Investigation of the Tus-ter Blocking Efficacy during the Chromosome Replication of Live Escherichia Coli Cells

Abstract

In E. coli, a circular chromosome is replicated in a bi-directional manner by two replisome complexes that assemble at a single origin of replication (oriC). The two replication forks are thought to terminate in the termination region, which is flanked by 10 ter sites. Each of these sites is bound by a Termination utilization substance (Tus) protein, thereby forming Tus-ter complexes. In vitro studies have shown that the replication forks are blocked when they approach a Tus-ter complex from the non-permissive side, but not from the permissive side. However, the blocking efficacy of the Tus-ter complex on a replication fork approaching from the non-permissive side, and the subsequent dynamics, have not been examined in live cells. To shed light on these processes in vivo, we utilize quantitative fluorescence microscopy combined with microfluidics to study four different E. coli strains that possess either a normal oriC or only an ectopic copy (oriZ) (inserted 344 kb in the E. coli genetic map along the chromosome), and have either wildtype Tus present or knocked-out (Δtus). In the oriZ strain, the clockwise replication fork (labelled via its sliding clamps) encounters the Tus-ter complex (via its non-permissive face) earlier than the counter-clockwise fork does. On tracking the progression of the replication forks in oriZ strain, we find that the presence of the Tus-ter complex reduces the rate of replication. By monitoring the fluorescence intensity of the fork as well as the duplication of a chromosomal locus, we can determine that a replication fork can nonetheless bypass the non-permissive side of a Tus-ter complex. The oriZ-Δtus strain exhibits replisome dynamics similar to that of oriC strain, whereas the chromosome dynamics differ substantially where the oriZ locus moves mostly towards cell-pole.