Abstract

Bacterial type IV secretion systems translocate virulence factors into eukaryotic cells1,2, distribute genetic material between bacteria and have shown potential as a tool for the genetic modification of human cells3. Given the complex choreography of the substrate through the secretion apparatus4, the molecular mechanism of the type IV secretion system has proved difficult to dissect in the absence of structural data for the entire machinery. Here we use electron microscopy to reconstruct the type IV secretion system encoded by the Escherichia coli R388 conjugative plasmid. We show that eight proteins assemble in an intricate stoichiometric relationship to form an approximately 3 megadalton nanomachine that spans the entire cell envelope. The structure comprises an outer membrane-associated core complex1 connected by a central stalk to a substantial inner membrane complex that is dominated by a battery of 12 VirB4 ATPase subunits organized as side-by-side hexameric barrels. Our results show a secretion system with markedly different architecture, and consequently mechanism, to other known bacterial secretion systems1,4,5,6.

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Accessions

Primary accessions

Electron Microscopy Data Bank

Data deposits

The electron microscope composite map of the T4SS3–10 complex has been deposited in the Electron Microscopy Data Bank under accession number EMD-2567.

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Acknowledgements

This work was financed by grant 098302 from the Wellcome Trust to G.W. and by equipment grant 079605 from the Wellcome Trust. R.F. and A.D. were financed by Institut Pasteur and the CNRS. F.G. was the recipient of ‘Bourse Roux’ from Institut Pasteur. We thank G. Péhau-Arnaudet for support with the electron microscopes at Institut Pasteur.

Author information

Author notes

    • Harry H. Low
    •  & Francesca Gubellini

    These authors contributed equally to this work.

    • Harry H. Low

    Present address: Faculty of Natural Sciences, Imperial College, London SW7 2AZ, UK.

Affiliations

  1. Institute of Structural and Molecular Biology, University College London and Birkbeck College, Malet Street, London WC1E 7HX, UK

    • Harry H. Low
    • , Angel Rivera-Calzada
    • , Nathalie Braun
    • , Sarah Connery
    • , Fang Lu
    • , Adam Redzej
    • , Elena V. Orlova
    •  & Gabriel Waksman
  2. Institut Pasteur, G5 Biologie structurale de la sécrétion bactérienne and UMR 3528-CNRS, 25 rue du Docteur Roux, 75015 Paris, France

    • Francesca Gubellini
    • , Annick Dujeancourt
    •  & Rémi Fronzes

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Contributions

H.H.L., F.G., R.F., E.V.O. and G.W. designed the experiments. H.H.L., F.G., S.C. and F.L. generated the clones. H.H.L. first purified the T4SS3–10 complex, and collected and processed the negative-stain electron microscope data for that complex. F.G., assisted by A.D., purified the T4SS3–10 complex and performed the stoichiometry, gold labelling and membrane wash experiments for that complex. A.R.-C. purified, collected electron microscope data and processed the TrwH/TrwF/TrwE complex. N.B., assisted by A.R., collected T4SS3–10 complex cryo-negative-stain data. H.H.L. and A.R.-C. purified, collected electron microscope data and processed TrwK and MBP–TrwK. A.R.-C. and F.L. purified the TrwM/TrwK complex and determined its stoichiometry. H.H.L. drafted the paper, and H.H.L., F.G., R.F., E.V.O. and GW finalized it.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Rémi Fronzes or Elena V. Orlova or Gabriel Waksman.

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DOI

https://doi.org/10.1038/nature13081

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