The Physiology of Oxidative Stress in Escherichia coli

大肠杆菌氧化应激的生理学

• 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
• 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

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）：本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。工作人员试图确定活性氧是如何在细胞内形成的，它们最迅速地破坏哪些生物分子，以及细胞如何保护自己免受它们的伤害。这些问题在大肠杆菌中可能最容易处理。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超敏突变体的能力。一种不能清除过氧化氢的突变菌株推动了几个方面的工作。因为它释放内源性H2O2到生长介质中，我们可以量化H2O2在有氧细胞内产生的速率。人们也可以很容易地在较长时间内施加低剂量的H2O2，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，人们可以识别出那些在保护大肠杆菌免受微摩尔H2O2胁迫方面至关重要的基因。在本应用中，我们建议通过追求四个目标来扩展这些研究：(1)确定大肠杆菌内最快速生成H2O2的氧化还原酶。(2)揭示过氧化氢和超氧化物使极易失活的转酮醇酶失活的机制。(3)探讨细胞内锰对大肠杆菌抗H2O2胁迫的保护作用。(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。大多数防御策略也分布广泛。因此，这项研究将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。