Cell boundary confinement sets the size and position of the E. coli chromosome

While the spatiotemporal structure of the genome is crucial to its biological function, many basic questions remain unanswered on the morphology and segregation of chromosomes. Here, we experimentally show in Escherichia coli that spatial confinement plays a dominant role in determining both the chromosome size and position. In non-dividing cells with lengths up to 10 times normal, single chromosomes are observed to expand more than 4 fold in size, an effect only modestly influenced by deletions of various nucleoid-associated proteins. Chromosomes show pronounced internal dynamics but exhibit a robust positioning where single nucleoids reside strictly at mid-cell, while two nucleoids self-organize at [1/4] and [3/4] cell positions. Molecular dynamics simulations of model chromosomes recapitulate these phenomena and indicate that these observations can be attributed to depletion effects induced by cytosolic crowders. These findings highlight boundary confinement as a key causal factor that needs to be considered for understanding chromosome organization.