The Physiology of Oxidative Stress in Escherichia coli

大肠杆菌氧化应激的生理学

基本信息

批准号：
7932504
负责人：
JAMES A. IMLAY
金额：
$ 14.58万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2011-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7932504
关键词：
Aerobic Anabolism Anaerobiosis Animal Model Aspartate Biochemistry Candidate Disease Gene Cell physiology Cells Characteristics Chemicals Coin Culture Media Data Defect Dose Electron Transport Electronics Enzymes Escherichia coli Fumarates Glutamate Synthase Goals Grant Growth Hydro-Lyases Hydrogen Peroxide Impairment In Vitro Investigation Iron Knock-out Laboratories Life Light Manganese Measures Metabolic Metals Modification Molecular Nature Organism Oxidants Oxidation-Reduction Oxidative Stress Oxidoreductase Oxygen Pathway interactions Pentosephosphate Pathway Phagocytes Physiological Physiology Process Reaction Reactive Oxygen Species Residual state Resistance Respiratory Chain Role Severities Source Stress Sulfur Superoxides System Testing Time Transketolase Work base cell injury cell type cofactor in vivo killings mutant oxidation polypeptide repaired

项目摘要

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.

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分子和生理学理解。研究人员试图确定活性氧是如何在细胞内形成的、它们对哪些生物分子的损害最快，以及细胞如何防御它们。这些问题在大肠杆菌中可能最容易解决。这种模型生物为这些研究提供了独特的优势，包括在缺氧情况下产生过敏突变体的能力。一种不能清除过氧化氢的突变菌株推动了多个方面的研究工作。由于它将内源性 H2O2 释放到生长培养基中，因此可以量化需氧细胞内生成 H2O2 的速率。人们还可以轻松地长时间施加低剂量的 H2O2，这种方法揭示了对 H2O2 损害最敏感的细胞过程。最后，通过敲除候选基因，我们可以识别那些对于保护大肠杆菌免受微摩尔 H2O2 应激至关重要的基因。在此应用中，我们建议通过追求四个目标来扩展这些研究：（1）查明大肠杆菌内最快速产生 H2O2 的氧化还原酶。 (2)揭示H2O2和超氧化物使转酮醇酶失活的机制，该酶似乎极易失活。 (3) 研究细胞内锰如何保护大肠杆菌免受 H2O2 应激。 (4) 确定保护铁硫酶免受氧化剂侵害的机制。氧化应激生物化学的大多数方面在所有生物体中都是保守的。大多数防御策略也广泛分布。因此，这项研究应该揭示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。