Dispensability of extrinsic DnaA regulators in Escherichia coli cell-cycle control
Investigating a long-standing conceptual question in bacterial physiology, we examine why DnaA, the bacterial master replication initiator protein, exists in both ATP and ADP forms, despite only the ATP form being essential for initiation. We engineered the D4 Escherichia coli strain, devoid of all known external elements facilitating the DnaA-ATP/ADP conversion and found that these cells display nearly wild-type behaviors under nonoverlapping replication cycles. However, during rapid growth with overlapping cycles,D4 cells exhibit initiation instability. This aligns with our model predictions, suggesting that the intrinsic ATPase activity of DnaA alone is sufficient for robust initiation control in E. coli and the DnaA-ATP/ADP conversion regulatory elements extend the robustness to multifork replication, indicating an evolutionary adaptation. Moreover, our experiments revealed constant DnaA concentrations during steady-state cell elongation in both wild-type and D4 cells. These insights not only advance our understanding of bacterial cell-cycle regulation and DnaA but also highlight a fundamental divergence from eukaryotic cell-cycle controls, emphasizing protein copy-number sensing in bacteria versus programmed protein concentration oscillations in eukaryotes.