Dissecting the Campylobacter jejuni flagellar motor using subtomogram averaging and rationally designed protein chimeras

Abstract

Bacterial cells must traverse a variety of environments to survive and thrive. To achieve motility, they have evolved the bacterial flagella motor. This complex proteinacious nano-machine is composed of up to 25 different proteins, which assemble together in a range of stoichiometries across multiple membranes. They are able to harness the flux of ions across inner membrane bound stators, to kick a cytoplasmic C-ring, thus generating torque. Rotational torque is then transmitted through the periplasmic axle, which can be augmented by additional protein scaffolds, to the external hook and filament, propelling the cell forwards. Through developments of in situ Electron Cryo-Tomography (ECT), increased structural complexity in the motors of species such as Campylobacter jejuni have been explored to nanometer resolutions. This has revealed additional, intricate complexes which enable increased base and novel functions. Using the C. Jejuni motor as a model, I have applied and developed a number of methods to study its complexity, in hopes of understanding the structures of functional proteinacious nanomachines in their native context. By dissecting the in situ structure of the cytoplasmic C. jejuni C-ring, I have revealed the domain placements of FliG C terminal, FliM, FliN and FliY. Combining this with phylogenetic and operon analysis, I have also explored the evolved structural role of FliNY intercalation at the distal tip of the C-ring, revealing the greater role FliY plays in the structural anchoring of the ATPase complex to the C-ring in C. jejuni. Furthermore, I’ve characterised a previously unknown component of the periplasmic scaffold dubbed Mot1, which acts as a stabilising outer brace by anchoring the proximal and sub-basal disks. To overcome the resolution limitations of ECT, which prevent unambiguous identification of protein components in nano-machines, I’ve developed software which identifies protein chains to insert into macromolecules without disrupting tertiary or quaternary structure to act as electron dense tags.