Abstract

Type IV secretion (T4S) systems form the most common and versatile class of secretion systems in bacteria, capable of injecting both proteins and DNAs into host cells. T4S systems are typically composed of 12 components that form 2 major assemblies: the inner membrane complex embedded in the inner membrane and the core complex embedded in both the inner and outer membranes. Here we present the 3.3 Å-resolution cryo-electron microscopy model of the T4S system core complex from Xanthomonas citri, a phytopathogen that utilizes this system to kill bacterial competitors. An extensive mutational investigation was performed to probe the vast network of protein–protein interactions in this 1.13-MDa assembly. This structure expands our knowledge of the molecular details of T4S system organization, assembly and evolution.

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Data availability

Density map is available at EMDB with accession code EMD-0089. Atomic model is available in Protein Data Bank with accession code 6GYB. NMR data are available at BMRB with accession number 27342. All other data supporting the findings of this study are available from the corresponding authors upon request.

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Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Acknowledgements

This work was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) grants 2011/07777-5 and 2017/17303-7 to C.S.F. and Wellcome Trust grant 098302 to G.W. G.G.S. and W.C. were recipients of FAPESP post-doctoral fellowship grants. D.P.S. and L.C.O. received post-doctoral and PhD scholarships, respectively, from the Conselho Nacional de Pesquisa e Desenvolvimento (CNPq). We thank the members of the Farah and Waksman laboratories for fruitful discussions. We thank A. Bruni-Cardoso for fluorescence microscope access. We thank LNNano/CNPEM for the access to the cryo-EM facility, proposals TEM-19470 and TEM-20247. We thank Diamond for access to and support of the Cryo-EM facilities at the UK national Electron Bio-Imaging Centre (eBIC), proposal EM14704, funded by the Wellcome Trust, MRC and BBSRC. We thank N. Lukoyanova for the use of the ISMB Polara microscope.

Author information

Author notes

    • Tiago R. D. Costa

    Present address: MRC Centre for Molecular Bacteriology and Infection, Department of Life Sciences, Imperial College London, London, UK

    • Diorge P. Souza

    Present address: MRC Laboratory for Molecular Cell Biology, University College London, London, UK

    • Luciana Coutinho de Oliveira

    Present address: Department of Medicinal Chemistry, Université du Québec, INRS - Institut Armand-Frappier, Laval, Québec, Canada

  1. These authors contributed equally: Germán G. Sgro, Tiago R. D. Costa.

Affiliations

  1. Departamento de Bioquímica, Instituto de Química, Universidade de São Paulo, São Paulo, Brazil

    • Germán G. Sgro
    • , William Cenens
    • , Diorge P. Souza
    • , Luciana Coutinho de Oliveira
    • , Roberto K. Salinas
    •  & Chuck S. Farah
  2. Institute of Structural and Molecular Biology at UCL and Birkbeck College, Department of Biological Sciences, Birkbeck College, London, UK

    • Germán G. Sgro
    • , Tiago R. D. Costa
    •  & Gabriel Waksman
  3. Laboratório Nacional de Nanotecnologia (LNNano), Centro Nacional de Pesquisa em Energia e Materiais (CNPEM), Campinas, Brazil

    • Alexandre Cassago
    •  & Rodrigo V. Portugal
  4. Institute of Structural and Molecular Biology at UCL and Birkbeck College, Research Department of Structural and Molecular Biology, Division of Biosciences, University College London, London, UK

    • Gabriel Waksman

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Contributions

G.G.S. cloned, expressed and purified the X. citri T4S system core complex. G.G.S., A.C. and R.V.P. prepared and collected negative-staining EM data. G.G.S., T.R.D.C., A.C. and R.V.P. determined the sample preparation conditions for cryo-EM. T.R.D.C. prepared cryo-EM grids used for data collection (with G.G.S.), collected cryo-EM data, performed the image analysis and carried out the EM reconstructions. G.G.S. built and refined the model. G.G.S., W.C. and D.P.S. carried out the mutagenesis work. W.C. performed and analysed the biological assays and the microscopy analysis. G.G.S. carried out the immunoblotting analysis. L.C.O., D.P.S., C.S.F. and R.K.S. performed the NMR analysis. G.G.S., T.R.D.C., W.C., C.S.F. and G.W. prepared the figures. G.G.S., C.S.F. and G.W. wrote the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding authors

Correspondence to Chuck S. Farah or Gabriel Waksman.

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DOI

https://doi.org/10.1038/s41564-018-0262-z