Energetic components VirD4, VirB11 and VirB4 mediate early DNA transfer reactions required for bacterial type IV secretion
Energetic components VirD4, VirB11 and VirB4 mediate early DNA transfer reactions required for bacterial type IV secretion
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DOI：
10.1111/j.1365-2958.2004.04345.x
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发表时间：
2004-12-01
影响因子：
3.6
通讯作者：
Christie, PJ
中科院分区：
文献类型：
--
作者：
Atmakuri, K;Cascales, E;Christie, PJ
细菌使用 IV 型分泌系统 (T4SS) 在细胞膜上转移 DNA (T-DNA) 和蛋白质底物。通过转移 DNA 免疫沉淀 (TrIP)，我们最近表明，T-DNA 通过依次与 VirD4 受体、VirB11 ATPase、内膜亚基 VirB6 和 VirB8，最后与 VirB2 菌毛蛋白和 VirB9 形成紧密接触，在根癌农杆菌 VirB/D4 T4SS 中易位。在这里，通过 TrIP，我们表明核苷三磷酸结合位点（Walker A 基序）突变不会破坏 VirD4 底物结合或转移到 VirB11，表明这些早期反应独立于 ATP 结合或水解进行。相比之下，VirD4、VirB11 和 VirB4 Walker A 突变均阻止底物向 VirB6 和 VirB8 的转移，表明这些亚基通过 ATP 依赖性机制为这种转移反应提供能量。通过免疫共沉淀，我们提供了 VirD4 与 VirB4 和 VirB11 独立于其他 T4SS 亚基或完整 Walker A 基序以及与双位内膜亚基 VirB10 相互作用的证据。我们通过共合成先前确定的 VirB/D4 T4SS 的“核心”成分，重建了从 VirD4 到 VirB11 以及 VirB6 和 VirB8 的底物转移。我们的研究结果定义了 DNA 底物结合和细菌 IV 型易位途径的早期转移反应的遗传要求。
Bacteria use type IV secretion systems (T4SS) to translocate DNA (T-DNA) and protein substrates across the cell envelope. By transfer DNA immunoprecipitation (TrIP), we recently showed that T-DNA translocates through the Agrobacterium tumefaciens VirB/D4 T4SS by forming close contacts sequentially with the VirD4 receptor, VirB11 ATPase, the inner membrane subunits VirB6 and VirB8 and, finally, VirB2 pilin and VirB9. Here, by TrIP, we show that nucleoside triphosphate binding site (Walker A motif) mutations do not disrupt VirD4 substrate binding or transfer to VirB11, suggesting that these early reactions proceed independently of ATP binding or hydrolysis. In contrast, VirD4, VirB11 and VirB4 Walker A mutations each arrest substrate transfer to VirB6 and VirB8, suggesting that these subunits energize this transfer reaction by an ATP-dependent mechanism. By co-immunoprecipitation, we supply evidence for VirD4 interactions with VirB4 and VirB11 independently of other T4SS subunits or intact Walker A motifs, and with the bitopic inner membrane subunit VirB10. We reconstituted substrate transfer from VirD4 to VirB11 and to VirB6 and VirB8 by co-synthesis of previously identified 'core' components of the VirB/D4 T4SS. Our findings define genetic requirements for DNA substrate binding and the early transfer reactions of a bacterial type IV translocation pathway.