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.