Diagnosing reactive oxygen species in bacteria

细菌学家提出，许多压力对细菌是有毒的，因为它们 刺激细胞内产生超氧化物和过氧化氢。在大多数情况下， 分子机制已经确定，没有一个是不言自明的。其中许多建议是 基于来自氧化还原活性荧光染料的数据，该染料被认为可以检测细胞内反应性的 氧物种（ROS）。然而，这些结果并不总是得到其他更直接的证据的支持。 方法.该提案包括初步证据，突出了对这些染料的两个担忧： 它们可以被细胞内活性氧以外的物质氧化， 进入细胞会受到压力的影响。目的1将系统地测试荧光素染料的效用， 被羟基自由基和与超氧化物反应的乙啶染料氧化。信号将 在E.大肠杆菌菌株，其中ROS在生物相关性的范围内被调节。染料 使用新的技术，信号将被标准化为它们的细胞内浓度。结果将 是对它们作为活性氧传感器有效性的严格测试。 目标2将开发一种预期可靠的氧化应激的替代标记物， 相关的，非专家也能接触到。丝氨酸转氨酶是一种广泛分布的酶， 铁硫簇被氧化剂转化为独特的[3Fe-4S]形式，可以通过 简单的体外方法。这个目标将优化这些测量，并测试酶是否 对体内相关的ROS水平有反应。 目标3将研究两种抗生素，其毒性作用已被提出依赖于 对细胞内ROS的影响。链黑菌素被认为通过氧化还原产生有毒量的H2 O2- 循环，然后H2 O2与药物结合的Fe（II）反应产生致命的羟基自由基。然而，在这方面， 这里考虑的另一种模型表明，氧化还原循环可能是最小的，游离H2 O2可能 不参与。实验将区分这两种假设。甲氧苄啶是一种 胸腺嘧啶合成的抑制剂，并且已发表的数据表明H2 O2可以促成其致死性。 作者指出，胸腺嘧啶饥饿的细胞产生有毒量的H2 O2。另一种可能性是 外源性H2 O2会造成DNA损伤，无胸腺嘧啶细胞难以修复。这两个亚- 介绍了ROS可能驱动抗生素作用的几种不同方式。这些概念 实验方法可以指导分析先导化合物，其抗生素作用是 怀疑与ROS有关

Bacteriologists have proposed that numerous stresses are toxic to bacteria because they stimulate the intracellular production of superoxide and hydrogen peroxide. In most cases, no molecular mechanism has been determined, and none is self-evident. Many of these proposals are based upon data from redox-active fluorescent dyes, which are believed to detect intracellular reactive oxygen species (ROS). However, those results have not always been supported by other more-direct methods. This proposal includes preliminary evidence that highlights two concerns about these dyes: that they can be oxidized by intracellular species other than ROS, and that the amount of dye that loads into cells can be affected by stress. Aim 1 will systematically test the utility of fluorescein dyes, which are oxidized by hydroxyl radicals, and ethidine dyes, which react with superoxide. Signals will be quantified in E. coli strains in which ROS are adjusted over the range of biological relevance. Dye signals will be normalized to their intracellular concentrations, using a novel technique. The result will be a rigorous test of their validity as ROS sensors. Aim 2 will develop an alternative marker of oxidative stress that is expected to be reliable, relevant, and accessible to non-experts. Serine dehydratase is a widely distributed enzyme whose iron-sulfur cluster is converted by oxidants to a unique [3Fe-4S] form that can be diagnosed through simple in vitro methods. This Aim will optimize these measurements and test whether the enzyme responds to the levels of ROS that are pertinent in vivo. Aim 3 will then investigate two antibiotics whose toxic actions have been proposed to depend upon intracellular ROS. Streptonigrin is thought to generate toxic amounts of H2O2 through redox- cycling, with the H2O2 then reacting with drug-bound Fe(II) to produce fatal hydroxyl radicals. However, an alternative model considered here suggests that redox-cycling may be minimal and free H2O2 may not participate. Experiments will distinguish between these two hypotheses. Trimethoprim is an inhibitor of thymine synthesis, and published data indicates that H2O2 can contribute to its lethality. Authors conjectured that thymine-starved cells produce toxic amounts of H2O2. A different possibility is that exogenous H2O2 creates DNA damage that thymineless cells struggle to repair. These two sub- aims introduce several distinct ways in which ROS may drive the action of antibiotics. These concepts and experimental approaches can guide the analysis of lead compounds whose antibiotic actions are suspected of involving ROS.