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Staphylococcus aureus is a versatile pathogen that can survive in diverse host environments. This versatility depends on its ability to sense nutrients and respond by modulating gene expression, including the synthesis of virulence determinants. In addition to its ability to synthesize virulence factors, the capacity of S. aureus to form biofilms is an important mediator of virulence in certain infections. Biofilms are a complex aggregation of bacteria commonly encapsulated by an adhesive exopolysaccharide matrix (polysaccharide intercellular adhesin; PIA). To study S. aureus biofilm formation, we assessed the metabolic requirements of S. aureus growing in a biofilm and found the bacteria extracted glucose and accumulated lactate, acetate, formate, and acetoin. Additionally, S. aureus selectively extracted six amino acids from the culture medium (serine, proline, arginine, glutamine, glycine, and threonine). The major staphylococcal exopolysaccharide, PIA, is synthesized when the tricarboxylic acid (TCA) cycle is repressed. To better understand TCA cycle-dependent regulation of PIA and virulence factor synthesis in S. aureus, we artificially induced the TCA cycle by limiting its ability to exogenously acquire a TCA cycle-derived amino acid (i.e., glutamine) by inactivating the glutamine permease gene (glnP) and assessed the effects on biofilm formation and virulence factor synthesis. We found that inactivation of this major glutamine transporter increased TCA cycle activity, transiently decreased PIA synthesis, and significantly reduced in vivo virulence in a rabbit endocarditis model, establishing a causal relationship between TCA cycle activity and virulence factor synthesis. This causal relationship between the TCA cycle and virulence factor synthesis suggests there are regulatory proteins connecting metabolism and the regulation of virulence factor synthesis. This regulation is likely to occur when a metabolite-responsive regulator responds to changes in TCA cycle associated biosynthetic intermediates, the redox status, and/or ATP. In related work, NMR metabolomic analysis of S. epidermidis indicated that TCA cycle stress altered the intracellular concentration of ribose. Using this information, three putative ribose-responsive RpiR-family regulators (orfs SAV0317, SAV0193 and SAV2315) were identified in S. aureus strain UAMS-1. The proteins encoded by sav0317 and sav0193 regulate hexose monophosphate shunt transcription and alter virulence factor synthesis by increasing the transcription or stability of RNAIII. These data confirm a close linkage of central metabolism and virulence factor synthesis in S. aureus and establish that this metabolic linkage can be manipulated to alter infectious outcomes.