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：
9384330
负责人：
JAMES A. IMLAY
金额：
$ 55.01万
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：
1994
资助国家：
美国
起止时间：
1994-05-01 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9384330
关键词：
Active Sites Address Aerobic Aerobic Bacteria Affect Affinity Anabolism Anaerobic Bacteria Anaerobiosis Bacteria Bacteroides thetaiotaomicron Biology Catabolism Cell Survival Cell physiology Cells Cellular Stress Charge DNA lesion Data Electrons Environment Enzymes Escherichia coli Evolution Exodeoxyribonuclease III Family Ferredoxin Fostering Fumarate Hydratase Funding Glucose Growth Habitats Health Human Human Pathology Hydro-Lyases Hydrogen Peroxide Hydrogenase In Vitro Innate Immune System Investigation Iron Isoenzymes Lactococcus lactis Lesion Life Manganese Metabolism Metals Methods Microbe Modeling Modification Molecular Molecular Chaperones Mononuclear Nature Organism Oxidants Oxidation-Reduction Oxidative Stress Oxides Oxygen Peripheral Pharmaceutical Preparations Phenotype Physiology Poison Poisoning Proteins Proteolysis Pyruvate synthase Reactive Oxygen Species Resistance Role Saccharomyces cerevisiae Solvents Source Staphylococcus aureus Stress Structure Study models Sulfur Superoxides Testing Work analog chelation cofactor cost divalent metal electron donor fascinate imprint improved in vivo microbial community mutant novel polypeptide repair enzyme repaired response stressor transcription factor transcriptomics unfoldase virtual

项目摘要

Oxidative stress has a large imprint upon biology: It determines the structure of microbial communities, is central to the action of the innate immune system, and is suspected of underlying a variety of human pathologies. Several basic questions frame the field: How are oxidants formed, and in what quantities? What are the specific biomolecules that they damage most rapidly? How do cells defend themselves? Why does oxidant sensitivity differ among organisms? Mechanistic details have primarily emerged from studies of the model bacterium E. coli. This application proposes to deepen and broaden the understanding. To date the reactive oxygen species (ROS) O2 and H2O2 are known to disrupt growth primarily by inactivating cluster-dependent dehydratases and non- - redox mononuclear iron enzymes. Dehydratases are vulnerable in virtually all organisms. However, it appears that the mononuclear enzymes of some aerobes acquired resistance by employing divalent metals other than iron. Such an evolutionary adaptation would be fascinating, as it would recapitulate a tactic that E. coli invokes during oxidative stress. Aim One will test whether this substitution is driven by changes in the intracellular metal pools or by modifications of the enzymes, and it will probe whether the shift in metallation exerts a cost in catalytic efficiency. Aim Two investigates the hypothesis that radical SAM enzymes comprise a novel third class of ROS- sensitive enzyme. Circumstantial data support the idea: they employ over-oxidizable peripheral iron-sulfur clusters, and E. coli responds to stress by replacing one of these enzymes with a cluster-free analogue. Yet those clusters might plausibly be protected in vivo either by bound SAM or by rapid re-reduction via their native electron donor. If this enzyme family is ROS-sensitive, then stress will affect a broad range of cell processes. Transcriptomic analyses have revealed surprising strategies by which E. coli copes with ROS, and Aim Three probes two newly discovered ones. First, the ClpSA and ClpX unfoldases somehow stabilize branched- chain biosynthesis during periods of H2O2 stress. The mechanism may be that by acting as chaperones the Clp proteins protect apo-dehydratases from aggregation and/or proteolysis. Thus damaged clusters can be re- built. Second, the data reveal that H2O2-stressed cells induce exonuclease III, a key enzyme that is specifically required to repair oxidative DNA lesions. The work will determine how its expression is controlled, whether other repair enzymes are co-regulated, and which enzymes are needed to allow growth in the face of environmental H2O2. Finally, Aim Four extends prior investigations of the molecular basis of obligate anaerobiosis. Previous work identified two key enzymes that are poisoned upon aeration of a model anaerobe; the next step is to resolve the underlying mechanisms. As proof of principle, this Aim will culminate in an effort to construct a derivative strain whose central metabolism is not blocked by oxygen. These projects are selected to fill in key pieces of the puzzle of oxidative stress. They are closely interconnected, with the mechanistic theme being the incompatibility of oxygen and iron-centered metabolism. Each Aim derives directly from data obtained during the current funding period.

氧化应激对生物学有很大的影响：它决定微生物群落的结构，对先天免疫系统的作用至关重要，并且被怀疑是多种人类免疫系统的基础病理学。该领域有几个基本问题：氧化剂是如何形成的，数量是多少？什么它们破坏最快的是哪些特定生物分子？细胞如何保护自己？为什么会不同生物体对氧化剂的敏感性不同？机制细节主要来自对模型细菌大肠杆菌的研究。这应用建议加深和拓宽理解。迄今为止，活性氧（ROS）众所周知，O2 和 H2O2 主要通过灭活簇依赖性脱水酶和非脱水酶来破坏生长。 - 氧化还原单核铁酶。几乎所有生物体中的脱水酶都很脆弱。然而，看来一些需氧菌的单核酶通过使用除铁。这种进化适应将是令人着迷的，因为它将重演大肠杆菌调用的策略在氧化应激期间。 Aim One 将测试这种替代是否是由细胞内的变化驱动的金属池或通过酶的修饰，它将探究金属化的转变是否会产生成本催化效率。目标二研究了自由基 SAM 酶构成新型第三类 ROS 的假设：敏感酶。环境数据支持这个想法：他们使用可过度氧化的外围铁硫簇，大肠杆菌通过用无簇的类似物替换其中一种酶来应对压力。然而这些簇可能在体内通过结合的 SAM 或通过其天然的快速再还原而受到保护电子供体。如果该酶家族对 ROS 敏感，那么压力将影响广泛的细胞过程。转录组分析揭示了大肠杆菌应对 ROS 和 Aim 的令人惊讶的策略三个探测器有两个是新发现的。首先，ClpSA 和 ClpX 解折叠酶以某种方式稳定支链- H2O2 胁迫期间的链生物合成。其机制可能是通过充当伴侣 Clp 蛋白保护脱辅基脱水酶免于聚集和/或蛋白水解。因此损坏的簇可以重新建造的。其次，数据表明，H2O2 应激的细胞会诱导核酸外切酶 III，这是一种特异性作用的关键酶。修复氧化DNA损伤所需的。这项工作将决定它的表达是如何被控制的，是否其他修复酶是共同调节的，并且需要哪些酶才能在面对环保双氧水。最后，目标四扩展了先前对专性厌氧的分子基础的研究。以前的工作确定了两种关键酶，它们在模型厌氧菌通气时会中毒；下一步是解决根本机制。作为原则证明，这一目标最终将努力构建一个其中心代谢不受氧气阻碍的衍生菌株。这些项目被选择来填补氧化应激难题的关键部分。他们紧密地相互关联，其机制主题是氧和以铁为中心的新陈代谢的不相容性。每个目标都直接源自当前资助期间获得的数据。