Cryo-EM structure of the bacteria-killing type IV secretion system core complex from Xanthomonas citri


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Biochemistry and EM map and model of the X. citri T4S system core complex.
Fig. 2: Structure of the X. citri T4S system core complex.
Fig. 3: The heterotrimer of the X. citri T4S system core complex structure and comparison with that of pKM101 from E. coli.
Fig. 4: Interactions between heterotrimers in the X. citri T4S system core complex.
Fig. 5: Effect of specific mutations in the core complex on T4S system-mediated cell lysis of neighbouring E. coli cells and VirB10 localization.
Fig. 6: Killing efficiency is correlated with T4S system assembly in X. citri.

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.


  1. 1.

    Alvarez-Martinez, C. E. & Christie, P. J. Biological diversity of prokaryotic type IV secretion systems. Microbiol. Mol. Biol. Rev. 73, 775–808 (2009).

    CAS  Article  Google Scholar 

  2. 2.

    Odenbreit, S. Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion. Science 287, 1497–1500 (2000).

    CAS  Article  Google Scholar 

  3. 3.

    Isaac, D. T. & Isberg, R. Master manipulators: an update on Legionella pneumophila Icm/Dot translocated substrates and their host targets. Future Microbiol. 9, 343–359 (2014).

    CAS  Article  Google Scholar 

  4. 4.

    Kotob, S. I., Hausman, S. Z. & Burns, D. L. Localization of the promoter for the ptl genes of Bordetella pertussis, which encode proteins essential for secretion of pertussis toxin. Infect. Immun. 63, 3227–3230 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    De Jong, M. F. & Tsolis, R. M. Brucellosis and type IV secretion. Future Microbiol. 7, 47–58 (2012).

    Article  Google Scholar 

  6. 6.

    Dehio, C. & Tsolis, R. M. Type IV effector secretion and subversion of host functions by Bartonella and Brucella species. Curr. Top. Microbiol. Immunol. 413, 269–295 (2017).

    CAS  PubMed  Google Scholar 

  7. 7.

    Siamer, S. & Dehio, C. New insights into the role of Bartonella effector proteins in pathogenesis. Curr. Opin. Microbiol. 23, 80–85 (2015).

    CAS  Article  Google Scholar 

  8. 8.

    Souza, D. P. et al. Bacterial killing via a type IV secretion system. Nat. Commun. 6, 6453 (2015).

    CAS  Article  Google Scholar 

  9. 9.

    Oliveira, L. C. et al. VirB7 and VirB9 interactions are required for the assembly and antibacterial activity of a type IV secretion system. Structure 24, 1707–1718 (2016).

    CAS  Article  Google Scholar 

  10. 10.

    Chandran, V. & Waksman, G. Structural biology of bacterial type IV secretion systems. Annu. Rev. Biochem. 84, 603–629 (2015).

    Article  Google Scholar 

  11. 11.

    Low, H. H. et al. Structure of a type IV secretion system. Nature 508, 550–553 (2014).

    CAS  Article  Google Scholar 

  12. 12.

    Costa, T. R. D. et al. Secretion systems in Gram-negative bacteria: structural and mechanistic insights. Nat. Rev. Microbiol. 13, 343–359 (2015).

    CAS  Article  Google Scholar 

  13. 13.

    Trokter, M., Felisberto-Rodrigues, C., Christie, P. J. & Waksman, G. Recent advances in the structural and molecular biology of type IV secretion systems. Curr. Opin. Struct. Biol. 27, 16–23 (2014).

    CAS  Article  Google Scholar 

  14. 14.

    Fronzes, R. et al. Structure of a type IV secretion system core complex. Science 323, 266–268 (2009).

    CAS  Article  Google Scholar 

  15. 15.

    Chandran, V. et al. Structure of the outer membrane complex of a type IV secretion system. Nature 462, 1011–1015 (2009).

    CAS  Article  Google Scholar 

  16. 16.

    Souza, D. P. et al. A component of the Xanthomonadaceae type IV secretion system combines a VirB7 motif with a N0 domain found in outer membrane transport proteins. PLoS Pathog. 7, e1002031 (2011).

    CAS  Article  Google Scholar 

  17. 17.

    Rivera-Calzada, A. et al. Structure of a bacterial type IV secretion core complex at subnanometre resolution. EMBO J. 32, 1195–1204 (2013).

    CAS  Article  Google Scholar 

  18. 18.

