Abstract
Escherichia coli coordinates replication and division cycles by initiating replication at a narrow range of cell sizes. By tracking replisomes in individual cells through thousands of division cycles in wild-type and mutant strains, we were able to compare the relative importance of previously described control systems. We found that accurate triggering of initiation does not require synthesis of new DnaA. The initiation size increased only marginally as DnaA was diluted by growth after dnaA expression had been turned off. This suggests that the conversion of DnaA between its active ATP- and inactive ADP-bound states is more important for initiation size control than the total free concentration of DnaA. In addition, we found that the known ATP/ADP converters DARS and datA compensate for each other, although the removal of them makes the initiation size more sensitive to the concentration of DnaA. Only disruption of the regulatory inactivation of DnaA mechanism had a radical impact on replication initiation. This result was corroborated by the finding that termination of one round of replication correlates with the next initiation at intermediate growth rates, as would be the case if RIDA-mediated conversion from DnaA-ATP to DnaA-ADP abruptly stops at termination and DnaA-ATP starts accumulating.