Spatial regulation of nucleoids and chromosome-partitioning proteins is important for proper chromosome partitioning in Escherichia coli. However, the underlying molecular mechanisms are unknown. In the present work, we showed that mutation or chemical perturbation of secretory A (SecA), an ATPase component of the membrane protein translocation machinery, SecY, a component of the membrane protein translocation channel and acyl carrier protein P (AcpP), which binds to SecA and MukB, a functional homologue of structural maintenance of chromosomes protein (SMC), resulted in a defect in chromosome partitioning. We further showed that SecA is essential for proper positioning of the oriC DNA region, decatenation and maintenance of superhelicity of DNA. Genetic interaction studies revealed that the topological abnormality observed in the secA mutant was due to combined inhibitory effects of defects in MukB, DNA gyrase and Topo IV, suggesting a role for the membrane protein translocation machinery in chromosome partitioning and/or structural maintenance of chromosomes.