Abstract

Bacterial cell envelope protein (CEP) complexes mediate a range of processes, including membrane assembly, antibiotic resistance and metabolic coordination. However, only limited characterization of relevant macromolecules has been reported to date. Here we present a proteomic survey of 1,347 CEPs encompassing 90% inner- and outer-membrane and periplasmic proteins of Escherichia coli. After extraction with non-denaturing detergents, we affinity-purified 785 endogenously tagged CEPs and identified stably associated polypeptides by precision mass spectrometry. The resulting high-quality physical interaction network, comprising 77% of targeted CEPs, revealed many previously uncharacterized heteromeric complexes. We found that the secretion of autotransporters requires translocation and the assembly module TamB to nucleate proper folding from periplasm to cell surface through a cooperative mechanism involving the β-barrel assembly machinery. We also establish that an ABC transporter of unknown function, YadH, together with the Mla system preserves outer membrane lipid asymmetry. This E. coli CEP 'interactome' provides insights into the functional landscape governing CE systems essential to bacterial growth, metabolism and drug resistance.

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Acknowledgements

We thank T. Silhavy, C. Whitfield, J.P. Côté, M. Mourez and P. Dersch for generously providing plasmids and reagents. We are also grateful to R. Reithmeier and M. Jessulat (Babu Lab) for advice. This work was supported by grants from the Canadian Institutes of Health Research to J.F.G., J.P. and A.E. (MOP-106449), J.P., A.E. and M.B. (PJT -148831) and T.F.M. (MOP-115182); the Ontario Ministry of Education and Innovation to T.F.M. and A.E.; the National Institutes of Health to P.U., M.H.S. and A.E. (GM109895); the Natural Sciences and Engineering Research Council of Canada to T.F.M. (DG-40197), A.Go. (DG-315735), J.P. (DG-06664), and M.B. (DG-20234); and the Canada Foundation for Innovation to T.F.M., M.B. and A.E.

Author information

Author notes

    • Mohan Babu
    • , Cedoljub Bundalovic-Torma
    •  & Charles Calmettes

    These authors contributed equally to this work.

    • Mohan Babu
    • , Milton Saier
    • , Peter Uetz
    • , Trevor F Moraes
    • , John Parkinson
    •  & Andrew Emili

    These authors jointly supervised this work.

Affiliations

  1. Department of Biochemistry, University of Regina, Regina, Saskatchewan, Canada.

    • Mohan Babu
    • , Sadhna Phanse
    • , Qingzhou Zhang
    • , Zoran Minic
    • , Sunyoung Kim
    • , Hiroyuki Aoki
    • , Viktor Deineko
    •  & James Vlasblom
  2. Hospital for Sick Children, Toronto, Ontario, Canada.

    • Cedoljub Bundalovic-Torma
    • , Yue Jiang
    •  & John Parkinson
  3. Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.

    • Cedoljub Bundalovic-Torma
    • , Charles Calmettes
    • , Christine Chieh-lin Lai
    • , Majida El Bakkouri
    • , Yogesh Hooda
    • , Megha Shah
    • , Trevor F Moraes
    •  & John Parkinson
  4. INRS-Institut Armand Frappier, Laval, Québec, Canada.

    • Charles Calmettes
  5. Donnelly Centre, University of Toronto, Toronto, Ontario, Canada.

    • Sadhna Phanse
    • , Hongbo Guo
    • , Olga Kagan
    • , Oxana Pogoutse
    • , Yogee Pandya
    • , Jack F Greenblatt
    •  & Andrew Emili
  6. Virginia Commonwealth University, Richmond, Virginia, USA.

    • Jitender Mehla
    • , J Harry Caufield
    •  & Peter Uetz
  7. Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.

    • Alla Gagarinova
    •  & Erik Holtzapple
  8. Department of Molecular Biology, UC San Diego, La Jolla, California, USA.

    • Irina Rodionova
    • , Erik Holtzapple
    • , Zhongge Zhang
    • , Ake Vastermark
    •  & Milton Saier
  9. Department of Computer Science, University of Regina, Regina, Saskatchewan, Canada.

    • Ashwani Kumar
  10. Department of Biology, Carleton University, Ottawa, Ontario, Canada.

    • Dan Burnside
    • , Mohsen Hooshyar
    •  & Ashkan Golshani
  11. J. Craig Venter Institute, Rockville, Maryland, USA.

    • Sessandra V Rajagopala
  12. Department of Computer Science and Department of Biology, University of Miami, Coral Gables, Florida, USA.

    • Stefan Wuchty
  13. Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

    • Jack F Greenblatt
    • , John Parkinson
    •  & Andrew Emili
  14. Department of Biology and Biochemistry, Boston University, Boston, Massachusetts, USA.

    • Andrew Emili

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Contributions

M.B. and A.E. designed and together supervised the project. Y.P. and O.P. constructed tagged strains. O.K., S.K. and V.D. performed protein purifications, while H.G. and Z.M. performed MS analyses. S.P. performed database searches and curation, and created the web portal. C.C., S.K., J.M., V.K., I.R., A.Ga., H.A., E.H., Z.Z., A.V., D.B., M.H., C.C.L., M.E.B., Y.H., M.S. and S.V.R. performed the follow-up and validation experiments. C.B.-T., S.P., Q.Z., Y.J., A.K., J.H.C. and S.W. performed the scoring and data analysis. M.B. and A.E. wrote the manuscript, with input from C.B.-T., C.C., J.V., A.Go., J.F.G., M.S., P.U., T.F.M. and J.P. All authors read and approved the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Mohan Babu or Andrew Emili.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–10

  2. 2.

    Life Sciences Reporting Summary

  3. 3.

    Supplementary Notes 1–2

Excel files

  1. 1.

    Supplementary Table 1

    Classification of the E. coli proteome and target cell envelope protein selection for AP/MS screens

  2. 2.

    Supplementary Table 2

    Co-purifying protein pairs compiled from reference Ecoyc database and from this study

  3. 3.

    Supplementary Table 3

    The physiological relevance and quality of PPIs by anecdotal supporting evidence, drug sensitivity profiles, and two-hybrid screens

  4. 4.

    Supplementary Table 4

    Putative (or novel) CEP complexes and their subunits identified by the core-attachment based clustering algorithm are indicated with their respective topology model

  5. 5.

    Supplementary Table 5

    Antibiotic susceptibility, evolutionary conservation, and paralogous analyses on CEPs or complexes, and bacterial strains/plasmids used in this study