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细胞内氧化应激的完整分子和生理学理解。工作人员试图确定活性氧是如何在细胞内形成的，它们最快破坏哪些生物分子，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，包括在缺氧条件下产生过敏突变体的能力。一种不能分解过氧化氢的突变菌株已经在几个方面推动了研究。因为它将内源性H2O2释放到生长培养基中，所以可以量化好氧细胞内产生H2O2的速率。人们也可以很容易地施加低剂量的H2O2延长一段时间，这种方法揭示了对H2O2损伤最敏感的细胞过程。最后，通过敲除候选基因，人们可以确定那些在防御大肠杆菌中起关键作用的基因。coli对微摩尔H_2O_2胁迫的耐受性。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）确定在E.杆菌(2)揭示H2O2和超氧化物歧化酶转酮醇酶的机制，这似乎是非常脆弱的失活。(3)探讨细胞内锰对大肠杆菌的保护作用。coli对H_2O_2胁迫的抗性。(4)确定保护铁硫酶免受氧化剂影响的机制。氧化应激生物化学的大多数方面在所有生物体中是保守的。大多数防御策略也分布广泛。因此，这项调查应阐明专性厌氧的分子基础，吞噬细胞的杀伤机制，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.