Project Summary Streptomyces are ubiquitous filamentous sporulating soil bacteria, important in the environment because they degrade and recycle the nutrients locked in many recalcitrant natural polymers in the soil (e.g., cellulose and chitin) and produce the majority of a wide range of biologically active compounds that are used extensively in human and veterinary medicine (e.g., antibiotic, antiviral, anticancer, antitumor, antifungal, immunosuppressant and anthelmintic pharmaceuticals). Streptomyces are also well known as one of the major producers of the compounds that give soil its characteristic earthy odor. These organisms have large linear genomes. Little is known about the architecture of the linear chromosome within the Streptomyces spore and the mechanisms which govern its development-associated genome segregation and nucleoid condensation during spore formation are poorly understood. The ultimate goal from the proposed basic research is to have a better understanding of the novel cellular processes of these distinctive organisms of tremendous medical importance. The unusual mycelial life cycle and linear structure of the Streptomyces genome are likely to require some unique solutions to the fundamental problems of genome segregation and condensation. The first specific aim of the proposal is to characterize a novel small coiled-coil protein that is exclusive to this group of organisms. A genetic approach was used to provide evidence that this protein is involved in development-associated genome segregation. A proximity-labeling approach, with a promiscuous biotin ligase fusion, is being used to identify proteins that interact with this novel protein in vivo. Alanine-scanning and random PCR-directed mutageneses are being used to identify residues important for protein-protein interaction with one known binding partner. The second specific aim is to characterize type VII secretion system ATPase proteins. In addition to their functions in protein secretion, the ATPase proteins are moonlighting in development-associated segregation and nucleoid condensation. A genetic approach will be used to dissect the first type VII secretion ATPase to identify the region(s) needed for genome segregation. A genetic approach will also be used to determine the parts of both type VII secretion ATPases to identify the regions necessary for correct nucleoid condensation in the spore. Mutants lacking genes encoding both type VII secretion ATPases have genomes organized against the spore periphery (doughnut-shaped) instead of being condensed as an ellipsoid nucleoid in the center of the spore.