Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins

  1. Victor M Hernández-Rocamora
  2. Natalia Baranova
  3. Katharina Peters
  4. Eefjan Breukink
  5. Martin Loose  Is a corresponding author
  6. Waldemar Vollmer  Is a corresponding author
  1. Newcastle University, United Kingdom
  2. Institute for Science and Technology Austria, Austria
  3. University of Utrecht, Netherlands

Abstract

Peptidoglycan is an essential component of the bacterial cell envelope that surrounds the cytoplasmic membrane to protect the cell from osmotic lysis. Important antibiotics such as β-lactams and glycopeptides target peptidoglycan biosynthesis. Class A penicillin binding proteins are bifunctional membrane-bound peptidoglycan synthases that polymerize glycan chains and connect adjacent stem peptides by transpeptidation. How these enzymes work in their physiological membrane environment is poorly understood. Here we developed a novel FRET-based assay to follow in real time both reactions of class A PBPs reconstituted in liposomes or supported lipid bilayers and we applied this assay with PBP1B homologues from Escherichia coli, Pseudomonas aeruginosa and Acinetobacter baumannii in the presence or absence of their cognate lipoprotein activator. Our assay will allow unravelling the mechanisms of peptidoglycan synthesis in a lipid-bilayer environment and can be further developed to be used for high throughput screening for new antimicrobials.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figures 1-5 and the corresponding figure supplements.

Article and author information

Author details

  1. Victor M Hernández-Rocamora

    Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle upon Tyne, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2517-5707
  2. Natalia Baranova

    Biophysics, Institute for Science and Technology Austria, Klosterneuburg, Austria
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3086-9124
  3. Katharina Peters

    Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle upon Tyne, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Eefjan Breukink

    Membrane Biochemistry and Biophysics, Bijvoet Centre for Biomolecular Research, University of Utrecht, Utrecht, Netherlands
    Competing interests
    The authors declare that no competing interests exist.
  5. Martin Loose

    Loose group, Institute for Science and Technology Austria, Klosterneuberg, Austria
    For correspondence
    mloose@ist.ac.at
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7309-9724
  6. Waldemar Vollmer

    Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle upon Tyne, United Kingdom
    For correspondence
    w.vollmer@ncl.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0408-8567

Funding

BBSRC (BB/R017409/1)

  • Waldemar Vollmer

European Research Council (ERC-2015-StG-679239)

  • Martin Loose

EMBO (EMBO ALTF 1163-2015)

  • Natalia Baranova

Human Frontiers Science Program (HFSP LT 000824/2016-L4)

  • Natalia Baranova

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Jie Xiao, Johns Hopkins University, United States

Version history

  1. Received: July 28, 2020
  2. Accepted: February 23, 2021
  3. Accepted Manuscript published: February 24, 2021 (version 1)
  4. Version of Record published: March 8, 2021 (version 2)

Copyright

© 2021, Hernández-Rocamora et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Victor M Hernández-Rocamora
  2. Natalia Baranova
  3. Katharina Peters
  4. Eefjan Breukink
  5. Martin Loose
  6. Waldemar Vollmer
(2021)
Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins
eLife 10:e61525.
https://doi.org/10.7554/eLife.61525

Share this article

https://doi.org/10.7554/eLife.61525

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    This work was supported by the Weizmann Krenter Foundation and the Weizmann – Ichilov (Tel Aviv Sourasky Medical Center) Collaborative Grant in Biomedical Research, by the Minerva Foundation, by the ISF KillCorona grant 3777/19.