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 demonstrate 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 allows 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.

中文翻译：

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膜重组青霉素结合蛋白实时监测肽聚糖的合成

肽聚糖是围绕细胞质膜的细菌细胞包膜的重要组成部分，以保护细胞免受渗透性裂解。重要的抗生素，例如β-内酰胺和糖肽靶向肽聚糖的生物合成。A类青霉素结合蛋白是双功能的膜结合肽聚糖合酶，可聚合聚糖链并通过转肽作用连接相邻的茎肽。这些酶如何在其生理膜环境中发挥作用尚不清楚。在这里，我们开发了一种新颖的基于FRET的检测方法，可以实时跟踪在脂质体或支持的脂质双层中重构的A类PBP的反应，并且我们用大肠杆菌，铜绿假单胞菌和鲍曼不动杆菌的PBP1B同源物证明了该检测方法是否存在其相关的脂蛋白激活剂。我们的测定方法可揭示脂双层环境中肽聚糖合成的机理，并可进一步开发用于高通量筛选新的抗菌剂。