Molecular basis of ionizing radiation resistance

项目摘要/摘要 在这个项目中，我们将系统地定义遗传和细胞适应 与细菌中的一种极端细菌表型有关--超常耐药性 对电离辐射(IR)的影响。而不是研究原型信息检索 耐药物种，如耐辐射球菌，我们正在产生抗IR 定向进化的大肠杆菌种群。由此产生的菌株接近， 在某些情况下，它的IR抗性水平超过了葡萄球菌的水平。分析 获得性表型背后的突变将使我们能够迅速 专注于关键的蜂窝创新。最终目标是定义所有 导致极端IR抵抗的过程和机制 表型。这种表型在大肠杆菌中的定向进化提供了一种 使这成为可能的窗口。 在这项工作中，我们将开发和扩展现有的四个资源 高度进化的大肠杆菌种群。使用定向进化，所有这些都具有 获得高水平的红外线抵抗力。这些种群被命名为IR-1-20，IR- 2-20、IR-3-20和IR-4-20。我们已经定义了最相关的突变 IR-2-20和IR-3-20的表型。此外，我们目前正在 从零开始产生四个新的进化种群，使用不同类型的 辐射源，并进一步进化现有的四个种群。确实有 四个具体目标： 目标1侧重于新人口和现有人口的演变，以及 定义对表型有重大贡献的突变。 具有关键突变的已确定菌株，分离自其他野生菌株 类型背景，将被构造。目标2代表了使用 现代系统生物学资源，彻底刻画进化者的特征 种群及其衍生的单菌落分离物。目标3将专注于 胰岛素抵抗的一种特殊致病机制探讨 参与复制重新启动的编码蛋白质的基因发生了遗传变化。 目标4是顶峰。我们将使用从目标1-3获得的信息来转移 通过引入一组定义的突变来保持IR抗性表型完整 肠沙门氏菌。

Project Summary/Abstract In this project, we will systematically define the genetic and cellular adaptations associated with an extremophile phenotype in bacteria – extraordinary resistance to the effects of ionizing radiation (IR). Instead of studying prototypical IR resistant species such as Deinococcus radiodurans, we are generating IR resistant Escherichia coli populations by directed evolution. The resulting strains approach, and in some cases exceed the levels of IR resistance seen in Deinococcus. Analysis of the mutations underlying the acquired phenotype will allow us to quickly home in on the key cellular innovations. The ultimate goal is to define ALL processes and mechanisms that contribute to an extreme IR resistance phenotype. The directed evolution of this phenotype in E. coli provides a window that makes this possible. In this work we will both exploit and expand an existing resource of four highly evolved populations of E. coli. Using directed evolution, all of these have acquired high levels of IR resistance. The populations are designated IR-1-20, IR- 2-20, IR-3-20, and IR-4-20. We have already defined the mutations most relevant to the phenotype in both IR-2-20 and IR-3-20. In addition, we are currently generating four new evolved populations from scratch, using a different type of radiation source, and further evolving the four existing populations. There are four specific aims: Aim 1 focuses on the evolution of new and existing populations, as well as definition of the mutations that make substantial contributions to the phenotype. Defined strains with key contributing mutations, isolated in an otherwise wild type background, will be constructed. Aim 2 represents a general effort to use the modern resources of systems biology to thoroughly characterize the evolved populations and single colony isolates derived from them. Aim 3 will focus on an exploration of one particular contributing mechanism of IR resistance involving genetic alterations in genes encoding proteins involved in replication restart. Aim 4 is the capstone. We will use information gained from aims 1-3 to transfer the IR resistance phenotype intact by introducing a defined set of mutations into Salmonella enterica.