PROJECT SUMMARY/ABSTRACT Bacillus anthracis spores are the infectious particles for the disease anthrax. Through abrasion, ingestion, or inhalation, spores access subcutaneous layers, gastrointestinal mucosa, or alveolar cavities where they are phagocytosed by macrophages, undergo germination, and cause cutaneous or systemic disease. While spores are required for transmission, their germination and outgrowth are critical for pathogenesis. However, owing to poor survival as vegetative cells in the environment, the cycle of germination, vegetative growth, and spore formation is essential for the pathogenic lifestyle of B. anthracis. Environmental changes due to global warming and deforestation have increased seasonal anthrax outbreaks in endemic areas and have led to the emergence of new zoonotic pathogens. Addressing these challenges will require a deeper understanding of the mechanisms of B. anthracis sporulation and germination. Targeting these processes will facilitate the development of strategies for more effective disease prevention and treatment. For years, the non-pathogenic soil bacterium Bacillus subtilis has served as the model for sporulation and germination of spore forming pathogens like B. anthracis. Only a handful of factors involved in these processes have been characterized in B. anthracis; in all cases these were homologs of B. subtilis sporulation/germination proteins. In preliminary studies, we used transposon sequencing (Tn-seq) to identify 156 sporulation genes in B. anthracis. Strikingly, 77 of these genes are either not required for B. subtilis sporulation or are not present in its genome. This grant proposal seeks to leverage modern molecular approaches to identify and characterize the complete set of sporulation and germination factors in B. anthracis. The aims are: (1) Characterize the complete set of B. anthracis sporulation factors. We will build an ordered library of Tn insertions in all nonessential B. anthracis genes using the Knockout Sudoku method. We will then use individual mutants to characterize all the sporulation genes we identified. We will perform quantitative sporulation assays and fluorescence microscopy to determine the stage at which development is blocked. Finally, we will initiate molecular characterization of those factors that have strong mutant phenotypes and are unique to B. anthracis. (2) Identify and characterize a comprehensive set of germination factors in B. anthracis. We will perform a complementary germination Tn-seq screen in B. anthracis that we have successfully performed in B. subtilis. Using the ordered knock out collection, we will then validate the hits and characterize those with the strongest germination defect that are unique to B. anthracis. If successful, this proposal will establish the extent to which B. subtilis can serve as a model for sporulation and germination in B. anthracis and will define a large set of factors unique to processes in this pathogen that will be subjec...