The bacterial chromosome is spatially organized through protein-mediated compaction, supercoiling, and cell-boundary confinement. Structural Maintenance of Chromosomes (SMC) complexes are a major class of chromosome-organizing proteins present throughout all domains of life. Here, we study the role of the Escherichia coli SMC complex MukBEF in chromosome architecture and segregation. Using quantitative live-cell imaging of shape-manipulated cells, we show that MukBEF is crucial to preserve the toroidal topology of the Escherichia coli chromosome and that it is non-uniformly distributed along the chromosome: it prefers locations toward the origin and away from the terminus of replication, and it is unevenly distributed over the origin of replication along the two chromosome arms. Using an ATP hydrolysis-deficient MukB mutant, we confirm that MukBEF translocation along the chromosome is ATP-dependent, in contrast to its loading onto DNA. MukBEF and MatP are furthermore found to be essential for sister chromosome decatenation. We propose a model that explains how MukBEF, MatP, and their interacting partners organize the chromosome and contribute to sister segregation. The combination of bacterial cell-shape modification and quantitative fluorescence microscopy paves way to investigating chromosome-organization factors in vivo.