Abstract Bacterial cells that form a division septum prior to completion of chromosome segregation face an extraordinary topological and mechanistic challenge to complete the DNA segregation process successfully. The cell division membranes, a complex of DNA-translocating membrane proteins, and the large, circular chromosome substrates all must come together in concert to successfully sort and separate the replicated chromosomes. Critical components of bacterial cell division have been studied for several decades, and yet fundamental features of the structure and function of the chromosome segregation machinery remain unexplored. The long-term goal of this project which combines a variety of biochemical structure-function studies with real-time, live-cell imaging is to understand in molecular mechanistic terms how chromosomal DNA is successfully partitioned across a cellular division septum. What is the path of the DNA substrate through the segregation machinery? How do the complexes function together to couple the ATP hydrolysis cycle with DNA movement? When the end of the circular chromosome is reached, how are the loops of DNA resolved across the division plane? Do these proteins truly function in isolation for their DNA translocation activity, or are there additional cellular components that are involved? Advances in these areas will produce a comprehensive model of the molecular architecture and associated biochemical events involved in this DNA segregation process.