Abstract

Peptidoglycan is the main component of the bacterial wall and protects cells from the mechanical stress that results from high intracellular turgor. Peptidoglycan biosynthesis is very similar in all bacteria; bacterial shapes are therefore mainly determined by the spatial and temporal regulation of peptidoglycan synthesis rather than by the chemical composition of peptidoglycan. The form of rod-shaped bacteria, such as Bacillus subtilis or Escherichia coli, is generated by the action of two peptidoglycan synthesis machineries that act at the septum and at the lateral wall in processes coordinated by the cytoskeletal proteins FtsZ and MreB, respectively1,2. The tubulin homologue FtsZ is the first protein recruited to the division site, where it assembles in filaments—forming the Z ring—that undergo treadmilling and recruit later divisome proteins3,4. The rate of treadmilling in B. subtilis controls the rates of both peptidoglycan synthesis and cell division3. The actin homologue MreB forms discrete patches that move circumferentially around the cell in tracks perpendicular to the long axis of the cell, and organize the insertion of new cell wall during elongation5,6. Cocci such as Staphylococcus aureus possess only one type of peptidoglycan synthesis machinery7,8, which is diverted from the cell periphery to the septum in preparation for division9. The molecular cue that coordinates this transition has remained elusive. Here we investigate the localization of S. aureus peptidoglycan biosynthesis proteins and show that the recruitment of the putative lipid II flippase MurJ to the septum, by the DivIB–DivIC–FtsL complex, drives peptidoglycan incorporation to the midcell. MurJ recruitment corresponds to a turning point in cytokinesis, which is slow and dependent on FtsZ treadmilling before MurJ arrival but becomes faster and independent of FtsZ treadmilling after peptidoglycan synthesis activity is directed to the septum, where it provides additional force for cell envelope constriction.

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Acknowledgements

We thank R. Sobral for the construction of pSG-murF plasmid; A. Jorge for the construction of the pSG-EzrA plasmid; T. Roemer for providing DMPI and strains AS-022 and AS-185; M. DeLisa for pTRC99a-P7; Richard Novick for pCN51; S. Foster for antibodies against DivIB and DivIC; A. Henriques for critical reading of the manuscript and L. Krippahl for help with image analysis tools. This study was funded by the European Research Council through grant ERC-2012-StG-310987 (to M.G.P.), by Project LISBOA-01-0145-FEDER-007660 Microbiologia Molecular, Estrutural e Celular (to ITQB-NOVA), by the National Institutes of Health through grant NIHGM113172 (to M.V.N.) and FCT fellowships SFRH/BD/71993/2010 (J.M.M.), SFRH/BD/86416/2012 (A.R.P.), SFRH/BPD/95031/2013 (N.T.R.), SFRH/BPD/87374/2012 (H.V.), SFRH/BD/52204/2013 (A.C.T.) and SFRH/BD/77849/2011 (M.T.F.).

Author information

Author notes

    • João M. Monteiro
    •  & Ana R. Pereira

    These authors contributed equally to this work.

Affiliations

  1. Bacterial Cell Biology, Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal

    • João M. Monteiro
    • , Ana R. Pereira
    • , Nathalie T. Reichmann
    • , Bruno M. Saraiva
    • , Pedro B. Fernandes
    • , Helena Veiga
    • , Andreia C. Tavares
    • , Margarida Santos
    • , Maria T. Ferreira
    • , Vânia Macário
    •  & Mariana G. Pinho
  2. Department of Chemistry, Indiana University, Bloomington, Indiana, USA

    • Michael S. VanNieuwenhze
  3. Instituto de Tecnologia Química e Biológica António Xavier and Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Oeiras, Portugal

    • Sérgio R. Filipe

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Contributions

J.M.M., A.R.P., N.T.R. and M.G.P. designed the research. J.M.M. and A.R.P. performed all experiments with the exception of the effect of antibiotics on the cell cycle which was performed by P.B.F. B.M.S. developed software for image analysis. J.M.M., A.R.P., N.T.R., P.B.F., H.V., A.C.T, M.S., M.T.F. and V.M. constructed strains. M.S.V. contributed new reagents (HADA). J.M.M., A.R.P., N.T.R., S.R.F. and M.G.P. analysed the overall data. B.M.S. analysed microscopy data quantified by eHooke software. P.B.F. analysed cell-cycle data. J.M.M., A.R.P, N.T.R. and M.G.P. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Mariana G. Pinho.

Reviewer Information Nature thanks J. Xiao and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Supplementary information

PDF files

  1. 1.

    Life Sciences Reporting Summary

  2. 2.

    Supplementary Tables 1-5

    This file contains Supplementary Table 1 – Strains used to study localisation of peptidoglycan synthesis in Staphylococcus aureus, Supplementary Table 2 – Sequences encoding RNA antisense fragments targeting divisome genes, Supplementary Table 3 – Strains and plasmids used in this study, Supplementary Table 4 – Oligonucleotides used in this study, and Supplementary Table 5 – Cloning strategies for the construction of strains using pMAD vector.

  3. 3.

    Supplementary Figure 1

    This file contains source data for western blots displayed in Extended Data Figure 2.

Videos

  1. 1.

    FtsZ55-56sGFP movement SR-SIM videos (left) followed by 15 min interval timelapse (right) in the absence (M9) or presence (M9+PC) of PC190723 5 µg/mL.

    FtsZ55-56sGFP movement SR-SIM videos (left) followed by 15 min interval timelapse (right) in the absence (M9) or presence (M9+PC) of PC190723 5 µg/mL. Scale bar 0.5 µm.

  2. 2.

    FtsZ55-56sGFP rings imaged every 5 min by SR-SIM.

    FtsZ55-56sGFP rings imaged every 5 min by SR-SIM. Scale bar 0.5 µm.

  3. 3.

    FtsZ55-56sGFP rings imaged every 5 min by SR-SIM in the presence of PC190723 5 µg/mL. The video shows a cell which constricts (1), a cell which constricts with defects (2) and a cell which does not constrict (3).

    FtsZ55-56sGFP rings imaged every 5 min by SR-SIM in the presence of PC190723 5 µg/mL. The video shows a cell which constricts (1), a cell which constricts with defects (2) and a cell which does not constrict (3). Scale bar 0.5 µm.

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https://doi.org/10.1038/nature25506

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