The architecture, assembly, and evolution of a complex flagellar motor

Bacterial flagella drive motility in many species, likely including the last bacterial common ancestor^1,2. Knowledge of flagellar assembly and function has mainly come from studies of Escherichia coli and Salmonella enterica, which have simple flagellar motors^3-7. However, most flagellated bacteria possess complex motors with unique, species-specific adaptations whose mechanisms and evolution remain largely unexplored^8-10. Here, we deploy a multidisciplinary approach to build a near-complete model of the flagellar motor in Campylobacter jejuni, revealing its remarkable complexity in architecture and composition. We identify an E-ring around the MS-ring, a periplasmic cage with two distinctive conformations, and an intricate interaction network between the E-ring and cage. These scaffolds play critical roles in stabilizing and regulating 17 torque-generating stator complexes for optimal motility. In-depth evolutionary analyses uncover the ancient origin and prevalence of the E-ring in flagellated species of the domain Bacteria as well as a unique exaptation of type IV pili components PilMNOPQF in the ancestral motor of the phylum Campylobacterota. Collectively, our studies reveal novel mechanisms of assembly and function in complex flagellar motors and shed light on the evolution of flagella and modern bacterial species.