The Escherichia coli replication initiator DnaA is titrated on the chromosome

Lorenzo Olivi (), Stephan Köstlbacher, Christina Ludwig, Mees Langendoen, Nico J. Claassens, Thijs J. G. Ettema, John Oost, Pieter Rein Wolde, Johannes Hohlbein () and Raymond H. J. Staals ()
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Lorenzo Olivi: Wageningen University & Research
Stephan Köstlbacher: Wageningen University & Research
Christina Ludwig: Technical University of Munich
Mees Langendoen: Wageningen University & Research
Nico J. Claassens: Wageningen University & Research
Thijs J. G. Ettema: Wageningen University & Research
John Oost: Wageningen University & Research
Pieter Rein Wolde: Institute AMOLF
Johannes Hohlbein: Wageningen University & Research
Raymond H. J. Staals: Wageningen University & Research

Nature Communications, 2025, vol. 16, issue 1, 1-18

Abstract: Abstract 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.

Date: 2025
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DOI: 10.1038/s41467-025-63147-1

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