The Escherichia coli replication initiator DnaA is titrated on the chromosome

DNA replication initiation is orchestrated in many prokaryotes by the replication initiator DnaA. Two models for regulation of DnaA activity in Escherichia coli have been proposed: the switch between an active and inactive form of DnaA, and the titration of DnaA on the E. coli chromosome. Although proposed decades ago, experimental evidence of a titration-based control mechanism is still lacking. Here, we first identified a conserved high-density region of binding motifs near the origin of replication, an advantageous trait for titration of DnaA. We then investigated the mobility of DnaA by single-particle tracking microscopy in wild-type and deletion mutants E. coli strains, while monitoring cellular size and DNA content. Our results indicate that the chromosome of E. coli controls the free amount of DnaA in a growth rate-dependent fashion. Finally, we provide insights on the relevance of DnaA titration in stabilising DNA replication by preventing re-initiation events during slow growth.