Molecular basis of ionizing radiation resistance

项目总结/摘要 在这个项目中，我们将系统地定义遗传和细胞适应 与细菌中的极端微生物表型相关-非凡的抵抗力 电离辐射（IR）的影响。而不是研究典型的IR 耐辐射球菌等耐红外线的菌种，我们正在生产耐红外线的 通过定向进化的大肠杆菌种群。产生的菌株接近， 并且在某些情况下超过了异常球菌中所见的IR抗性水平。分析 获得性表型背后的突变将使我们能够快速地 专注于关键的蜂窝创新。最终目标是定义所有 有助于极端IR抗性的过程和机制 表型该表型在E.大肠杆菌提供了一种 这扇窗户使这一切成为可能。 在这项工作中，我们将利用和扩大现有的资源， 高度进化的E.杆菌使用定向进化，所有这些都有 获得了高水平的抗红外线能力。种群被指定为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 与IR-2-20和IR-3-20中的表型相关。此外，我们目前 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。有 四个具体目标： 目标1侧重于新的和现有的人口的演变，以及 对表型有实质性贡献的突变的定义。 具有关键贡献突变的确定菌株，在其他野生环境中分离 的背景，将建立。目标2代表了使用 系统生物学的现代资源来彻底描述进化的 群体和单菌落分离物。目标3将侧重于 探索了一种特殊的IR抗性贡献机制， 基因编码蛋白质的基因改变参与复制重新启动。 目标4是顶点。我们将使用从目标1-3中获得的信息来转移 IR抗性表型通过将一组定义的突变引入 肠道沙门氏菌

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.