Bacteria communicate and cooperate to dictate a variety of cellular behaviors including pathogenesis and antibiotic resistance. While researchers have gained insight into the mechanisms involved in these processes, it remains unclear how microbial cooperation is affected by periodic spatial disturbance (any relatively discrete event in space and time that disrupts the spatial distribution of the microbial population). Periodic spatial disturbances have been linked to key ecological and evolutionary processes. The lack of understanding of how these events tie into communication and cooperation within the microbial system limits our understanding of the interactions within microbial communities. It is with this greater understanding that we can address exposures of personnel during wartime efforts, gain insight in how to better control microbial communities, and address mechanisms of infectious disease. Bacteria use small molecules to cooperate. The majority of the small molecules used for cooperation are not retained by the bacterium that produces them. Therefore, the concentration of small molecules detected by each bacterium is dictated by the positions of neighboring bacteria. Accordingly, the spatial distribution of bacteria and small molecules is critical for cooperation. Previous research has evaluated cooperation and spatial distribution within undisturbed or wellmixed environments, which do not accurately reflect natural conditions where systems are periodically disturbed. Periodic spatial disturbance will unequally alter the positioning of the bacteria and small molecules as they travel at different timescales, thus affecting access to the small molecules, and perturbing cooperation. Our research has demonstrated that intermediate frequencies (e.g., 3, 6, and 9 disturbance events per hour) of periodic spatial disturbances can either enhance or detract from cooperation in engineered bacteria. It remains unclear whether periodic spatial disturbance will affect cooperation in biofilms composed of opportunistic pathogens. Our central hypothesis is that intermediate frequencies of periodic spatial disturbance will alter cooperative cell-cell interactions mediated by small molecules. Over the long term, our aim is to develop an understanding of how to manipulate bacterial cooperation to either enhance, or reduce, microbial growth. Over the short term, the objective of this grant proposal is to determine how periodic spatial disturbances caused by physical force affects cooperation in Pseudomonas aeruginosa and Staphylococcus aureus, two species that infect military personnel and have application in bioremediation. We propose two specific aims. 1. Determine how periodic spatial disturbance affects cooperation in Pseudomonas aeruginosa and Staphylococcus aureus. Our hypothesis is that intermediate periodic spatial disturbance will change the expression of virulence factors driven by small diffusible molecules. 2. Investigate how periodic spatial disturbance affects interspecies interactions in a mixed species biofilm. Our hypothesis is that periodic spatial disturbance performed at intermediate frequencies will alter small molecule mediate cooperative and competitive interactions in a biofilm composed of S. aureus and P. aeruginosa. Students will use microplate reader assays, qPCR, and flow cytometry to measure changes in expression of select virulence factors that are activated through cooperation. They will use agent based modeling and ordinary differential equations to understand the mechanisms that drive changes in gene expression. Once we have an understanding of how periodic spatial disturbance affects cooperation, we can rationally disturb bacterial populations to enhance cooperation, which will have implications in bioremediation efforts. We will also reduce cooperation, which will have implications in the protection of warfighters from infection.