Article

Bacterial cell wall biogenesis is mediated by SEDS and PBP polymerase families functioning semi-autonomously

  • Nature Microbiology 1, Article number: 16172 (2016)
  • doi:10.1038/nmicrobiol.2016.172
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Abstract

Multi-protein complexes organized by cytoskeletal proteins are essential for cell wall biogenesis in most bacteria. Current models of the wall assembly mechanism assume that class A penicillin-binding proteins (aPBPs), the targets of penicillin-like drugs, function as the primary cell wall polymerases within these machineries. Here, we use an in vivo cell wall polymerase assay in Escherichia coli combined with measurements of the localization dynamics of synthesis proteins to investigate this hypothesis. We find that aPBP activity is not necessary for glycan polymerization by the cell elongation machinery, as is commonly believed. Instead, our results indicate that cell wall synthesis is mediated by two distinct polymerase systems, shape, elongation, division, sporulation (SEDS)-family proteins working within the cytoskeletal machines and aPBP enzymes functioning outside these complexes. These findings thus necessitate a fundamental change in our conception of the cell wall assembly process in bacteria.

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Acknowledgements

The authors thank all members of the Bernhardt, Rudner and Garner laboratories for advice and discussions. The authors thank P. de Boer and C. Hale for the gift of the mreB::galK strain for constructing sandwich fusions and L. Lavis for his gift of JF dyes. This work was supported by the National Institutes of Health (R01AI083365 to T.G.B., AI099144 to T.G.B., CETR U19 AI109764 to T.G.B. and DP2AI117923 to E.C.G.). E.C.G. was also supported by a Smith Family Award and a Searle Scholar Fellowship. P.D.A.R. was supported in part by a pre-doctoral fellowship from CHIR. J.A.M. was supported by the Dana-Farber Strategic Research Initiative.

Author information

Author notes

    • Hongbaek Cho
    • , Carl N. Wivagg
    •  & Mrinal Kapoor

    These authors contributed equally to this work.

Affiliations

  1. Department of Microbiology and Immunobiology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Hongbaek Cho
    • , Patricia D. A. Rohs
    •  & Thomas G. Bernhardt
  2. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, USA

    • Carl N. Wivagg
    • , Mrinal Kapoor
    • , Zachary Barry
    •  & Ethan C. Garner
  3. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA

    • Hyunsuk Suh
    •  & Jarrod A. Marto
  4. Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, Boston, Massachusetts 02215, USA

    • Jarrod A. Marto

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Contributions

T.G.B., E.C.G., H.C., C.N.W., M.K., Z.B., P.D.A.R. and H.S. designed the experiments and wrote/edited the manuscript. H.C. performed the radiolabelling studies and constructed E. coli strains for physiological labelling and imaging studies. C.N.W. and M.K. performed imaging studies and analysis. Z.B. performed CDF analysis. H.C., H.S. and J.A.M. performed and analysed data from the liquid chromatography–mass spectrometry study of MSPBP1b modification. M.K. constructed B. subtilis strains. P.D.A.R. constructed and characterized the dominant-negative RodA variants and made E. coli PBP1a fusion strains for imaging.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Ethan C. Garner or Thomas G. Bernhardt.

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    Supplementary Figures 1–11, Original Gel Images,Legends for Supplementary Videos 1–10, Supplementary Tables 1–4, Supplementary Methods, Supplementary References

Videos

  1. 1.

    Supplementary Video 1

    Inhibition of MS 15 PBP1b does not affect MreB motion

  2. 2.

    Supplementary Video 2

    RodA moves circumferentially around the cell axis in E. coli.

  3. 3.

    Supplementary Video 3

    PBP2 moves circumferentially around the cell axis in E. coli.

  4. 4.

    Supplementary Video 4

    PBP2 exhibits both diffusive and directional motion

  5. 5.

    Supplementary Video 5

    Induction of RodA(D262N) inhibits MreB motion

  6. 6.

    Supplementary Video 6A

    PBP1b exhibits fast diffusive motion in E. coli.

  7. 7.

    Supplementary Video 6B

    PBP1b exhibits fast diffusive motion in E. coli

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    Supplementary Video 7

    PBP1a does not exhibit directional motion in E. coli

  9. 9.

    Supplementary Video 8

    B. subtilis mNeon-PBP1 does not exhibit MreB-like motion.

  10. 10.

    Supplementary Video 9

    B. subtilis mNeon-PBP1 is predominantly in the slower diffusive state at low induction levels

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    Supplementary Video 10

    The slower diffusive state of B. subtilis mNeon-PBP1 is saturable