The spatial organization of replication is determined by cell size independently of chromosome copy number

Escherichia coli coordinates replication and division cycles by initiating replication at approximately the same size per chromosome at all growth rates. By tracking replisomes in individual cells through thousands of division cycles, we have dissected the mechanism behind this precise process. We have characterized wild-type cells grown under different conditions and also many mutants related to the expression and binding states of the initiator protein DnaA. This rich data set allowed us to compare the relative importance of all previously described control systems. We found that the replication initiation size regulation is not strongly dependent on the absolute concentration of DnaA, nor does it depend on active dnaA expression. Replication initiation is also not consistently triggered by cell division or replication termination. In contrast, some of the factors that convert DnaA between its ATP- and ADP-bound states have a strong effect on initiation size. We suggest a plausible model for DnaA-ATP mediated triggering of initiation at fast growth, where regulatory inactivation of DnaA (RIDA) is the main system for monitoring the number of chromosomes during active replication.