    Gordon, J. E. et al. Use of chimeric type IV secretion systems to define contributions of outer membrane subassemblies for contact-dependent translocation. Mol. Microbiol. 105, 273–293 (2017).

    CAS  Article  Google Scholar 

  19. 19.

    Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017).

    CAS  Article  Google Scholar 

  20. 20.

    Zhang, K. Gctf: real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016).

    CAS  Article  Google Scholar 

  21. 21.

    Scheres, S. H. W. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012).

    CAS  Article  Google Scholar 

  22. 22.

    Punjani, A., Rubinstein, J. L., Fleet, D. J. & Brubaker, M. A. CryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat. Methods 14, 290–296 (2017).

    CAS  Article  Google Scholar 

  23. 23.

    Pettersen, E.F. et al. UCSF Chimera—a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).

    CAS  Article  Google Scholar 

  24. 24.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010).

    CAS  Article  Google Scholar 

  25. 25.

    Adams, P.D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010).

    CAS  Article  Google Scholar 

  26. 26.

    Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010).

    CAS  Article  Google Scholar 

  27. 27.

    The PyMOL Molecular Graphics System v.1.8. (Schrödinger, LLC, 2017).

  28. 28.

    Cavanagh, J., Fairbrother, W. J., Palmer, A. G., Rance, M. & Skelton, N. J. Protein NMR Spectroscopy: Principles and Practice (Academic Press, San Diego, 2007).

  29. 29.

    Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    CAS  Article  Google Scholar 

  30. 30.

    Vranken, W. F. et al. The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins Struct. Funct. Genet. 59, 687–696 (2005).

    CAS  Article  Google Scholar 

  31. 31.

    Marsh, J. A., Singh, V. K., Jia, Z. & Forman-Kay, J. D. Sensitivity of secondary structure propensities to sequence differences between α- and γ-synuclein: implications for fibrillation. Protein Sci. 15, 2795–2804 (2006).

    CAS  Article  Google Scholar 

  32. 32.

    Shen, Y. & Bax, A. Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks. J. Biomol. NMR 56, 227–241 (2013).

    CAS  Article  Google Scholar 

  33. 33.

    da Silva, A. C. R. et al. Comparison of the genomes of two Xanthomonas pathogens with differing host specificities. Nature 417, 459–463 (2002).

    Article  Google Scholar 

  34. 34.

    Ke, N., Landgraf, D., Paulsson, J. & Berkmen, M. Visualization of periplasmic and cytoplasmic proteins with a self-labeling protein tag. J. Bacteriol. 198, 1035–1043 (2016).

    CAS  Article  Google Scholar 

  35. 35.

    Sawitzke, J. A. et al. Probing cellular processes with oligo-mediated recombination and using the knowledge gained to optimize recombineering. J. Mol. Biol. 407, 45–59 (2011).

    CAS  Article  Google Scholar 

  36. 36.

    Vettiger, A., Basler, M. Type VI secretion system substrates are transferred and reused among sister cells. Cell 167, 99–110 (2016).

    CAS  Article  Google Scholar 

  37. 37.

    Clark, D. J. & Maaloe, O. DNA replication and the division cycle in Escherichia coli. J. Mol. Biol. 23, 99–112 (1967).

    CAS  Article  Google Scholar 

  38. 38.

    RStudio: Integrated Development for R (RStudio Team, 2016);

  39. 39.

    Wickham, H. ggplot2 (Springer-Verlag, New York, 2009).

  40. 40.

    Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2013).

    Article  Google Scholar 

  41. 41.

    Jiang, C., Brown, P. J. B., Ducret, A. & Brun, Y. V. Sequential evolution of bacterial morphology by co-option of a developmental regulator. Nature 506, 489–493 (2014).

    CAS  Article  Google Scholar 

  42. 42.

    Schägger, H. Tricine–SDS–PAGE. Nat. Protoc. 1, 16–22 (2006).

    Article  Google Scholar 

Download references


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




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.

Corresponding authors

Correspondence to Chuck S. Farah or Gabriel Waksman.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

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

Supplementary information

Supplementary Information

Supplementary Figures 1–9, Supplementary Tables 1–5, Supplementary Notes, Supplementary Discussion, Supplementary References

Reporting Summary

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Sgro, G.G., Costa, T.R.D., Cenens, W. et al. Cryo-EM structure of the bacteria-killing type IV secretion system core complex from Xanthomonas citri. Nat Microbiol 3, 1429–1440 (2018).

Download citation

Further reading