Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins
- Newcastle University, United Kingdom
- Institute for Science and Technology Austria, Austria
- 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
Victor M Hernández-Rocamora
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
- Jie Xiao, Johns Hopkins University, United States
Version history
- Received: July 28, 2020
- Accepted: February 23, 2021
- Accepted Manuscript published: February 24, 2021 (version 1)
- 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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Real time monitoring of peptidoglycan synthesis by membrane-reconstituted penicillin binding proteins
eLife 10:e61525.
https://doi.org/10.7554/eLife.61525
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