ACIDIFICATION TOLERANCE RESPONSE OF SALMONELLA

沙门氏菌的酸化耐受反应

• 批准号: 3307444
• 负责人: JOHN W FOSTER
• 金额: $ 16.11万
• 依托单位: UNIVERSITY OF SOUTH ALABAMA
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-08-15 至 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3307444
• 关键词: DNA damage; Salmonella infections; Salmonella typhimurium; acidity /alkalinity; adenosinetriphosphatase; environmental stressor; gene expression; genetic operator element; genetic promoter element; homeostasis; host organism interaction; lac operon; membrane permeability; microorganism genetics; molecular cloning; molecular genetics; mutant; nucleic acid sequence; regulatory gene; sensory signal detection; stress proteins; structural genes; transposon /insertion element; virulence

Salmonella must endure intervals of acid stress in widely diverse environments ranging from pond water to fused phagolysosomes in infected macrophages. We have discovered a unique survival response that effectively protects the cells from extreme low pH. This acid tolerance response (ATR) is composed of two distinct stages. A pre-acid shock stage triggered below pH 6 and a post-acid shock stage induced below pH 4.5. Both stages are required for the cell to survive low pH (pH 3.3) These findings present a challenging biological question. How does a neutralophillic organism protect itself from severe acid stress? Preliminary results indicate preshock produces an inducible pH homeostasis system that augments the housekeeping system as external pH decreases below pH 4 while acid shock appears to generate a set of proteins designed to minimize acid damage to macromolecules. An adapted culture in which both systems are engaged can survive pH 3.3 stress approximately 1000 fold better than unadapted cultures. Biochemical and genetic approaches have revealed integral roles for the Mg+2 dependent proton-translocating ATPase (atp) and the iron-regulatory locus, fur, in the ATR system. This proposal has three aims designed to investigate the ATR phenomenon at the biochemical, genetic and molecular levels. They are to (1) define the genetic components of the ATR system, (2) reveal the protective mechanisms used and (3) assign specific functions to specific atr loci. Genetic characterization will occur through the generation of acid-sensitive atr insertions (including lacZ operon fusion) and the identification of new regulatory loci. These genes will be cloned and the most intriguing subjected to molecular analysis. As part of this aim, the Salmonella fur product will be scrutinized for properties commensurate to its role in acid tolerance. We will examine the preshock inducible pH homeostasis mechanism by measuring buffering capacity, proton influx and proton efflux in adapted and unadapted cells. Insights regarding post shock protection of cellular macromolecules will be gained by using acid-sensitive indicator proteins and observing acid-damaged DNA repair. The unifying theme throughout this project is to correlate specific atr mutations with the loss of a given repair or protection system. This proposal presents a comprehensive research plan designed to reveal the elegant molecular response of salmonella to low pH. On a higher level, the knowledge gained will address fundamental questions concerning general defense strategies used by micro-organisms to survive harsh environments.

沙门氏菌必须在广泛不同的环境中耐受一段时间的酸胁迫 环境范围从池塘水到受感染的融合吞噬溶酶体 巨噬细胞。 我们发现了一种独特的生存反应 有效保护细胞免受极低pH的影响。 这种耐酸 响应（ATR）由两个不同的阶段组成。 酸前休克 pH 值低于 6 时触发阶段和 pH 值以下引发酸冲击后阶段 4.5. 细胞在低 pH 值 (pH 3.3) 下生存需要这两个阶段 这些发现提出了一个具有挑战性的生物学问题。 一个如何 嗜中性生物体保护自己免受严重的酸应激？ 初步结果表明预电击产生可诱导的 pH 值 作为外部 pH 值增强内务管理系统的稳态系统 pH 值降至 4 以下，而酸休克似乎会产生一系列 旨在最大限度地减少酸对大分子的损害的蛋白质。 一个改编的 两个系统同时参与的培养物可以在 pH 3.3 压力下生存 比未适应的培养物好大约 1000 倍。 生化和 遗传方法揭示了 Mg+2 依赖性的不可或缺的作用 质子转位 ATP 酶 (atp) 和铁调节位点，毛皮， ATR 系统。 该提案有三个目的旨在研究 ATR 现象 在生化、遗传和分子水平上。 他们要 (1) 定义 ATR 系统的遗传组成部分，(2) 揭示 使用的保护机制和（3）将特定功能分配给特定的 atr 基因座。 遗传特征将通过产生 酸敏感的 atr 插入（包括 lacZ 操纵子融合）和 识别新的调控位点。 这些基因将被克隆并 最有趣的是分子分析。 作为本次活动的一部分 目的是对沙门氏菌毛皮产品的特性进行严格审查 与其耐酸作用相称。 我们将检查 通过测量缓冲来实现休克前诱导 pH 稳态机制 适应和未适应的容量、质子流入和质子流出 细胞。 关于细胞冲击后保护的见解 使用酸敏感指示蛋白可以获得大分子 并观察酸损伤的 DNA 修复。 贯穿始终的统一主题 该项目旨在将特定的 atr 突变与 atr 的缺失联系起来 给予修复或保护系统。 该提案提出了一项全面的研究计划，旨在揭示 沙门氏菌对低 pH 值的优雅分子反应。 在更高的 水平，所获得的知识将解决基本问题 关于微生物生存的一般防御策略 恶劣的环境。