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Molecular mechanism for rotational switching of the bacterial flagellar motor

Nature Structural & Molecular Biology volume 27, pages 1041–1047 (2020)Cite this article

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Abstract

The bacterial flagellar motor can rotate in counterclockwise (CCW) or clockwise (CW) senses, and transitions are controlled by the phosphorylated form of the response regulator CheY (CheY-P). To dissect the mechanism underlying flagellar rotational switching, we use Borrelia burgdorferi as a model system to determine high-resolution in situ motor structures in cheX and cheY3 mutants, in which motors are locked in either CCW or CW rotation. The structures showed that CheY3-P interacts directly with a switch protein, FliM, inducing a major remodeling of another switch protein, FliG2, and altering its interaction with the torque generator. Our findings lead to a model in which the torque generator rotates in response to an inward flow of H+ driven by the proton motive force, and conformational changes in FliG2 driven by CheY3-P allow the switch complex to interact with opposite sides of the rotating torque generator, facilitating rotational switching.

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Fig. 1: Structure of the flagellar motor in constantly flexing ∆cheX cells.
Fig. 2: CheY3-P binding to the flagellar motor.
Fig. 3: Stator–rotor interactions in constantly running ∆cheY3 cells.
Fig. 4: Molecular architectures of the flagellar motors without and with CheY3-P.
Fig. 5: Model for the mechanism of rotational switching.

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Data availability

Cryo-EM density maps that support the local refined ∆cheX (EMD-21885) and ∆cheY3 (class-1: EMD-21884, class-2: EMD-21886) motor structures determined by cryo-electron tomography have been deposited in the Electron Microscopy Data Bank (EMDB). Other maps and models are available upon request.

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Acknowledgements

We thank M. Manson, J. Radolf, S. Kojima and M. Homma for critical reading and suggestions. This work was supported by grants from the National Institute of Allergy and Infectious Diseases (R01AI087946, R01AI078958, R01AI132818 and R01AI59048) and the National Institute of Dental and Craniofacial Research (R01DE023080). This work is dedicated to the memory of Professor Fanghua Li, who was an incredible scientist and a trusted mentor for Y.C., J.L. and many others in the field of electron microscopy.

Author information

Author notes

  1. Xiaowei Zhao

    Present address: Howard Hughes Medical Institute, Janelia Research Campus, Ashburn, VA, USA

  2. These authors contributed equally: Yunjie Chang, Kai Zhang.

Authors and Affiliations

  1. Department of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT, USA

    Yunjie Chang, Brittany L. Carroll & Jun Liu

  2. Microbial Sciences Institute, Yale University, West Haven, CT, USA

    Yunjie Chang, Brittany L. Carroll & Jun Liu

  3. Philips Institute for Oral Health Research, Virginia Commonwealth University, Richmond, VA, USA

    Kai Zhang & Chunhao Li

  4. Department of Pathology and Laboratory Medicine, University of Texas Health Science Center, Houston, TX, USA

    Xiaowei Zhao & Steven J. Norris

  5. Department of Microbiology, Immunology, and Cell Biology, Robert C. Byrd Health Sciences Center, West Virginia University, Morgantown, WV, USA

    Nyles W. Charon

  6. Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, NC, USA

    Md A. Motaleb

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Contributions

J.L. and C.L. conceived the project. Y.C. performed cryo-ET experiments, data analysis, modeling and wrote the manuscript draft. K.Z. performed genetic and biochemical experiments and analysis. B.L.C. and X.Z. contributed structural analysis. J.L. and C.L. supervised all work. M.A.M., S.J.N. and N.W.C. provided B. burgdorferi strains. Y.C., C.L. and J.L. prepared the manuscript with input from all authors.

Corresponding authors

Correspondence to Chunhao Li or Jun Liu.

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Extended data

Extended Data Fig. 1 Swimming motility modes and flagellar switching in E. coli and B. burgdorferi.

a, b, Cartoon of the swimming motility modes in E. coli: run and tumble. c, The motor rotates CCW as a default state. d, When the level of CheY-P becomes high enough, CheY-P binds to the C-ring, and the motor switches to CW rotation. The chemotaxis protein CheZ dephosphorylates CheY-P to return the motor to CCW rotation. e, f, Swimming motility modes in B. burgdorferi: run and flex. Periplasmic flagella (PF) are located between the inner membrane (IM) and outer membrane (OM). The flagellar motors are attached near each cell pole. Spirochetes run when the anterior flagella rotate CCW and the posterior flagella rotate CW (e). When the flagella at both poles rotate in the same direction (CW), the spirochetes flex in place and fail to move translationally. The swimming motility of B. burgdorferi is also controlled by a chemotaxis system. The homologs of CheY and CheZ in B. burgdorferi are CheY3 and CheX.

Extended Data Fig. 2 Cryo-ET imaging of the flagellar motors in ∆cheX and ∆cheY3 mutants.

a, A representative tomographic section from a ∆cheX cell tip reconstruction. Outer membrane (OM), inner membrane (IM), peptidoglycan layer (PG), and motors are clearly resolved in the tomogram. b, A representative section of a tomogram from a ∆cheY3 cell tip. Multiple motors with different orientations can be found at the cell tip. The insertions in (a, b) are the dark-field images showing a ∆cheX cell constantly flexing and a constantly running ∆cheY3 cell, respectively. c, A medial cross-section of an averaged map of the ∆cheX motor. d, The unrolled map refined using the stator region densities shows 16 stator complexes are embedded in the inner membrane (IM), while the C-ring subunits are unresolved due to symmetry mismatch between the C-ring and the stator. e, The unrolled map refined using the C-ring region densities shows 46-fold symmetric features, while the stator becomes blurry. Scale bar, 20 nm.

