Hu Longhua, Vecchiarelli Anthony G, Mizuuchi Kiyoshi, Neuman Keir C, Liu Jian
Biochemistry and Biophysics Center, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland.
Department of Molecular, Cellular, and Developmental Biology (MCDB), University of Michigan, Ann Arbor, Michigan.
Biophys J. 2017 Apr 11;112(7):1489-1502. doi: 10.1016/j.bpj.2017.02.039.
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.
细菌质粒是染色体外的DNA，为细菌生存提供选择性优势。质粒分配可以非常稳健。对于高拷贝数质粒，扩散确保两个子细胞在细胞分裂后都能继承质粒。相比之下，大多数低拷贝数质粒需要由保守的三方ParA类型系统进行主动分配。ParA是一种与染色体DNA结合的ATP酶；ParB是ParA ATP酶的刺激物，特异性地在类似着丝粒的位点parS与质粒结合。ParB对ParA ATP酶的刺激会使ParA从细菌染色体上释放出来，之后需要很长时间才能重置其DNA结合亲和力。我们之前在体外证明，ParA系统可以利用这种生化不对称性进行定向货物运输。多个ParA-ParB键可以将一个被parS包被的货物连接到一个DNA地毯上，它们可以共同作为一个布朗棘轮发挥作用，引导货物持续移动，后面跟着一个ParA耗尽区。通过扩展这个模型，我们认为类似的布朗棘轮机制概括了体内观察到的所有主动分离的质粒运动。我们证明，随着ParA耗尽区的补充速率相对于货物速度逐渐增加，质粒运动性会发生调整，从扩散演变为极到极振荡、局部偏移，最终变为静止。当质粒复制时，子代在很大程度上表现出与其母代相似的运动性，只是当单焦点祖代进行局部偏移时，子代焦点会进行定向分离。我们表明，定向分离使质粒分配的保真度最大化。鉴于局部偏移和定向分离是体内最常见的质粒运动模式，我们认为ParA类型分配系统的运作在进化过程中已被塑造，以实现高保真的质粒分离。
