The Physiology of Oxidative Stress in Escherichia coli

描述（由申请人提供）：本领域的目标是开发细胞内氧化应激的完整分子和生理模型。我们想知道超氧化物和过氧化氢是如何在细胞内形成的，它们会破坏哪些生物分子，以及细胞防御它们的策略。目前，我们正在努力解决大肠杆菌中的这些问题。这种模式生物提供了独特的实验优势，包括在没有氧气的情况下产生和培养无防御突变体的能力。在过去的15年里，人们对超氧化物胁迫的认识取得了巨大的进步，主要是通过对一株大肠杆菌的分析。大肠杆菌超氧化物歧化酶缺失突变株。我们最近培育出一种类似的菌株，它不能分解过氧化氢。这种突变体使我们能够量化细胞内H2 O2形成的速率，并检测这种H2 O2引起的生长和存活缺陷。在本申请中，我们提出利用这种和其他突变体，以及一组实验方法，为E。大肠杆菌，以确定氧化应激机制和防御的细节。(1)我们将精确定位H2 O2在细胞质中形成的位点和O2-在完整的需氧细胞的胞质中形成的位点。(2)我们将确定H2 O2抑制对数期细胞生物合成和杀死早期稳定期细胞的机制。(3)我们将确定Dps保护DNA的机制，以及一个未定义的因子保护乌头酸酶A的铁硫簇。我们认为，对E.大肠杆菌的研究将为解决氧化应激的关键问题提供一个蓝图：专性厌氧，吞噬细胞的杀伤机制，以及高等生物的内源性氧化应激。

DESCRIPTION (provided by applicant): A goal of our field is to develop a complete molecular and physiological model of intracellular oxidative stress. We want to know how superoxide and hydrogen peroxide are formed inside cells, which biomolecules they damage, and the strategies by which cells defend themselves against them. We are currently trying to solve these problems in Escherichia coli. This model organism provides unique experimental advantages, including the ability to generate and culture defenseless mutants in the absence of oxygen. In the past 15 years enormous strides were made in understanding superoxide stress, largely from the analysis of an E. coli mutant that lacked superoxide dismutase. We have recently generated an analogous strain that cannot scavenge hydrogen peroxide. This mutant has allowed us to quantify the rate of intracellular H202 formation and to detect the growth and survival defects that this H202 causes. In this application we propose to exploit this and other mutants, as well as a battery of experimental methodologies that were developed for E. coli, in order to nail down the details of oxidative stress mechanisms and defenses. (1) We will pinpoint the sites at which H202 is formed in the cytosol and 02- is formed in the eriplasm of intact, aerobic cells.(2) We will determine the mechanisms by which H202 inhibits biosynthesis in log-phase cells and kills early-stationary-phase cells.(3) We will identify mechanisms by which Dps protects DNA and an undefined factor protects the iron-sulfur cluster of aconitase A. We believe that knowledge of oxidative damage and defense in E. coli will provide a blueprint for efforts to solve key issues in oxidative stress: obligate anaerobiosis, the killing mechanism of) hagocytes, and endogenous oxidative stress in higher organisms.