Prevention of RK2 plasmid replication initiation in starved Escherichia coli cells

Bacterial adaptation to environmental stress involves stringent regulation of DNA replication. While the mechanisms controlling chromosomal replication under adverse conditions, such as amino acid starvation, are relatively well characterized, the molecular basis for stress-induced inhibition of plasmid replication remains largely unknown. In this study, we investigated how amino acid starvation affects the replication of the broad-host-range RK2 plasmid in Escherichia coli, focusing on the plasmid-encoded replication initiator TrfA and host-encoded initiator DnaA. We found that the RK2 plasmid origin of replication (oriV) occupation by TrfA and DnaA is prevented in stress conditions. We also did not detect increase of plasmid DNA level showing that new rounds of RK2 replication are not initiated. The replication-inactive state persisted even in cells expressing a hyperactive monomeric TrfA variant that is incapable of handcuffing, indicating that other regulatory mechanisms, beyond handcuffing, contribute to the prevention of the plasmid replication. The reduction of initiators binding to the plasmid origin during stress coincided with a substantial decrease in the intracellular levels of TrfA, as shown in this study, and of DnaA, as reported previously. Given that cell division is arrested during stress, the only explanation for the observed gradual decrease of TrfA levels is proteolysis. Our findings demonstrate that during amino acids starvation, RK2 plasmid replication in E. coli is likely prevented by a significant drop in initiator proteins concentrations. This uncovers a previously underappreciated layer of plasmid replication control under stress conditions.