PROJECT SUMMARY/ABSTRACT The proposed studies are part of our long-term effort to elucidate fundamental mechanisms underlying chromosome dynamics and bacterial envelope biogenesis in the bacterium Bacillus subtilis. Compaction of replicated chromosomes into morphologically and spatially distinct sister chromatids is essential for faithful DNA segregation in all organisms. The goal of our research is to broadly elucidate how B. subtilis organizes and segregates its chromosomes but we have placed particular emphasis on defining the activity and function of the broadly conserved SMC condensin complex in these processes. Our studies have revealed that these ring-shaped ATPases extrude DNA loops. Loop extrusion ensures that these complexes act in cis drawing DNA in on itself and away from its sister chromosome. This activity can explain how SMC complexes can resolve newly replicated origins in bacteria and sister chromatids in eukaryotes. Our future research seeks to establish whether loop extrusion mediates origin resolution and the extent to which it drives the dynamic rearrangements of the chromosome during the replication-segregation cycle. Separately, we will define the molecular basis for site-specific unloading of SMC complexes by XerD at the replication terminus. Analysis of chromosome dynamics in this simple bacterial system will provide mechanistic insight and a level of resolution not possible in more complex organisms. The bacterial cell wall peptidoglycan (PG) is composed of long glycan strands cross-linked together by short peptides. This three-dimensional meshwork protects the cell from osmotic lysis, determines shape, and its assembly is the target of some of our most successful antibiotics. Accordingly, a deeper understanding of cell wall biogenesis has broad implications for both basic bacterial cell biology and therapeutic development. Research over the last half century has identified virtually all the factors involved in envelope assembly. A major gap in our knowledge is how these enzymes are coordinated with each other and how the cell monitors the envelope for defects and directs their repair. Our future research is focused on three signal transduction pathways that play central roles in these processes. The WalR-WalK two-component signaling system is involved in the homeostatic control of cell wall hydrolases required for growth. The SigI-RsgI pathway monitors the cell wall meshwork and induces PG remodeling enzymes when it identifies defects. Finally, the SigM-YhdLK pathway monitors the universal lipid carrier undecaprenyl-phosphate and prioritizes its use for cell wall synthesis. We seek to define how these pathways function in molecular detail. Our findings will provide broadly relevant principles for envelope assembly and maintenance in all bacteria.