Diversification of Campylobacter jejuni Flagellar C-Ring Composition Impacts Its Structure and Function in Motility, Flagellar Assembly, and Cellular Processes.

Bacterial flagella are reversible rotary motors that rotate external filaments for bacterial propulsion. Some flagellar motors have diversified by recruiting additional components that influence torque and rotation, but little is known about the possible diversification and evolution of core motor components. The mechanistic core of flagella is the cytoplasmic C ring, which functions as a rotor, directional switch, and assembly platform for the flagellar type III secretion system (fT3SS) ATPase. The C ring is composed of a ring of FliG proteins and a helical ring of surface presentation of antigen (SPOA) domains from the switch proteins FliM and one of two usually mutually exclusive paralogs, FliN or FliY. We investigated the composition, architecture, and function of the C ring of Campylobacter jejuni, which encodes FliG, FliM, and both FliY and FliN by a variety of interrogative approaches. We discovered a diversified C. jejuni C ring containing FliG, FliM, and both FliY, which functions as a classical FliN-like protein for flagellar assembly, and FliN, which has neofunctionalized into a structural role. Specific protein interactions drive the formation of a more complex heterooligomeric C. jejuni C-ring structure. We discovered that this complex C ring has additional cellular functions in polarly localizing FlhG for numerical regulation of flagellar biogenesis and spatial regulation of division. Furthermore, mutation of the C. jejuni C ring revealed a T3SS that was less dependent on its ATPase complex for assembly than were other systems. Our results highlight considerable evolved flagellar diversity that impacts motor output, biogenesis, and cellular processes in different species.IMPORTANCE The conserved core of bacterial flagellar motors reflects a shared evolutionary history that preserves the mechanisms essential for flagellar assembly, rotation, and directional switching. In this work, we describe an expanded and diversified set of core components in the Campylobacter jejuni flagellar C ring, the mechanistic core of the motor. Our work provides insight into how usually conserved core components may have diversified by gene duplication, enabling a division of labor of the ancestral protein between the two new proteins, acquisition of new roles in flagellar assembly and motility, and expansion of the function of the flagellum beyond motility, including spatial regulation of cell division and numerical control of flagellar biogenesis in C. jejuni Our results highlight that relatively small changes, such as gene duplications, can have substantial ramifications on the cellular roles of a molecular machine.

High-Throughput Electron Cryo-tomography of Protein Complexes and Their Assembly.

Electron cryo-tomography and subtomogram averaging enable visualization of protein complexes in situ, in three dimensions, in a near-native frozen-hydrated state to nanometer resolutions. To achieve this, intact cells are vitrified and imaged over a range of tilts within an electron microscope. These images can subsequently be reconstructed into a three-dimensional volume representation of the sample cell. Because complexes are visualized in situ, crucial insights into their mechanism, assembly process, and dynamic interactions with other proteins become possible. To illustrate the electron cryo-tomography workflow for visualizing protein complexes in situ, we describe our workflow of preparing samples, imaging, and image processing using Leginon for data collection, IMOD for image reconstruction, and PEET for subtomogram averaging.