Many bacteria move using a complex, self-assembling nanomachine, the bacterial flagellum. Biosynthesis of the flagellum depends on a flagellar-specific type III secretion system (T3SS), a protein export machine homologous to the export machinery of the virulence-associated injectisome. Six cytoplasmic (FliH/I/J/G/M/N) and seven integral-membrane proteins (FlhA/B FliF/O/P/Q/R) form the flagellar basal body and are involved in the transport of flagellar building blocks across the inner membrane in a proton motive force-dependent manner. However, how the large, multi-component transmembrane export gate complex assembles in a coordinated manner remains enigmatic. Specific for most flagellar T3SSs is the presence of FliO, a small bitopic membrane protein with a large cytoplasmic domain. The function of FliO is unknown, but homologs of FliO are found in >80% of all flagellated bacteria. Here, we demonstrate that FliO protects FliP from proteolytic degradation and promotes the formation of a stable FliP–FliR complex required for the assembly of a functional core export apparatus. We further reveal the subcellular localization of FliO by super-resolution microscopy and show that FliO is not part of the assembled flagellar basal body. In summary, our results suggest that FliO functions as a novel, flagellar T3SS-specific chaperone, which facilitates quality control and productive assembly of the core T3SS export machinery.

许多细菌使用复杂的自组装纳米机器细菌鞭毛移动。鞭毛的生物合成取决于鞭毛特异的III型分泌系统（T3SS），该蛋白分泌机与毒力相关注射体的分泌机同源。六个细胞质（FliH / I / J / G / M / N）和七个完整膜蛋白（FlhA / B FliF / O / P / Q / R）形成鞭毛基体，并参与鞭毛构件的运输质子动力依赖的方式穿过内膜。但是，大型的多组分跨膜出口门复合体如何以协调的方式组装仍然是个谜。对于大多数鞭毛T3SS而言，特异的是FliO的存在，FliO是具有较大胞质结构域的小双位膜蛋白。FliO的功能未知，但是在所有鞭毛细菌中，> 80％都发现了FliO的同系物。在这里，我们证明FliO保护FliP免受蛋白水解降解，并促进功能性核心出口装置组装所需的稳定FliP–FliR复合物的形成。我们通过超分辨率显微镜进一步揭示了FliO的亚细胞定位，并显示FliO不是组装的鞭毛基体的一部分。总而言之，我们的研究结果表明FliO可以充当新型的，T3SS特有的鞭毛伴侣，从而促进T3SS核心出口机械的质量控制和生产组装。我们通过超分辨率显微镜进一步揭示了FliO的亚细胞定位，并显示FliO不是组装的鞭毛基体的一部分。总而言之，我们的研究结果表明FliO可以充当新型的，T3SS特有的鞭毛伴侣，从而促进T3SS核心出口机械的质量控制和生产组装。我们通过超分辨率显微镜进一步揭示了FliO的亚细胞定位，并显示FliO不是组装的鞭毛基体的一部分。总而言之，我们的研究结果表明FliO可以充当新型的，T3SS特有的鞭毛伴侣，从而促进T3SS核心出口机械的质量控制和生产组装。