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

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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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.

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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.

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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.

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Correspondence to Chuck S. Farah or Gabriel Waksman.

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

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

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