Characterisation of ShigellaSpa33 and ThermotogaFliM/N reveals a new model for C-ring assembly in T3SS

Flagellar type III secretion systems (T3SS) contain an essential cytoplasmic-ring (C-ring) largely composed of two proteins FliM and FliN, whereas an analogous substructure for the closely related non-flagellar (NF) T3SS has not been observed in situ. We show that the spa33 gene encoding the putative NF-T3SS C-ring component in Shigella flexneri is alternatively translated to produce both full-length (Spa33-FL) and a short variant (Spa33-C), with both required for secretion. They associate in a 1:2 complex (Spa33-FL/C-2) that further oligomerises into elongated arrays in vitro. The structure of Spa33-C-2 and identification of an unexpected intramolecular pseudodimer in Spa33-FL reveal a molecular model for their higher order assembly within NF-T3SS. Spa33-FL and Spa33-C are identified as functional counterparts of a FliM-FliN fusion and free FliN respectively. Furthermore, we show that Thermotoga maritimaFliM and FliN form a 1:3 complex structurally equivalent to Spa33-FL/C-2, allowing us to propose a unified model for C-ring assembly by NF-T3SS and flagellar-T3SS.