Project Summary While microbes are single-celled organisms, they naturally form densely packed communities known as biofilms. Biofilms pose a major public health challenge as they often cause difficult-to-treat infections that exhibit properties such as antimicrobial resistance and persistence even with long courses of antibiotics. Since microbiology research has often been conducted using domesticated strains under controlled laboratory conditions where biofilms do not form, there is a need for understanding emergent behavior that only exists in biofilms. Integrating bacterial gene regulation and metabolism with biofilm morphology and behavior requires a model system with sufficiently mapped genetic and metabolic pathways, established genetic tractability, and known biofilm growth conditions. For these reasons, Bacillus subtilis is considered a model organism for biofilm studies. Bacillus subtilis biofilms are composed of a network of resident cells and tightly regulated extracellular matrix. Initial biofilm formation and matrix expression is controlled by the expression of master regulators that respond to traditional quorum-sensing molecules. However, newly appreciated ion-based signaling in Bacillus subtilis is critical for community level fitness by coordinating nutrient sharing within a biofilm. This discovery highlights signaling pathways that are critical for the formation and overall fitness of biofilms yet remain undiscovered. To help close this gap in knowledge, we will focus on discovering the synthesis genes for acetylcholine and acetylcholine-based cell-to-cell signaling phenotypes. Our leading hypothesis is acetylcholine acts as a signaling molecule in cell-to-cell communications within biofilms. Aim 1 will identify acetylcholine biofilm signaling phenotypes using microscopy and Aim 2 will identify the genes responsible for acetylcholine synthesis. The overarching goal of this project is to provide the first description of genetic, metabolic, and physiological mechanisms for acetylcholine in all bacterial species. Additionally, the function of and synthesis gene(s) responsible for acetylcholine production will provide the scientific community with the foundational knowledge to explore bacterial homologs in other species and how signaling within bacteria may also impact complex systems like the human gut-brain-axis in development and disease.