Type IV secretion systems (T4SSs) are sophisticated nanomachines used by many bacterial pathogens to translocate protein and DNA substrates across a host cell membrane. Although T4SSs have important roles in promoting bacterial infections, little is known about the biogenesis of the apparatus and the mechanism of substrate transfer. Here, high-throughput cryoelectron tomography (cryo-ET) was used to visualize Legionella pneumophila T4SSs (also known as Dot/Icm secretion machines) in both the whole-cell context and at the cell pole. These data revealed the distribution patterns of individual Dot/Icm machines in the bacterial cell and identified five distinct subassembled intermediates. High-resolution in situ structures of the Dot/Icm machine derived from subtomogram averaging revealed that docking of the cytoplasmic DotB (VirB11-related) ATPase complex onto the DotO (VirB4-related) ATPase complex promotes a conformational change in the secretion system that results in the opening of a channel in the bacterial inner membrane. A model is presented for how the Dot/Icm apparatus is assembled and for how this machine may initiate the transport of cytoplasmic substrates across the inner membrane.IMPORTANCE Many bacteria use type IV secretion systems (T4SSs) to translocate proteins and nucleic acids into target cells, which promotes DNA transfer and host infection. The Dot/Icm T4SS in Legionella pneumophila is a multiprotein nanomachine that is known to translocate over 300 different protein effectors into eukaryotic host cells. Here, advanced cryoelectron tomography and subtomogram analysis were used to visualize the Dot/Icm machine assembly and distribution in a single L. pneumophila cell. Extensive classification and averaging revealed five distinct intermediates of the Dot/Icm machine at high resolution. Comparative analysis of the Dot/Icm machine and subassemblies derived from wild-type cells and several mutants provided a structural basis for understanding mechanisms that underlie the assembly and activation of the Dot/Icm machine.

中文翻译：

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通过低温电子层析成像分析Dot / Icm型IVB分泌系统子组件，揭示了由DotB结合引起的构象变化。

IV型分泌系统（T4SS）是许多细菌病原体使用的复杂的纳米机器，可在宿主细胞膜上转运蛋白质和DNA底物。尽管T4SS在促进细菌感染中具有重要作用，但关于该装置的生物发生和底物转移的机制知之甚少。在这里，高通量冷冻电子断层扫描（cryo-ET）用于在全细胞环境和细胞极处可视化嗜肺军团菌T4SS（也称为Dot / Icm分泌机）。这些数据揭示了细菌细胞中单个Dot / Icm机器的分布方式，并鉴定出五个不同的组装中间体。从子图平均得出的高分辨率Dot / Icm机器原位结构显示，胞质DotB（与VirB11相关）ATPase复合物与DotO（与VirB4相关）ATPase复合物的对接促进了分泌系统的构象变化，从而导致在细菌内膜通道的开口处。提出了一个模型，说明如何组装Dot / Icm装置以及该机器如何启动细胞质底物穿过内膜的运输。要点许多细菌使用IV型分泌系统（T4SS）将蛋白质和核酸转运到靶细胞中，促进DNA转移和宿主感染。嗜肺军团菌中的Dot / Icm T4SS是一种多蛋白质纳米机器，已知可以将300多种不同的蛋白质效应子转运到真核宿主细胞中。这里，先进的低温电子断层扫描和子图分析被用于可视化Dot / Icm机器的组装和在单个嗜肺乳杆菌细胞中的分布。广泛的分类和平均显示了高分辨率的Dot / Icm机器的五个不同中间体。对Dot / Icm机器及其衍生自野生型细胞和几个突变体的子组件的比较分析，为理解构成Dot / Icm机器的组装和激活机制的机制提供了结构基础。