Bacterial flagella grow through an injection-diffusion mechanism

  1. Thibaud T Renault
  2. Anthony O Abraham
  3. Tobias Bergmiller
  4. Guillaume Paradis
  5. Simon Rainville
  6. Emmanuelle Charpentier
  7. Călin C Guet
  8. Yuhai Tu
  9. Keiichi Namba
  10. James P Keener  Is a corresponding author
  11. Tohru Minamino
  12. Marc Erhardt  Is a corresponding author
  1. Helmholtz Centre for Infection Research, Germany
  2. Osaka University, Japan
  3. Institute of Science and Technology Austria, Austria
  4. Laval University, Canada
  5. Max Planck Institute for Infection Biology, Germany
  6. IBM Thomas J. Watson Research Center, United States
  7. University of Utah, United States

Abstract

The bacterial flagellum is a self-assembling nanomachine. The external flagellar filament, several times longer than a bacterial cell body, is made of a few tens of thousands subunits of a single protein: flagellin. A fundamental problem concerns the molecular mechanism of how the flagellum grows outside the cell, where no discernible energy source is available. Here, we monitored the dynamic assembly of individual flagella using in situ labelling and real-time immunostaining of elongating flagellar filaments. We report that the rate of flagellum growth, initially ∼1,700 amino acids per second, decreases with length and that the previously proposed chain mechanism does not contribute to the filament elongation dynamics. Inhibition of the proton motive force-dependent export apparatus revealed a major contribution of substrate injection in driving filament elongation. The combination of experimental and mathematical evidence demonstrates that a simple, injection-diffusion mechanism controls bacterial flagella growth outside the cell.

Article and author information

Author details

  1. Thibaud T Renault

    Junior Research Group Infection Biology of Salmonella, Helmholtz Centre for Infection Research, Braunschweig, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Anthony O Abraham

    Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8710-1351
  3. Tobias Bergmiller

    Institute of Science and Technology Austria, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  4. Guillaume Paradis

    Department of Physics, Engineering Physics and Optics, Laval University, Quebec City, Canada
    Competing interests
    The authors declare that no competing interests exist.
  5. Simon Rainville

    Department of Physics, Engineering Physics and Optics, Laval University, Quebec City, Canada
    Competing interests
    The authors declare that no competing interests exist.
  6. Emmanuelle Charpentier

    Max Planck Institute for Infection Biology, Berlin, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Călin C Guet

    Institute of Science and Technology Austria, Klosterneuburg, Austria
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6220-2052
  8. Yuhai Tu

    IBM Thomas J. Watson Research Center, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
  9. Keiichi Namba

    Graduate School of Frontier Biosciences, Osaka University, Suita, Japan
    Competing interests
    The authors declare that no competing interests exist.
  10. James P Keener

    Department of Mathematics, University of Utah, Salt Lake City, United States
    For correspondence
    keener@math.utah.edu
    Competing interests
    The authors declare that no competing interests exist.
  11. Tohru Minamino

    Graduate School of Frontier Biosciences, Osaka University, Suita, Japan
    Competing interests
    The authors declare that no competing interests exist.
  12. Marc Erhardt

    Junior Research Group Infection Biology of Salmonella, Helmholtz Centre for Infection Research, Braunschweig, Germany
    For correspondence
    marc.erhardt@helmholtz-hzi.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6292-619X

Funding

Helmholtz-Gemeinschaft (VH-NG-932)

  • Marc Erhardt

Japan Society for the Promotion of Science (15H01640)

  • Tohru Minamino

Max-Planck-Gesellschaft

  • Emmanuelle Charpentier

National Institutes of Health (R01GM081747)

  • Yuhai Tu

European Commission (334030)

  • Marc Erhardt

Japan Society for the Promotion of Science (25000013)

  • Keiichi Namba

Natural Sciences and Engineering Research Council of Canada

  • Simon Rainville

Alexander von Humboldt-Stiftung

  • Thibaud T Renault

Japan Society for the Promotion of Science (26293097)

  • Tohru Minamino

Japan Society for the Promotion of Science (24117004)

  • Tohru Minamino

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2017, Renault et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 6,271
    views
  • 1,142
    downloads
  • 74
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Thibaud T Renault
  2. Anthony O Abraham
  3. Tobias Bergmiller
  4. Guillaume Paradis
  5. Simon Rainville
  6. Emmanuelle Charpentier
  7. Călin C Guet
  8. Yuhai Tu
  9. Keiichi Namba
  10. James P Keener
  11. Tohru Minamino
  12. Marc Erhardt
(2017)
Bacterial flagella grow through an injection-diffusion mechanism
eLife 6:e23136.
https://doi.org/10.7554/eLife.23136

Share this article

https://doi.org/10.7554/eLife.23136

Further reading

    1. Chromosomes and Gene Expression
    2. Structural Biology and Molecular Biophysics
    Liza Dahal, Thomas GW Graham ... Xavier Darzacq
    Research Article

    Type II nuclear receptors (T2NRs) require heterodimerization with a common partner, the retinoid X receptor (RXR), to bind cognate DNA recognition sites in chromatin. Based on previous biochemical and overexpression studies, binding of T2NRs to chromatin is proposed to be regulated by competition for a limiting pool of the core RXR subunit. However, this mechanism has not yet been tested for endogenous proteins in live cells. Using single-molecule tracking (SMT) and proximity-assisted photoactivation (PAPA), we monitored interactions between endogenously tagged RXR and retinoic acid receptor (RAR) in live cells. Unexpectedly, we find that higher expression of RAR, but not RXR, increases heterodimerization and chromatin binding in U2OS cells. This surprising finding indicates the limiting factor is not RXR but likely its cadre of obligate dimer binding partners. SMT and PAPA thus provide a direct way to probe which components are functionally limiting within a complex TF interaction network providing new insights into mechanisms of gene regulation in vivo with implications for drug development targeting nuclear receptors.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Angel D'Oliviera, Xuhang Dai ... Jeffrey S Mugridge
    Research Article

    The SARS-CoV-2 main protease (Mpro or Nsp5) is critical for production of viral proteins during infection and, like many viral proteases, also targets host proteins to subvert their cellular functions. Here, we show that the human tRNA methyltransferase TRMT1 is recognized and cleaved by SARS-CoV-2 Mpro. TRMT1 installs the N2,N2-dimethylguanosine (m2,2G) modification on mammalian tRNAs, which promotes cellular protein synthesis and redox homeostasis. We find that Mpro can cleave endogenous TRMT1 in human cell lysate, resulting in removal of the TRMT1 zinc finger domain. Evolutionary analysis shows the TRMT1 cleavage site is highly conserved in mammals, except in Muroidea, where TRMT1 is likely resistant to cleavage. TRMT1 proteolysis results in reduced tRNA binding and elimination of tRNA methyltransferase activity. We also determined the structure of an Mpro-TRMT1 peptide complex that shows how TRMT1 engages the Mpro active site in an uncommon substrate binding conformation. Finally, enzymology and molecular dynamics simulations indicate that kinetic discrimination occurs during a later step of Mpro-mediated proteolysis following substrate binding. Together, these data provide new insights into substrate recognition by SARS-CoV-2 Mpro that could help guide future antiviral therapeutic development and show how proteolysis of TRMT1 during SARS-CoV-2 infection impairs both TRMT1 tRNA binding and tRNA modification activity to disrupt host translation and potentially impact COVID-19 pathogenesis or phenotypes.