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.

Faithful chromosome and plasmid segregation is essential for bacterial survival, yet the underlying physical principles remain unclear. We show that ParB partition complexes are bona fide biomolecular condensates whose fusion-fission dynamics are tightly regulated to prevent collapse into a single droplet. Using quantitative imaging with inducible chromosome degradation, we demonstrate that ParB condensates operate near the fusion-separation boundary, where minimal energy allows splitting. We further uncover a dual role of the ATPase ParA: tethering condensates to the nucleoid and promoting their assembly. These findings establish partition complexes as phase-separated organelles and reveal how their dynamics are tuned to ensure robust DNA segregation, underscoring phase separation as a key principle of bacterial cell organization.

Cell function relies on liquid-like organelles formed through phase transitions, yet the mechanisms ensuring their specificity and rapid assembly remain poorly understood. In bacterial chromosome segregation via the ParABS system, hundreds of ParB proteins are recruited around the centromere-like parS sequence forming the partition complex. Recent studies have shown that ParB binds CTP and undergoes cycles of binding and unbinding near parS, however, this accounts for the recruitment of only a small fraction of ParB molecules, leaving its role unclear. Separately, a lattice gas model with fixed interaction energy has been proposed to describe ParB phase separation, but it fails to explain key experimental observations, including the absence of droplets in ParB variants. We reconcile these two perspectives by proposing that the ParB-CTP cycle acts as a molecular switch that enhances ParB-ParB interactions, triggering phase transition from vapor to liquid-like condensates. Our hypothesis is supported by numerical simulations of droplet formation and experiments showing that ParB variants disrupting the CTP cycle fail to undergo phase separation. These findings establish a mechanistic framework for ParB-CTP-mediated phase transitions and may have broader implications for understanding the spatial control of intracellular condensate formation.