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Title: "Bridging Intracellular Signaling and Multicellular Development Programs in Bacillus subtilis Biofilms"

Advisory Committee: Allyson Sgro, PhD – BME (Advisor) Mary Dunlop, PhD – BME (Chair) Ahmad Khalil, PhD – BME Joe Larkin, PhD – Biology and Physics

Abstract: Most bacteria in nature live not as single, planktonic cells but in structured communities known as biofilms. Within a biofilm, bacteria differentiate into a variety of phenotypes that perform specialized roles such as production of the protective matrix that encases the biofilm or secreting a surfactant molecule to allow the biofilm to spread over surfaces. These roles are especially well-characterized in biofilms formed by the gram-positive soil bacterium Bacillus subtilis. What remains largely unknown is how the cells in a biofilm make the decisions to differentiate into a specified phenotype and how this differentiation is regulated in different parts of the biofilm and at different points of its development. Additionally, it is known that bacteria use a suite of cyclic nucleotides as intracellular signaling molecules and, while there are some studies linking different levels of cyclic nucleotides to different phenotypes in biofilms, these are done using bacterial species with fewer defined biofilm phenotypes. Here, we propose a series of aims to interrogate the link between two cyclic nucleotides known to affect B. subtilis biofilm formation, cyclic di-GMP and cyclic di-AMP, and phenotype differentiation during early biofilm formation. By using a combination of computational modeling and quantitative fluorescent microscopy, we aim to create quantitative predictions of cyclic nucleotide levels and then to test those predictions using a fluorescent genetically-encoded sensor for the well studied cyclic di-GMP. We will then take these approaches to study a less-characterized molecule, cyclic di-AMP. In doing so, we aim to not only fully characterize links between cyclic nucleotide signaling and phenotypic differentiation in early biofilm development, but to provide broader understanding on how independent cell units coordinate complex behaviors over large spatial and temporal scales.