Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold

Abstract

Although it is known that diverse bacterial flagellar motors produce different torques, the mechanism underlying torque variation is unknown. To understand this difference better, we combined genetic analyses with electron cryo-tomography subtomogram averaging to determine in situ structures of flagellar motors that produce different torques, from Campylobacter and Vibrio species. For the first time, to our knowledge, our results unambiguously locate the torque-generating stator complexes and show that diverse high-torque motors use variants of an ancestrally related family of structures to scaffold incorporation of additional stator complexes at wider radii from the axial driveshaft than in the model enteric motor. We identify the protein components of these additional scaffold structures and elucidate their sequential assembly, demonstrating that they are required for stator-complex incorporation. These proteins are widespread, suggesting that different bacteria have tailored torques to specific environments by scaffolding alternative stator placement and number. Our results quantitatively account for different motor torques, complete the assignment of the locations of the major flagellar components, and provide crucial constraints for understanding mechanisms of torque generation and the evolution of multiprotein complexes.

Keywords: Campylobacter; bacterial flagellar motors; electron cryo-tomography; macromolecular evolution; torque.

Fig. 1.

High-torque bacterial flagellar motors assemble large periplasmic disk complexes. (A–C) Tomographic slices through intact cells of Salmonella (A), V. fischeri (B), and C. jejuni (C) showing individual flagellar motors. Height of all image panels is 100 nm. (D–F) Slices (100 × 100 × 0.81 nm) through subtomogram averages of hundreds of motors. Color keys indicate the regions of the motor (named in G–I), of Salmonella (average of 286 motors) (D), V. fischeri (average of 302 motors) (E), and C. jejuni (average of 156 motors) (F). (G–I) Isosurface renderings of motors shown in D–F. FliI and FlhA_C are components of the flagellar type III secretion system.

Fig. 2.

Disk complexes scaffold alternative stator-complex locations and numbers. (A) WT V. fischeri motor highlighting the putative MotB (red arrow) and the putative FliG_C:MotA interface (blue arrow). (B) Close-up of putative MotB density and FliG_C:MotA interface. (C and D) Thirteen-fold symmetry through the stator and C-ring planes shown by white dotted lines in A. (E–H) Images of V. fischeri ΔmotB equivalent to the images in A–D confirming the identity of proposed stator complexes. (I–P) Equivalent images for WT C. jejuni (I–L) and C. jejuni ΔmotB (M–P). Note 17 stator-complex densities observed in C. jejuni. Yellow arrows in I and J indicate an additional density on the inner lobe of the C. jejuni C-ring as compared with Salmonella and V. fischeri. Note that the odd-numbered stator counts result in an asymmetric appearance of motor cross-sections.

Fig. S1.

Gold-standard FSC curves of subtomogram averages. Datasets were fully separated into two half datasets, and two independent reconstructions were calculated using different template subtomograms from each dataset (59). FSC curves were calculated between the two aligned averages. Subsequently these two averages were averaged together and symmetrized to form the final averaged structure. Resolutions in Angstroms at a 0.143 threshold are Salmonella WT: 69.4; V. fischeri WT: 63.0; C. jejuni WT: 44.7; V. fischeri ΔmotB: 70.3; C. jejuni ΔmotB: 45.8; C. jejuni ΔflgQ: 60.1; C. jejuni ΔflgP: 61.2; C. jejuni ΔpflA: 85.3; C. jejuni ΔpflB: 61.9; and V. fischeri ΔflgP: 81.0.

Fig. S2.

Unsymmetrized motors showing clear symmetries. C. jejuni motors (A, Left) exhibited clear 17-fold symmetry (A, Right), which was applied to improve the signal-to-noise ratio (B). V. fischeri exhibited 13-fold symmetry (C), which was applied to improve the signal-to-noise ratio (D).

Fig. 3.

Assembly of the stator complexes in V. fischeri requires FlgP. (A) V. fischeri flagellar motors from ΔflgP (Left), ΔmotB (Center), and WT (Right) strains. (B) Model of assembly of the V. fischeri disk complex. Early-stage disk-complex components MotXY and FlgOT assemble before FlgP incorporation, followed by the assembly of the stator-complex ring. Images show 100 × 100 × 0.81 nm slices through subtomogram averages.

Fig. 4.

Protein location within the C. jejuni disk complex and hierarchical assembly pathway revealed by subtomogram averaging of WT and mutant motors. (A) Subtomogram averages arranged to illustrate the hierarchical assembly dependencies of the C. jejuni disk complex. Note that the outer membrane is poorly resolved in ΔflgQ and ΔflgP mutants because without the basal disk as an anchor the membrane is in a different position in each component subtomogram. Images are 100 × 100 × 0.81 nm slices through subtomogram averages. (B) Model of the hierarchical assembly of the C. jejuni disk complex.

Fig. 5.

Localization interdependencies of five components of the C. jejuni disk complex. Immunoblot analysis of localization of FlgP, PflA, PflB, MotA, and MotB in various WT C. jejuni and mutant strains. (A) FlgP subcellular localization in flagellar T3SS, rod, and ring mutants. (B) Subcellular localization of FlgP, PflA, and PflB in specific flagellar disk mutants. (C) Localization of MotA and MotB stator proteins in flagellar disk-complex mutants. All proteins were detected with specific antiserum. Cyt, cytoplasm; IM, inner membrane; OM, outer membrane; Peri, periplasm; WCL, whole-cell lysate. For all immunoblots, equivalent amounts of cell cultures were lysed and subcellular compartments fractionated.

Fig. 6.

Wider stators featuring more stator complexes quantitatively account for torque diversity. (A) Disk complexes scaffold stators to increase the number of stators and the radius of the stator-complex ring. The stator number and increasing wider C-ring in each flagellar motor correlate directly with the torque produced by each motor. (B) Comparison of predicted and observed torques of flagellar motors from various species. Filled circles represent enteric bacteria; open circles represent Vibrio spp.; open squares represent ε-proteobacteria; and filled squares represent spirochetes. Where there was no direct torque measurement, C. jejuni and B. burgdorferi torques are inferred from closely related species with similar motors (H. pylori and Leptospira, respectively). Relative torque strengths are validated by swimming speeds and the ability of different bacteria to swim through viscous fluids (1, 4, 7). The dotted line represents a perfect prediction.