The type VI secretion system (T6SS) is a multiprotein weapon used by bacteria to destroy competitor cells. The T6SS contractile sheath wraps an effector-loaded syringe that is injected into the target cell. This tail structure assembles onto the baseplate that is docked to the membrane complex. In enteroaggregative Escherichia coli, TssA plays a central role at each stage of the T6SS assembly pathway by stabilizing the baseplate and coordinating the polymerization of the tail. Here we adapted an assay based on APEX2-dependent biotinylation to identify the proximity partners of TssA in vivo. By using stage-blocking mutations, we define the temporal contacts of TssA during T6SS biogenesis. This proteomic mapping approach also revealed an additional partner of TssA, TagA. We show that TagA is a cytosolic protein tightly associated with the membrane. Analyses of sheath dynamics further demonstrate that TagA captures the distal end of the sheath to stop its polymerization and to maintain it under the extended conformation.
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Excel spradsheets with the raw mass-spectrometry data have been provided as Supplementary Datasheets 1–9. Plasmid pKD4-Nter-APEX2 has been deposited in the Addgene plasmid repository under accession number 112868. All data that support the findings of this study are available from the corresponding author on request.
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This work was funded by the Centre National de la Recherche Scientifique, the Aix-Marseille Université and grants from the Agence Nationale de la Recherche (ANR-14-CE14-0006-02, ANR-17-CE11-0039-01). Y.G.S. is supported by a doctoral fellowship from the French ministry of research. We thank H. Le Guenno of the IMM microscopy facility for helpful advice regarding deconvolution analyses; J. Sturgis (IMM), E. A. Rucks and S. Ouellette (University of South Dakota, Vermillion, USA) for initial discussions on biotin-dependent ligation; the members of the Cascales, Lloubès, Sturgis and Bouveret research groups for discussions, Y. A. Nadal-Sitron for encouragements and M. Ba, I. Bringer, A. Brun and O. Uderso for technical assistance.
Y.G.S. and E.C. designed and conceived the experiments. Y.G.S., T.D., R.L. and L.E. performed the experiments. Y.G.S. performed all of the experiments, with the help of T.D. and L.E. for fluorescence microscopy. R.L. performed the mass spectrometry analyses. E.C supervised the execution of the experiments. L.J. and E.C. provided tools. E.C. wrote the paper with contributions from Y.G.S, T.D., R.L. and L.J.
The authors declare no competing interests.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Tables 1–3, Supplementary Figures 1–7.
Raw mass spectrometry datasheet for 17-2 wild-type cells producing APEX2–TssA.
Raw mass spectrometry datasheet for 17-2 wild-type cells producing APEX2–TssA (without membrane solubilization by detergent).
Raw mass spectrometry datasheet for 17-2 wild-type cells.
Raw mass spectrometry datasheet for 17-2 wild-type cells producing APEX2.
Raw mass spectrometry datasheet for 17-2 wild-type cells producing APEX2–GspE.
Raw mass spectrometry datasheet for 17-2 wild-type cells producing APEX2–GspE.
Raw mass spectrometry datasheet for 17-2 ∆tssK cells.
Raw mass spectrometry datasheet for 17-2 ∆tssK cells producing APEX2–TssA.
Raw mass spectrometry datasheet for 17-2 ∆hcp cells producing APEX2–TssA.
Time-lapse fluorescence microscopy recording of cells.
Time-lapse fluorescence microscopy recording of ΔtagA cells producing TssB-sfGFP.
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Santin, Y.G., Doan, T., Lebrun, R. et al. In vivo TssA proximity labelling during type VI secretion biogenesis reveals TagA as a protein that stops and holds the sheath. Nat Microbiol 3, 1304–1313 (2018). https://doi.org/10.1038/s41564-018-0234-3
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