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

Nature Microbiology volume 1, Article number: 16172 (2016) Cite this article

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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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Figure 1: The Rod system and an in vivo assay of peptidoglycan (PG) polymerase activity.
Figure 2: PG polymerization by the Rod complex does not require aPBP activity.
Figure 3: PBP2 and RodA display directed, circumferential motions similar to MreB.
Figure 4: aPBPs can function independently from the cytoskeletal machinery.

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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.

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Author notes

  1. Hongbaek Cho, Carl N. Wivagg and Mrinal Kapoor: These authors contributed equally to this work.

Authors and Affiliations

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

    Hongbaek Cho, Patricia D. A. Rohs & Thomas G. Bernhardt

  2. Department of Molecular and Cellular Biology, Harvard University, Cambridge, 02138, Massachusetts, USA

    Carl N. Wivagg, Mrinal Kapoor, Zachary Barry & Ethan C. Garner

  3. Department of Biological Chemistry and Molecular Pharmacology Harvard Medical School, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Hyunsuk Suh & Jarrod A. Marto

  4. Department of Cancer Biology and Blais Proteomics Center Dana-Farber Cancer Institute, Dana-Farber Cancer Institute, Boston, 02215, Massachusetts, 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.

Corresponding authors

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

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Supplementary information

Supplementary Information

Supplementary Figures 1–11, Original Gel Images,Legends for Supplementary Videos 1–10, Supplementary Tables 1–4, Supplementary Methods, Supplementary References (PDF 3916 kb)

Supplementary Video 1

Inhibition of MS 15 PBP1b does not affect MreB motion (MOV 843 kb)

Supplementary Video 2

RodA moves circumferentially around the cell axis in E. coli. (MOV 812 kb)

Supplementary Video 3

PBP2 moves circumferentially around the cell axis in E. coli. (MOV 1346 kb)

Supplementary Video 4

PBP2 exhibits both diffusive and directional motion (MOV 486 kb)

Supplementary Video 5

Induction of RodA(D262N) inhibits MreB motion (MOV 9245 kb)

Supplementary Video 6A

PBP1b exhibits fast diffusive motion in E. coli. (MOV 657 kb)

Supplementary Video 6B

PBP1b exhibits fast diffusive motion in E. coli (MOV 8634 kb)

Supplementary Video 7

PBP1a does not exhibit directional motion in E. coli (MOV 2976 kb)

Supplementary Video 8

B. subtilis mNeon-PBP1 does not exhibit MreB-like motion. (MOV 2018 kb)

Supplementary Video 9

B. subtilis mNeon-PBP1 is predominantly in the slower diffusive state at low induction levels (MOV 1174 kb)

Supplementary Video 10

The slower diffusive state of B. subtilis mNeon-PBP1 is saturable (MOV 1983 kb)

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Cho, H., Wivagg, C., Kapoor, M. et al. Bacterial cell wall biogenesis is mediated by SEDS and PBP polymerase families functioning semi-autonomously. Nat Microbiol 1, 16172 (2016). https://doi.org/10.1038/nmicrobiol.2016.172

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