This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Stress response is the ability of an organism to adjust to drastic changes in environmental parameters. All living organisms have genetically encoded stress response and adaptation systems. Oxidative stress is a common stress form at the cellular level. Recent revolutionary progress in high-throughput experimental and computational approaches offer an opportunity to characterize the molecular mechanisms of oxidative stress response at the level of organisms, cells, genomes, regulatory networks and individual components. We propose to characterize stress response and regulation by focusing on oxidative stress response mechanisms. We will examine Pseudomonas bacterial species under conditions of oxidative stress to (i) identify and quantify gene expression changes, (ii) analyze proteomic and metabolic changes, (iii) build a global transcriptomic and proteomics networks, and (iv) characterize oxidative stress response networks. Much of the proposed research is cutting edge, has not been performed in any biological system, and it will allow for a new avenue of research for the Alfano and Becker research groups and new grant funding opportunities. Research into the mechanisms by which model organisms maintain redox homeostasis have revealed it to be an intricate and complex process. Bacterial antioxidant mechanisms are best understood in commensal Escherichia coli and the Gram-positive model organism Bacillus subtilis (saprophyte) (41), however, there are significant gaps in our understanding of redox homeostasis in non-model organisms. Here we seek to use systems biology approaches to determine if the mechanisms by which bacteria that are exposed to the intense oxidative stress response of the innate immune system vary from that of free-living or commensal bacteria. Specifically, we will use the animal pathogen Pseudomonas aeruginosa and the plant pathogen P. syringae. Both species are exposed to endogenous oxidative stress and exposed to oxidative stress from their host's innate immune response. It will be informative to compare and contrast the importance of oxidative stress responses in pathogenicity of plants and animals. A systems biology approach will allow for a greater understanding of the divergent and convergent evolutionary traits that these bacteria have acquired. We anticipate that we will identify oxidative stress response mechanisms that are common to both species. Our long-term goal is to understand how redox signals from biotic stress are mediated in Gram-negative pathogens of plants and animals to elucidate mechanisms of oxidative stress protection. The Specific Aims of this application are as follows: Identify and quantitate gene expression changes in P. aeruginosa and P. syringae under different oxidative stress conditions; Analyze proteomic and metabolic changes during oxidative stress in P. aeruginosa and P. syringae; Build global network of transcriptomic and proteomic changes induced by oxidative stress; and test role of gene products in oxidative stress response.