PROJECT ABSTRACT Infections due to antibiotic-resistant bacteria are becoming costlier and deadlier, and drug development efforts to expand our antibiotic arsenal cannot keep up with the rapid pace of bacterial evolution. New approaches are needed to address this pressing problem. Targeting the SOS response, a key mechanism by which bacteria adapt to antibiotics and evolve resistance, is an emerging approach with significant promise. The SOS response is a network of genes that are turned on when the DNA damage ‘SOS signal’ is activated. Across many bacterial pathogens, SOS pathway effectors have been shown to serve critical roles in the evasion of antibiotics, including heightened mutation rates, transfer of mobile resistance genes, and phenotypic conversion to antibiotic-resistant states. These findings highlight the potential for small molecule SOS inhibitors to function as antibiotic adjuvants, preventing bacteria from adapting or evolving resistance to antibiotics. The core of the ‘SOS signal’ is well-conserved across pathogens and is encapsulated in the interaction of two proteins, LexA and RecA. RecA binds to DNA fragments to form activated RecA* and this complex stimulates the auto-proteolysis of LexA. This process is pivotal to turning on the SOS response as LexA is a repressor of the SOS response. Upon auto-proteolysis, LexA is no longer able to function as a repressor and the SOS response is activated. The overall goal of this project is to reveal how the ‘SOS signal’ is generated, with a particular focus on three major unknowns: 1) the molecular composition of the complex between LexA and RecA*, 2) how conformational dynamics and allosteric modulation permits transmission of the signal between these two proteins, and 3) how the generation of the SOS signal can be inhibited by small molecules. To address these unresolved and important questions, protein engineering will be employed to reduce the SOS signal to its minimal components; novel minimally-perturbing fluorescent probes will be utilized to report on structural dynamics; and newly-discovered small molecule SOS antagonists will be exploited to dissect the critical steps in generation of the SOS signal. This proposal offers fundamental biochemical and biophysical insights into the conserved LexA/RecA* signaling axis and will fuel a novel approach to the problem of antibiotic resistance, rooted in targeting an adaptive pathway that promotes evasion and the evolution of resistance itself.