Extended Data Fig. 3 Comparison between in situ stator complex and the purified stator components.

a, Structure of purified MotA complex from A. aeolicus resolved by single particle EM (EMD 3417)27. b, The in situ stator complex in the ∆cheX motor has a bell-shaped structure embedded in the inner membrane (IM) and a periplasmic domain. The top part of the periplasmic domain matches well with the crystal structure of the S. enterica MotB periplasmic domain (PDB 2ZVY)28 (middle panel). The bell-shaped structure has similar size and shape as the structure of EMD 3417 (right panel). c, The in situ stator complex in the ∆cheY3-Class-2 motor is similar to that in the ∆cheX motor.

Extended Data Fig. 4 Schematic diagram for the in-frame replacement of cheX-cheY3 genes with cheY3-gfp and FLAG affinity purification using FLAG-FliM/FliN to pull down HisCheY3/CheY3*.

a, aadA, a streptomycin resistance gene was used as a selection marker. pcheX(F) and GFP (R) are oligonucleotide primers utilized to verify the occurrence of the allelic exchange of the recombinant construct (bottom) into the targeted region in the B. burgdorferi chromosome (top). b, Ni-NTA affinity purification using FLAG-tagged FliM (FliM-FLAG) and FLAG-tagged FliN (FliN-FLAG) to pull down HisCheY3 or HisCheY3* (CheY3D79A), respectively. HisCheY3 was co-purified with FliM-FLAG (b), but not with FliN-FLAG c, suggesting that CheY3 does not bind on FliN. In contrast, more HisCheY3 protein was co-purified with FliM-FLAG with acetyl phosphate (b), and HisCheY3* was not co-purified with FliM-FLAG (b) or FliN-FLAG (c). These results indicate that CheY3 binds to FliM protein in a phosphorylation-dependent manner.

Extended Data Fig. 5 Motor structures in constantly running ∆cheY3 cells.

a, A medial cross-section of an averaged structure in ∆cheY3 B. burgdorferi cell. b, c, Cross-sections show the stator ring and the C-ring, respectively. d, Focused structure of the C-ring (unrolled along the central rod). Two distinct classes in the ∆cheY3 cells are named as ∆cheY3-Class-1 e-h, and ∆cheY3-Class-2 i-l. Class-1 and Class-2 account ~45% and ~55% of all the ∆cheY3 motors we used for current work, respectively. The stator structures in Class-1 and Class-2 f and j, are quite similar, while the C-ring subunits (compare h with l) are tilted in different directions.

Extended Data Fig. 6 Motors adopt distinct conformations at the two cell poles in the same ∆cheY3 cell.

a, A tomographic section from one cell tip showed in panel b. Scale bar, 200 nm. (b) An overview of one intact ∆cheY3 cell. Scale bar, 1 µm. c, A tomographic section from another tip of the same cell in panel b. Scale bar, 200 nm. The motors at each cell tip were aligned separately, then focused refined to the C-ring. d-f, The motors from one cell tip have CCW conformation. g-t, The motors from another tip appear to adopt CW conformation. j-l, Averaged structure from motors located at one tip of five cells shows a better structure with CCW conformation. m-o, Averaged structure from motors located at another tip of five cells shows a better structure with CW conformation.

Extended Data Fig. 7 CheY3-P binding triggers conformational change.

a-d, Comparison between the C-ring models before (grey, top left in each panel) and after (colored, top right in each panel) CheY3-P binding. e, The dash framed regions in panel a are overlapped to show their differences. The N-terminal domain of FliM (FliMN) folds out ~154˚ to interact with CheY3-P. f, Binding of CheY3-P induces ~27˚ tilt of the FliM middle domain (FliMM). g, h, FliG2 undergoes a large tilt and alters the interactions between FliG2 and MotA. The charged residues (Lys275, Arg292, Glu299, and Asp300) in the C-terminal domain of FliG2 (FliG2C) are colored in red.

Extended Data Fig. 8 Comparison of the C-ring structures in CCW and CW rotation.

a, b, Diameters of the FliG2C, FliM and FliN rings in the C-ring with CCW rotation (∆cheY3-class-2). c, d, Diameters of the FliG2, FliM and FliN rings in the C-ring with CW rotation (∆cheX).

Extended Data Fig. 9 Motility model for B. burgdorferi.

a, b, In the default state, the concentration of CheY3-P is low, and the cell runs. The motors at the anterior cell pole rotate CCW, and the motors at the posterior cell pole rotate CW. Binding of unidentified proteins (grey circles at the inner side of the C-ring) to the C-ring at the posterior cell pole likely changes the motor to a CW conformation. c, d, At high concentrations of CheY3-P, the CCW rotating motors switch to CW rotation, while the CW rotating motors keep turning CW. Thus, the motors at both cell poles rotate CW and the cell flexes. After the flex, the direction of flagellar rotation at the two poles can switch so that the cell reverses the direction of its run.

Supplementary information

Supplementary Information

Supplementary Table 1 and Fig. 1.

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Supplementary Video 1

∆cheX cells flex in the video.

Supplementary Video 2

A refined structure of the flagellar motor in the ∆cheX mutant.

Supplementary Video 3

∆cheY3 cells constantly run in the video.

Supplementary Video 4

One class average of the flagellar motor in the ∆cheY3 mutant.

Supplementary Video 5

Another class average of the flagellar motor in the ∆cheY3 mutant.

Supplementary Video 6

An animation showing flagellar rotational switching in the Lyme disease spirochete.

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Chang, Y., Zhang, K., Carroll, B.L. et al. Molecular mechanism for rotational switching of the bacterial flagellar motor. Nat Struct Mol Biol 27, 1041–1047 (2020). https://doi.org/10.1038/s41594-020-0497-2

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