iiinova — where your digital self keeps evolving

Lorenzo Olivi @loreoliv.bsky.social · 11/05/2026

🧵 New pre-print out from my time at @mib-wur.bsky.social showing how the titration of the initiator protein DnaA on the chromosome of E. coli can generate stable DNA replication @hohlbeinlab.bsky.social @nicoc-micsynmet.bsky.social doi.org/10.64898/202...

163

Lorenzo Olivi @loreoliv.bsky.social · 22/08/2025

12 Bonus! DnaA is also a transcription factor, so together with @baybioms.bsky.social we also investigated how changes in its regulation alter E. coli proteome! Surprisingly, impacting titration or switch alone seems to have no impact on the genes known to be regulated by DnaA...

020

Lorenzo Olivi @loreoliv.bsky.social · 22/08/2025

10 More active DnaA in the cell? The chromosome titrates DnaA more, minimizing re-initiations. Less active DnaA? More DnaA is free, mitigating delays in replication. Even more interestingly, severely hampering titration leads to frequent re-initiation events!

120

Lorenzo Olivi @loreoliv.bsky.social · 22/08/2025

9 We then deleted known DnaA control loci and measured the DNA content of cells through flow cytometry, while we used microscopy 🔬 to measure their size and to determine the bound fraction of DnaA. We could then suggest a role of titration in stabilizing DNA replication in slow-growing E. coli.

120

Lorenzo Olivi @loreoliv.bsky.social · 22/08/2025

8 Next, using single-particle tracking 🔬, we observed the binding state of DnaA in live E. coli cells during steady-state growth at different growth rates. Consistent with previous modelling, we observed that the chromosome of E. coli controls DnaA bound fraction in a growth rate-dependent fashion!

120

Lorenzo Olivi @loreoliv.bsky.social · 22/08/2025

7 We started by checking the distribution of DnaA boxes on the chromosome of E. coli. Glad to say that @stephkoe.bsky.social‬ totally knocked it out of the park with this analysis! He showed that DnaA boxes tend to be enriched around oriC, a favorable feature for initiator titration.

130

Lorenzo Olivi @loreoliv.bsky.social · 22/08/2025

5 We now know that DnaA exists in two forms: DnaA-ATP, competent in unwinding oriC and DnaA-ADP, inactive in oriC unwinding. In this switch model, it is the accumulation of DnaA-ATP that determines replication initiation. As a result, the switch model almost replaced initiator titration.

140

Lorenzo Olivi @loreoliv.bsky.social · 22/08/2025

4 During early days of characterization of DNA replication control, it was hypothesized that the chromosome could act as a sponge 🧽 for DnaA, retarding DnaA binding to oriC by first titrating it on the high affinity boxes. This form of control is what is known as initiator titration model.

140

Lorenzo Olivi @loreoliv.bsky.social · 14/11/2024

Starting to think I should use our dog Ombra as a metaphor to explain students how fluorescent proteins allow you to tell apart things of interest from the background

041