Fast assembly and in vivo coalescence of ParB 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 and ParA, an ATPase, spatially organizes these complexes. Yet how ParB condensates preserve their dynamic behavior without collapsing into a single droplet has remained unclear. Here, we combined inducible 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 diffuse freely and coalesce within seconds upon encounter, following Brownian first-encounter statistics. Strikingly, condensates operate near the fusion-separation boundary, such that minimal energy is sufficient to split droplets after replication, thereby preventing irreversible coalescence. Using different mutants, we further show that proper condensate assembly is essential for coalescence. These findings uncover a dual role of ParA: beyond tethering condensates to the nucleoid to limit mobility and prevent uncontrolled fusion, ParA also promotes a ParB state competent for condensate assembly and thus coalescence, likely by enhancing ParB-ParB interactions. Finally, condensates rapidly disassemble and reassemble upon 1,6-hexanediol treatment, underscoring their reversible, dynamic nature and the stabilizing contribution of ParB-DNA interactions. Together, our results establish ParB partition complexes as bona fide biocondensates and reveal how their dynamics are finely tuned by ParA to ensure robust and faithful DNA segregation. More broadly, these findings highlight regulated phase separation as a key organizing principle of bacterial replicons.