The objectives of this proposal are to determine a) the factors and mechanisms that modulate biofilm formationand dispersal as a function of cellular respiration in Staphylococcus aureus, b) determine how the expressionof these factors is regulated, and c) determine the molecular stimuli for a regulatory system that responds toalterations in the cellular respiratory status. Staphylococcus aureus is a human commensal and a leadingcause of hospital and community acquired infections. For S. aureus to colonize and invade host tissues, it mustsuccessfully sense and respond to environmental perturbations and transition between individualistic andmulticellular behaviors. S. aureus has the ability to form multicellular communities called biofilms, which are theetiologic agents of recurrent staphylococcal infections. We have found that S. aureus modulates biofilmformation as a function of electron flux though respiratory pathways. We have found that when S. aureus isgrowing without a terminal electron acceptor (fermentative growth) there is an increase in cell lysis, whichleads to the release of intracellular components including DNA. The DNA that is released is vital to biofilmintegrity where it aids in holding the cells together in the biofilm matrix. We also discovered that the addition ofa terminal electron acceptor to fermentative biofilms resulted in biofilm dispersal. We have identified two globalregulatory systems that mediate fermentative biofilm formation. Both of these regulatory systems have beenshown to be necessary for pathogenesis, but their molecular stimuli are currently unknown. The overarchinggoal of this project is to understand the physiological changes that occur within S. aureus upon changes incellular respiration, and examine how these changes affect the community structure. We will use genetic,physiologic, biochemical, and molecular techniques to investigate an understudied variable that causes S.aureus to modulate cell lysis, and thereby affect biofilm formation and biofilm dispersal. We will further definethe factors involved in fermentative cell lysis and biofilm formation. We will also define the factors andmolecular mechanisms involved in the dispersal of fermentative biofilms. We will examine the molecular stimuliof one regulatory system (Sae) and examine how another regulatory system (Srr) controls the expression offactors that alter cell lysis and biofilm formation. Ultimately, we would like to apply our findings to modulate thebehavior of S. aureus using molecules that alter cell signaling, and thereby, positively affect infectionoutcomes. Completion of the studies will help provide knowledge about two requirements for staphylococcalpathogenesis: environmental sensing and biofilm formation.