PROJECT SUMMARY/ABSTRACT Bacteria in the orders Bacillales and Clostridiales cause over a million infections each year and are responsible for huge monetary losses to the food industry. These bacteria can resist antibiotics and sterilization by entering a highly durable spore state. Spores are metabolically inactive and can remain dormant for decades, yet upon exposure to nutrients they rapidly resume vegetative growth and cause food spoilage, food-borne illness, or life-threatening disease. The exit from dormancy, germination, is a key target in addressing these diseases. The germination program of most spore-forming bacteria involves a common series of chemical steps and a small set of highly conserved factors. GerA-family receptors embedded in the spore membrane are required for nutrient sensing. The presence of germinants triggers the release of monovalent cations from the spore core, which is rapidly followed by the expulsion of large stores of dipicolinic acid (DPA) likely mediated by a putative transporter complex encoded by the spoVA (5A) operon. This activates cell wall hydrolases that degrade the specialized spore cortex peptidoglycan, allowing rehydration of the spore core, macromolecular synthesis, and resumption of growth. The mechanisms behind each of these steps are almost entirely unknown. We seek to define the germination signal transduction pathway in molecular terms, taking an integrative approach that combines genetic, biochemical, computational, and structural methods. The aims are: (1) Elucidate the mechanisms of nutrient detection and signal transduction. We will determine how GerA- family receptors detect amino acids, sugars, and inorganic cations to trigger germination. We will test the hypothesis that the germination receptors oligomerize forming a membrane pore that functions as a ligand- gated ion channel that releases monovalent cations in response to nutrients. (2) Determine the mechanism of DPA release from the spore core. We will investigate the model that two subunits encoded by the 5A locus form a membrane channel and a third component functions a cytosolic plug that keeps the channel closed. We will test the model that this complex transports DPA and is activated by cation release. If successful, the proposed work will provide molecular-level insight into how spores detect nutrients, trigger ion release, and activate export of DPA, providing the mechanistic and structural framework needed for discovery and optimization of small molecule modulators of the germination pathway. Our work will enable the development of treatments that either inappropriately induce germination, leaving cells vulnerable to standard antibacterial therapies, or block it, directly preventing disease.