Staphylococcus aureus is a major opportunistic pathogen. Due to its metabolic diversity and virulence factor arsenal, it can colonize almost every facet of the human body. The Accessory Gene Regulator (Agr) quorum sensing system has been shown to be critical for promoting biofilm dispersal and significantly contributes to in vivo virulence in animal infection models. In comparison, much less is mechanistically understood regarding how environmental changes in parameters such as pH and oxygenation affect Agr quorum sensing. Unpublished data from our lab suggests that the RNAIII P3 promoter, directly induced by Agr quorum sensing, is induced in aerobic cultures containing glucose and remains uninduced during low-O2 growth. Blood hemolysis and production of phenol soluble modulins (PSMs) were also completely repressed under low-O2 growth. Although these data suggest that low-O2 growth represses Agr expression and function, the mechanism behind this regulation is unclear. Furthermore, previously published studies have found that the expression of many metabolic genes is altered by agr mutation. Based on these observations, our central hypothesis is that Agr expression and activity have a reciprocal relationship to cellular metabolism, in which perturbation of either will have a direct impact on the virulence potential of S. aureus. The overall goal of this proposal is to interrogate the influence of metabolism on Agr function and virulence gene regulation in defined hypoxic and altered pH environments, and in turn, fully assess the impact of Agr disruption on metabolism. This proposal seeks to: 1) identify mechanisms by which low-O2 and pH regulate human blood hemolysis by validating and characterizing mutants from the Nebraska Transposon Mutant Library (NTML) with increased human blood hemolysis during hypoxic growth and making clean deletion mutants to assess growth, acid production, and gene expression of Agr under anaerobic and hypoxic conditions; 2) interrogate the influence of metabolism on Agr function and virulence factor expression to determine how respiratory status affects Agr expression in S. aureus in both pH adjusted and buffered media under defined O2 partial pressures; and 3) elucidate the contribution of Agr and oxygenation to S. aureus metabolism using RNA-seq and targeted metabolomics to monitor the impact of Agr on wildtype and isogenic agr mutants, under defined hypoxic conditions. These data will allow us to resolve strain-dependent effects on Agr-mediated gene expression in response to hypoxic growth under defined metabolic states and will address a current gap in our mechanistic understanding of how S. aureus virulence factor production and its metabolic state cross-regulate through Agr. The data obtained in these studies will generate further hypotheses for future undergraduate research projects and will serve as an important springboard for continued undergraduate research opportunities in biomedical research.