Bacterial conjugation mediates contact-dependent transfer of DNA from donor to recipient bacteria, thus facilitating the spread of virulence and resistance plasmids. Here we describe how variants of the plasmid-encoded donor outer membrane (OM) protein TraN cooperate with distinct OM receptors in recipients to mediate mating pair stabilization and efficient DNA transfer. We show that TraN from the plasmid pKpQIL (Klebsiella pneumoniae) interacts with OmpK36, plasmids from R100-1 (Shigella flexneri) and pSLT (Salmonella Typhimurium) interact with OmpW, and the prototypical F plasmid (Escherichia coli) interacts with OmpA. Cryo-EM analysis revealed that TraN_pKpQIL interacts with OmpK36 through the insertion of a β-hairpin in the tip of TraN into a monomer of the OmpK36 porin trimer. Combining bioinformatic analysis with AlphaFold structural predictions, we identified a fourth TraN structural variant that mediates mating pair stabilization by binding OmpF. Accordingly, we devised a classification scheme for TraN homologues on the basis of structural similarity and their associated receptors: TraNα (OmpW), TraNβ (OmpK36), TraNγ (OmpA), TraNδ (OmpF). These TraN-OM receptor pairings have real-world implications as they reflect the distribution of resistance plasmids within clinical Enterobacteriaceae isolates, demonstrating the importance of mating pair stabilization in mediating conjugation species specificity. These findings will allow us to predict the distribution of emerging resistance plasmids in high-risk bacterial pathogens.

细菌结合介导 DNA 从供体到受体细菌的接触依赖性转移，从而促进毒力和抗性质粒的传播。在这里，我们描述了质粒编码的供体外膜 (OM) 蛋白 TraN 的变体如何与受体中不同的 OM 受体合作，以介导交配对稳定和有效的 DNA 转移。我们显示来自质粒 pKpQIL（肺炎克雷伯菌）的 TraN 与 OmpK36 相互作用，来自 R100-1（福氏志贺菌）和 pSLT（鼠伤寒沙门氏菌）的质粒与 OmpW 相互作用，原型 F 质粒（大肠杆菌）与 OmpA 相互作用。冷冻电镜分析显示 TraN _pKpQIL通过将 TraN 尖端的 β-发夹插入 OmpK36 孔蛋白三聚体的单体中，与 OmpK36 相互作用。将生物信息学分析与 AlphaFold 结构预测相结合，我们确定了第四个 TraN 结构变体，它通过结合 OmpF 来调节交配对稳定性。因此，我们根据结构相似性及其相关受体设计了 TraN 同系物的分类方案：TraNα (OmpW)、TraNβ (OmpK36)、TraNγ (OmpA)、TraNδ (OmpF)。这些 TraN-OM 受体配对具有现实意义，因为它们反映了耐药质粒在临床肠杆菌科分离株中的分布，证明了交配对稳定在介导结合物种特异性中的重要性。