Metabolic labeling of the bacterial peptidoglycan by functionalized glucosamine
Yang Xu,
Víctor M. Hernández-Rocamora,
Joseph H. Lorent,
Ruud Cox,
Xiaoqi Wang,
Xue Bao,
Marjon Stel,
Gaël Vos,
Ramon M. van den Bos,
Roland J. Pieters,
Joe Gray,
Waldemar Vollmer,
Eefjan Breukink
Affiliations
Yang Xu
Membrane Biochemistry and Biophysics, Bijvoet Centre for Biomolecular Research, Department of Chemistry, Utrecht University, Padualaan 8, 3584 Utrecht, the Netherlands
Víctor M. Hernández-Rocamora
Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle Upon Tyne, UK
Joseph H. Lorent
Membrane Biochemistry and Biophysics, Bijvoet Centre for Biomolecular Research, Department of Chemistry, Utrecht University, Padualaan 8, 3584 Utrecht, the Netherlands
Ruud Cox
Membrane Biochemistry and Biophysics, Bijvoet Centre for Biomolecular Research, Department of Chemistry, Utrecht University, Padualaan 8, 3584 Utrecht, the Netherlands
Xiaoqi Wang
Membrane Biochemistry and Biophysics, Bijvoet Centre for Biomolecular Research, Department of Chemistry, Utrecht University, Padualaan 8, 3584 Utrecht, the Netherlands
Xue Bao
Membrane Biochemistry and Biophysics, Bijvoet Centre for Biomolecular Research, Department of Chemistry, Utrecht University, Padualaan 8, 3584 Utrecht, the Netherlands
Marjon Stel
Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 Utrecht, the Netherlands
Gaël Vos
Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 Utrecht, the Netherlands
Ramon M. van den Bos
Membrane Biochemistry and Biophysics, Bijvoet Centre for Biomolecular Research, Department of Chemistry, Utrecht University, Padualaan 8, 3584 Utrecht, the Netherlands
Roland J. Pieters
Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 Utrecht, the Netherlands
Joe Gray
Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle Upon Tyne, UK
Waldemar Vollmer
Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle Upon Tyne, UK
Eefjan Breukink
Membrane Biochemistry and Biophysics, Bijvoet Centre for Biomolecular Research, Department of Chemistry, Utrecht University, Padualaan 8, 3584 Utrecht, the Netherlands; Corresponding author
Summary: N-Acetylglucosamine (GlcNAc) is an essential monosaccharide required in almost all organisms. Fluorescent labeling of the peptidoglycan (PG) on N-acetylglucosamine has been poorly explored. Here, we report on the labeling of the PG with a bioorthogonal handle on the GlcNAc. We developed a facile one-step synthesis of uridine diphosphate N-azidoacetylglucosamine (UDP-GlcNAz) using the glycosyltransferase OleD, followed by in vitro incorporation of GlcNAz into the peptidoglycan precursor Lipid II and fluorescent labeling of the azido group via click chemistry. In a PG synthesis assay, fluorescent GlcNAz-labeled Lipid II was incorporated into peptidoglycan by the DD-transpeptidase activity of bifunctional class A penicillin-binding proteins. We further demonstrate the incorporation of GlcNAz into the PG layer of OleD-expressed bacteria by feeding with 2-chloro-4-nitrophenyl GlcNAz (GlcNAz-CNP). Hence, our labeling method using the heterologous expression of OleD is useful to study PG synthesis and possibly other biological processes involving GlcNAc metabolism in vivo.
