Brownian Ratchet Mechanism for Faithful Segregation of Low-Copy-Number Plasmids

Abstract

Bacterial plasmids are extrachromosomal DNA that provides selective advantages for bacterial survival. Plasmid partitioning can be remarkably robust. For high-copy-number plasmids, diffusion ensures that both daughter cells inherit plasmids after cell division. In contrast, most low-copy-number plasmids need to be actively partitioned by a conserved tripartite ParA-type system. ParA is an ATPase that binds to chromosomal DNA; ParB is the stimulator of the ParA ATPase and specifically binds to the plasmid at a centromere-like site, parS. ParB stimulation of the ParA ATPase releases ParA from the bacterial chromosome, after which it takes a long time to reset its DNA-binding affinity. We previously demonstrated in vitro that the ParA system can exploit this biochemical asymmetry for directed cargo transport. Multiple ParA-ParB bonds can bridge a parS-coated cargo to a DNA carpet, and they can work collectively as a Brownian ratchet that directs persistent cargo movement with a ParA-depletion zone trailing behind. By extending this model, we suggest that a similar Brownian ratchet mechanism recapitulates the full range of actively segregated plasmid motilities observed in vivo. We demonstrate that plasmid motility is tuned as the replenishment rate of the ParA-depletion zone progressively increases relative to the cargo speed, evolving from diffusion to pole-to-pole oscillation, local excursions, and, finally, immobility. When the plasmid replicates, the daughters largely display motilities similar to that of their mother, except that when the single-focus progenitor is locally excursive, the daughter foci undergo directed segregation. We show that directed segregation maximizes the fidelity of plasmid partition. Given that local excursion and directed segregation are the most commonly observed modes of plasmid motility in vivo, we suggest that the operation of the ParA-type partition system has been shaped by evolution for high fidelity of plasmid segregation.

Brownian ratchet mechanisms of ParA-mediated partitioning

“…The conceptual underpinning of different ParA-mediated Brownian ratchet models, including the “DNA-relay model” (6), “hitch-hiking model” (5), and our mechanochemical coupling-mediated Brownian ratchet model (3, 4, 14–16), is essentially the same. All three can be considered as burnt-bridge Brownian ratchets, as originally proposed in (3, 4, 15).…”

“…As proposed by our original ParA-mediated Brownian ratchet model (3, 4, 15), this free energy gradient results in a so-called “chemophoresis” force (energy descent/distance) (19) that could direct the cargo movement (3, 4, 15). From the standpoint of chemical bonds, this asymmetric distribution of the ParA–ParB bonds bridging the cargo with the substrate provides a net force that pulls the cargo forward (6, 14, 16). Regardless of the mode of driving force, all the ParA-mediated Brownian ratchet models agree that the asymmetric ParA distribution is necessary for directed and persistent cargo movement (3–6, 14–16), as originally proposed (3, 4, 15).…”

“…From the standpoint of chemical bonds, this asymmetric distribution of the ParA–ParB bonds bridging the cargo with the substrate provides a net force that pulls the cargo forward (6, 14, 16). Regardless of the mode of driving force, all the ParA-mediated Brownian ratchet models agree that the asymmetric ParA distribution is necessary for directed and persistent cargo movement (3–6, 14–16), as originally proposed (3, 4, 15). The differences among these models lie in the details concerning exactly how this asymmetry in ParA distribution emerges and is harnessed to drive directed cargo movements, the dimensionality over which ratcheting takes place, and the effect of ParA-ParB bonds on the diffusive mobility of the cargo.…”

“…Both the mechanochemical Brownian ratchet model (14, 16) and the DNA-relay model (6) invoke chemical bond-mediated tethering to exert mechanical forces that direct persistent movement. The chemical bond herein refers to DNA-ParA-ParB-cargo linkage, which, for brevity, we refer as a ParA-ParB bond thereafter.…”

“…Certainly, the quenching of cargo diffusion due to bond tethering hinges on bond elasticity: If the bond tether stiffness is insufficient to quench cargo diffusion, as assumed in the DNA-relay model, then it will be equally incapable of maintaining directed and persistent cargo movement. The corresponding movement trajectories would be much more diffusive (6), as opposed to relatively straight lines (3, 14, 16). …”