Fast assembly and in vivo coalescence of ParBF biocondensates involved in bacterial DNA partition

Faithful DNA segregation in bacteria relies on ParABS systems, in which ParB assembles into condensates at centromere-like parS sites, while the ATPase ParA spatially organizes these complexes. How these ParB condensates maintain dynamic behavior without collapsing into a single structure has remained unclear. Here, we combine chromosome degradation with quantitative imaging to dissect the kinetics and physical principles governing ParB condensate dynamics in vivo. In the absence of the nucleoid, ParB condensates from the plasmid F diffuse freely and coalesce within seconds upon encounter. Strikingly, quantitative analyses indicate that condensates may operate near the fusion-separation boundary, such that minimal energy is sufficient to split them after replication, preventing irreversible coalescence. Using mutants, we demonstrate that condensate assembly is required for coalescence and uncover a dual role for ParAF: nucleoid tethering restricts condensate mobility and limits fusion, while ParAF also promotes a ParBF state competent for assembly and coalescence, likely by enhancing ParB-ParB interactions. Finally, condensates rapidly disassemble and reassemble upon 1,6-hexanediol treatment, underscoring their reversibility and the stabilizing contribution of ParB-DNA interactions. Together, our results establish ParBF complexes as bona-fide biocondensates tuned by ParAF to ensure robust DNA segregation. More broadly, these findings highlight regulated phase separation as a key organizing principle of bacterial replicons.