Secretion systems are protein export machines that enable bacteria to exploit their environment through the release of protein effectors. The Type 9 Secretion System (T9SS) is responsible for protein export across the outer membrane (OM) of bacteria of the phylum Bacteroidota. Here we trap the T9SS of Flavobacterium johnsoniae in the process of substrate transport by disrupting the T9SS motor complex. Cryo-EM analysis of purified substrate-bound T9SS translocons reveals an extended translocon structure in which the previously described translocon core is augmented by a periplasmic structure incorporating the proteins SprE, PorD and a homologue of the canonical periplasmic chaperone Skp. Substrate proteins bind to the extracellular loops of a carrier protein within the translocon pore. As transport intermediates accumulate on the translocon when energetic input is removed, we deduce that release of the substrate–carrier protein complex from the translocon is the energy-requiring step in T9SS transport.

分泌系统是蛋白质输出机器，使细菌能够通过释放蛋白质效应物来利用其环境。 9 型分泌系统 (T9SS) 负责跨拟杆菌门细菌的外膜 (OM) 输出蛋白质。在这里，我们通过破坏 T9SS 运动复合体，在底物运输过程中捕获约氏黄杆菌的 T9SS 。对纯化的底物结合 T9SS 易位子的冷冻电镜分析揭示了一种扩展的易位子结构，其中先前描述的易位子核心通过包含蛋白 SprE、PorD 和典型周质伴侣 Skp 同源物的周质结构而增强。底物蛋白与易位子孔内载体蛋白的细胞外环结合。当能量输入被移除时，转运中间体在易位子上积聚，我们推断从易位子释放底物-载体蛋白复合物是 T9SS 转运中的能量需求步骤。