Novel transient cytoplasmic rings stabilize assembling bacterial flagellar motors

The process by which bacterial cells build their intricate flagellar motility apparatuses has long fascinated scientists. Our understanding of this process comes mainly from studies of purified flagella from two species, Escherichia coli and Salmonella enterica . Here, we used electron cryo‐tomography (cryo‐ET) to image the assembly of the flagellar motor in situ in diverse Proteobacteria: Hylemonella gracilis , Helicobacter pylori , Campylobacter jejuni , Pseudomonas aeruginosa , Pseudomonas fluorescens , and Shewanella oneidensis . Our results reveal the in situ structures of flagellar intermediates, beginning with the earliest flagellar type III secretion system core complex (fT3SScc) and MS‐ring. In high‐torque motors of Beta‐, Gamma‐, and Epsilon‐proteobacteria, we discovered novel cytoplasmic rings that interact with the cytoplasmic torque ring formed by FliG. These rings, associated with the MS‐ring, assemble very early and persist until the stators are recruited into their periplasmic ring; in their absence the stator ring does not assemble. By imaging mutants in Helicobacter pylori , we found that the fT3SScc proteins FliO and FliQ are required for the assembly of these novel cytoplasmic rings. Our results show that rather than a simple accretion of components, flagellar motor assembly is a dynamic process in which accessory components interact transiently to assist in building the complex nanomachine. Synopsis The assembly of bacterial flagellar motors is an “inside‐out” process, starting from the inner membrane. Cryo‐ET reveals novel structures that surround the cytoplasmic C‐ring of high‐torque motors that intriguingly, are only associated with certain assembly stages and are not present in the fully assembled motor. Transient cytoplasmic rings surround the motors of various Beta‐, Gamma‐, and Epsilon‐proteobacteria during assembly. These rings appear to surround the FliG‐ring of the assembling motors. In Helicobacter pylori, these rings are dependent on components of the flagellar type III secretion system. Graphical Abstract Cryo‐ET imaging of the assembly process of the flagellum in a diverse array of Proteobacteria reveals the presence of transient cytoplasmic rings required for the final assembly of high‐torque motors.