The Escherichia coli replication initiator DnaA is titrated on the chromosome.

DNA replication initiation is orchestrated in bacteria 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, and the titration of DnaA on the 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 visualising single proteins inside single cells of 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. Moreover, they address long-standing questions on the relevance of DnaA titration in stabilising DNA replication by preventing re-initiation events during slow growth.