The Physiology of Oxidative Stress in Escherichia coli

描述(由申请人提供)：该领域的目标是获得对细胞内氧化应激的完整的分子和生理理解。工作人员试图确定细胞内活性氧物种是如何形成的，它们最快地破坏哪些生物分子，以及细胞如何防御它们。这些问题在大肠杆菌中可能是最容易处理的。这种模式生物为这些研究提供了独特的优势，包括在缺乏氧气的情况下产生超敏突变的能力。一种不能清除过氧化氢的突变菌株推动了几个方面的工作。因为它将内源性过氧化氢释放到生长介质中，所以人们可以量化在有氧细胞内产生过氧化氢的速度。人们还可以很容易地在较长的一段时间内施加低剂量的过氧化氢，这种方法揭示了对过氧化氢损伤最敏感的细胞过程。最后，通过敲除候选基因，人们可以识别那些对保护大肠杆菌免受微摩尔过氧化氢胁迫至关重要的基因。在本申请中，我们建议通过追求四个目标来扩展这些研究：(1)准确定位在大肠杆菌中最快速产生过氧化氢的氧化还原酶。(2)揭示H_2O_2和超氧化物歧化转酮醇酶的机制，转酮醇酶似乎极易失活。(3)研究细胞内锰对H_2O_2胁迫的保护作用。(4)确定保护铁硫酶免受氧化剂伤害的机制。氧化应激的生物化学的大部分方面在所有生物体中都是保守的。大多数防御策略也是广泛分布的。因此，这项研究将有助于阐明专性厌氧的分子基础，吞噬细胞的杀伤机制，以及内源性氧化应激的性质和严重程度。

DESCRIPTION (provided by applicant): The goal of this field is to attain a complete molecular and physiological understanding of intracellular oxidative stress. Workers seek to determine how reactive oxygen species are formed inside cells, which biomolecules they most rapidly damage, and how cells defend themselves against them. These problems may be most tractable in Escherichia coli. This model organism provides unique advantages for these studies, including the ability to generate hypersensitive mutants in the absence of oxygen. A mutant strain that cannot scavenge hydrogen peroxide has pushed work forward on several fronts. Because it releases endogenous H2O2 into the growth medium, one can quantify the rate at which H2O2 is generated inside aerobic cells. One can also easily impose low doses of H2O2 for an extended period of time, an approach that has revealed the cellular processes that are most sensitive to impairment by H2O2. Finally, by knocking out candidate genes, one can identify those that are critical in defending E. coli against micromolar H2O2 stress. In this application we propose to extend these studies by pursuing four aims: (1) To pinpoint the redox enzymes that most rapidly generate H2O2 inside E. coli. (2) To reveal the mechanism by which H2O2 and superoxide inactivate transketolase, which appears to be extremely vulnerable to inactivation. (3) To investigate how intracellular manganese protects E. coli against H2O2 stress. (4) To identify mechanisms that protect iron-sulfur enzymes from oxidants. Most aspects of the biochemistry of oxidative stress are conserved among all organisms. Most defensive strategies are widely distributed, too. Therefore, this investigation should shed light upon the molecular bases of obligate anaerobiosis, the killing mechanisms of phagocytes, and the nature and severity of endogenous oxidative stress.