A research program will be undertaken to study agr signal transduction in the commensal pathogen, Staphylococcus aureus. The accessory gene regulator (agr) locus found in all staphylococci encodes a quorum sensing (QS) circuit that controls the expression of virulence and other accessory genes. It consists of two oppositely oriented transcriptional units, of which one encodes four proteins, AgrBDCA, involved in production and sensing of an autoinducer peptide (AIP), and the other encodes a regulatory RNA that is the effector of target gene regulation. The finding that staphylococcal virulence can be inhibited through antagonism of this QS pathway has fueled tremendous interest in understanding the detailed mechanisms at play throughout the circuit. Building on recent breakthroughs that have allowed us to reconstitute much as the quorum sensing circuit using purified components, we propose to integrate chemical, biochemical, biophysical and genetic tools for the purpose of obtaining a deeper understanding into the molecular processes underlying the production and sensing of the autoinducer peptide (AIP) pheromone that is central to agr regulation. The program will move forward in three directions: Aim 1, identifying the key missing players in AIP biosynthesis; Aim 2, understanding how agonism and antagonism of the QS system relates to newly discovered conformational changes in the AIP receptor, AgrC, and; Aim 3, identifying novel modulators of agr through sophisticated target-based screens. These studies will lay the groundwork for the development of therapeutic strategies targeting agr, but also contribute to a fundamental understanding of QS systems of this type, which are pervasive in the low-GC bacterial phylum, Firmicutes.