剂AlgT和DNA结合/弯曲的作用 将研究algB表达中的蛋白质整合宿主因子。 响应于这些蛋白质的algB启动子的特征在于 核糖核酸酶保护作图和引物延伸分析。 生物化学和遗传学方法的结合， 研究应该提供对这种复杂调节的见解， 重要的毒力因子，最终目标是提高 对于用产藻酸盐的P. 铜绿。