Toxin–antitoxin (TA) loci were initially identified on conjugative plasmids, and one function of plasmid-encoded TA systems is to stabilize plasmids or increase plasmid competition via postsegregational killing. Here, we discovered that the type II TA system, Pseudoalteromonas rubra plasmid toxin–antitoxin PrpT/PrpA, on a low-copy-number conjugative plasmid, directly controls plasmid replication. Toxin PrpT resembles ParE of plasmid RK2 while antitoxin PrpA (PF03693) shares no similarity with previously characterized antitoxins. Surprisingly, deleting this prpA-prpT operon from the plasmid does not result in plasmid segregational loss, but greatly increases plasmid copy number. Mechanistically, the antitoxin PrpA functions as a negative regulator of plasmid replication, by binding to the iterons in the plasmid origin that inhibits the binding of the replication initiator to the iterons. We also demonstrated that PrpA is produced at a higher level than PrpT to prevent the plasmid from overreplicating, while partial or complete degradation of labile PrpA derepresses plasmid replication. Importantly, the PrpT/PrpA TA system is conserved and is widespread on many conjugative plasmids. Altogether, we discovered a function of a plasmid-encoded TA system that provides new insights into the physiological significance of TA systems.

毒素-抗毒素 (TA) 基因座最初是在结合质粒上鉴定的，质粒编码的 TA 系统的一个功能是通过分离后杀伤来稳定质粒或增加质粒竞争。在这里，我们发现 II 型 TA 系统，即 Pseudoalteromonas rubra质粒毒素 - 抗毒素 PrpT/PrpA，在低拷贝数共轭质粒上，直接控制质粒复制。毒素 PrpT 类似于质粒 RK2 的 ParE，而抗毒素 PrpA (PF03693) 与先前表征的抗毒素没有相似之处。令人惊讶的是，删除这个prpA - prpT来自质粒的操纵子不会导致质粒分离丢失，但会大大增加质粒拷贝数。从机制上讲，抗毒素 PrpA 通过与质粒起源中的 iterons 结合，抑制复制起始者与 iterons 的结合，起到质粒复制的负调节剂的作用。我们还证明了 PrpA 的产生水平高于 PrpT，以防止质粒过度复制，而不稳定 PrpA 的部分或完全降解会抑制质粒复制。重要的是，PrpT/PrpA TA 系统是保守的并且广泛存在于许多接合质粒中。总之，我们发现了质粒编码的 TA 系统的功能，它为 TA 系统的生理意义提供了新的见解。