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

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 beta-lactams and glycopeptides target peptidoglycan biosynthesis. Class A penicillin-binding proteins (PBPs) 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 Forster resonance energy transfer-based assay to follow in real time both reactions of class A PBPs reconstituted in liposomes or supported lipid bilayers and 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.

Automated summary

Peptidoglycan is a crucial part of the bacterial cell envelope, and the Class A penicillin-binding proteins are essential enzymes involved in its synthesis. Researchers have developed a new assay to study the mechanisms of these enzymes in a lipid-bilayer environment, opening up possibilities for new directions in peptidoglycan synthesis research.