Tracking the Location and Short-Time Dynamics of the Origin of Chromosomal Replication in E. coli

Abstract

The location of the origin of chromosomal replication (oriC) in E. coli has been previously studied along the long axis of the cell, but little is known about its radial position. It is thought to interact with the cell membrane in a manner which helps regulate the initiation of replication. oriC was labeled with GFP using a ParB-parS system which was a gift from the Gourse lab at UW Madison. For cells taken from a stationary phase culture, the oriC radial distribution peaks ∼250 nm from the center of the cell and not at the inner membrane, which is ∼400 nm from the center. Cells in other stages of growth will be examined to determine the position of oriC throughout the growth cycle and in various media.The dynamics of oriC on the several second timescale were also investigated. oriC was tracked at 10 Hz for 15 seconds. Mean squared displacement versus time along the long and short axes of the cell were fit with a confined diffusion model including a term for diffusion of the confinement domain. Diffusion analysis shows that short-term diffusion (1-2 s) along the long axis (5.2 x 10−3 μm2s−1) is slightly faster than along the short axis (4.0 x 10−3 μm2s−1); the confinement domain is slightly larger along the long axis as well. We suggest that the highly compacted DNA is stiffer along the short axis than along the long axis, as befits a large polymer confined within a spherocylindrical geometry. The diffusion constant on the 2-5 s time scale is 3.3 x 10−4 μm2s−1 in both dimensions, representing movement of the confinement domain.