A three-dimensional ParF meshwork assembles through the nucleoid to mediate plasmid segregation

Abstract

Genome segregation is a fundamental step in the life cycle of every cell. Most bacteria rely on dedicated DNA partition proteins to actively segregate chromosomes and low copy-number plasmids. Here, by employing super resolution microscopy, we establish that the ParF DNA partition protein of the ParA family assembles into a three-dimensional meshwork that uses the nucleoid as a scaffold and periodically shuttles between its poles. Whereas ParF specifies the territory for plasmid trafficking, the ParG partner protein dictates the tempo of ParF assembly cycles and plasmid segregation events by stimulating ParF adenosine triphosphate hydrolysis. Mutants in which this ParG temporal regulation is ablated show partition deficient phenotypes as a result of either altered ParF structure or dynamics and indicate that ParF nucleoid localization and dynamic relocation, although necessary, are not sufficient per se to ensure plasmid segregation. We propose a Venus flytrap model that merges the concepts of ParA polymerization and gradient formation and speculate that a transient, dynamic network of intersecting polymers that branches into the nucleoid interior is a widespread mechanism to distribute sizeable cargos within prokaryotic cells.

Structures of partition protein ParA with nonspecific DNA and ParB effector reveal molecular insights into principles governing Walker-box DNA segregation

“…The bacterial TP228 ParA-GFP fusion protein was used as a control and, as previously reported, localized with the E. coli chromosome (Supplemental Fig. S5; Ringgaard et al 2009;McLeod et al 2016). Colocalization was also revealed between wild-type archaeal pNOB8 ParA-GFP and the bacterial nucleoid (Fig.…”

Plasmid Localization and Partition in Enterobacteriaceae

“…Recent 3D-SIM and single-particle tracking PALM imaging indicated that ParA F does not form filaments in vivo but follows two distinct behaviors: it appears as a few concentrated patches bound to some dense regions of the nucleoid, and it oscillates within the nucleoid (22). Current subcellular imaging supports biochemical data that Brownian ratchet mechanisms (described below) explain dynamic ParA localization and activity in partition (20,22,23,(39)(40)(41)(42)(43).…”

“…They have also allowed visualization of plasmid and protein dynamics with high temporal resolution (for review on fluorescence imaging techniques, see reference 17). In addition, evidence indicates that the bacterial chromosome compacted in the nucleoid contributes both passively (as the major structure occupying space inside a bacterial cell) and actively (as a support and/or matrix for plasmid movement) to plasmid partition mechanisms (18)(19)(20)(21)(22)(23); also see below).…”

“…3D fluorescence microscopy, namely, structured illumination microscopy (3D-SIM) with resolution to ∼80 nm, revealed that F plasmid partition complexes are intimately associated with the bacterial nucleoid and localized within its mass (22) rather than simply on its surface. Recently, the RHH 2 CBP of the type I system of plasmid TP228 has also been imaged, and it forms large foci with plasmid DNA, which dynamically associate with the cognate ParA in live E. coli cells (23).…”

“…Some, such as ParA pB171 , ParA F , and ParA TP228 , displayed an oscillatory behavior ( Fig. 2B and C) (12,23,(34)(35)(36)(37). Others, such as ParA P1 , did not oscillate but did form dynamic gradients (38).…”