AbstractOxidative stress is concomitant with aerobic metabolism. Thus, bacterial genomes encode elaborate mechanisms to achieve redox homeostasis. Here we report that the peroxide-sensing transcription factor, oxyR, is a common mutational target using bacterial species belonging to two genera, Escherichia coli and Vibrio natriegens, in separate growth conditions implemented during laboratory evolution. The mutations clustered in the redox active site, dimer interface, and flexible redox loop of the protein. These mutations favor the oxidized conformation of OxyR that results in constitutive expression of the genes it regulates. Independent component analysis of the transcriptome revealed that the constitutive activity of OxyR reduces DNA damage from reactive oxygen species, as inferred from the activity of the SOS response regulator LexA. This adaptation to peroxide stress came at a cost of lower growth, as revealed by calculations of proteome allocation using genome-scale models of metabolism and macromolecular expression. Further, identification of similar sequence changes in natural isolates of E. coli indicates that adaptation to oxidative stress through genetic changes in oxyR can be a common occurrence.