Robust control of replication initiation in the absence of DnaA-ATP ? DnaA-ADP regulatory elements in Escherichia coli*

Replication initiation, governed by the widely conserved master initiator protein DnaA, is one of the most well-studied biological processes in bacteria. Specifically, conversion between the ATP and ADP form of DnaA during growth is critical for initiation control. In Escherichia coli, the regulatory elements for conversion have been discovered and extensively characterized over decades. However, they are not widely conserved in bacteria, raising questions on how to generalize the findings in E. coli to other organisms. In this work, we show that the intrinsic ATPase activity of DnaA itself is sufficient for robust and precise initiation control. We constructed and studied E. coli mutants lacking the extrinsic control of either DnaA-ATP → DnaA-ADP (by hda and datA) or DnaA-ADP → DnaA-ATP (by DARS1 and DARS2). These cells showed distinct and opposing characteristics in initiation timing, intrinsic noise (the degree of initiation asynchrony), and extrinsic noise (cell-to-cell variability). Strikingly, when all four regulatory elements were deleted, E. coli exhibited a near wild-type phenotype, with only mildly increased intrinsic and extrinsic initiation noise. By further characterizing the DnaA variants with increased and decreased ATPase activity, we conclude that DnaA is the only requirement for robust initiation, shedding new evolutionary light on cell-cycle control in bacteria.