A large subfamily of the type IV secretion systems (T4SSs), termed the conjugation systems, transmit mobile genetic elements (MGEs) among many bacterial species. In the initiating steps of conjugative transfer, DNA transfer and replication (Dtr) proteins assemble at the origin‐of‐transfer (oriT) sequence as the relaxosome, which nicks the DNA strand destined for transfer and couples the nicked substrate with the VirD4‐like substrate receptor. Here, we defined contributions of the Dtr protein TraK, a predicted member of the Ribbon‐Helix‐Helix (RHH) family of DNA‐binding proteins, to transfer of DNA and protein substrates through the pKM101‐encoded T4SS. Using a combination of cross‐linking/affinity pull‐downs and two‐hybrid assays, we determined that TraK self‐associates as a probable tetramer and also forms heteromeric contacts with pKM101‐encoded TraI relaxase, VirD4‐like TraJ receptor, and VirB11‐like and VirB4‐like ATPases, TraG and TraB, respectively. TraK also promotes stable TraJ–TraB complex formation and stimulates binding of TraI with TraB. Finally, TraK is required for or strongly stimulates the transfer of cognate (pKM101, TraI relaxase) and noncognate (RSF1010, MobA relaxase) substrates. We propose that TraK functions not only to nucleate pKM101 relaxosome assembly, but also to activate the Tra_pKM101 T4SS via interactions with the ATPase energy center positioned at the channel entrance.

中文翻译：

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TraK 辅助因子通过 pKM101 IV 型分泌系统激活底物转移，与其在松弛体组装中的作用无关。

IV 型分泌系统 (T4SS) 的一个大亚科，称为接合系统，在许多细菌物种之间传递移动遗传元件 (MGE)。在接合转移，DNA转移和复制的启动步骤（DTR）的蛋白质聚集在原点的转移的（ORIT) 序列作为松弛体，它会切割用于转移的 DNA 链，并将带切口的底物与类似 VirD4 的底物受体偶联。在这里，我们定义了 Dtr 蛋白 TraK（DNA 结合蛋白的 Ribbon-Helix-Helix（RHH）家族的预测成员）对通过 pKM101 编码的 T4SS 转移 DNA 和蛋白质底物的贡献。使用交联/亲和力下拉和双杂交测定的组合，我们确定 TraK 自缔合为可能的四聚体，并与 pKM101 编码的 TraI 松弛酶、VirD4 样 TraJ 受体和 VirB11-形成异聚接触分别为类似和 VirB4 样 ATP 酶 TraG 和 TraB。TraK 还促进稳定的 TraJ-TraB 复合物形成并刺激 TraI 与 TraB 的结合。最后，TraK 是同源物（pKM101，TraI 松弛酶）和非同源（RSF1010，MobA 松弛酶）底物。我们提出 TraK 不仅可以使 pKM101 松弛体组装成核，还可以激活 Tra_{pKM101 T4SS}通过与位于通道入口处的 ATPase 能量中心相互作用。