Detection of Lipid-Linked Peptidoglycan Precursors by Exploiting an Unexpected Transpeptidase Reaction

Penicillin-binding proteins (PBPs) are involved in the synthesis and remodeling of bacterial peptidoglycan (PG). <i>Staphylococcus aureus</i> expresses four PBPs. Genetic studies in <i>S. aureus</i> have implicated PBP4 in the formation of highly cross-linked PG, but biochemical studies have not reached a consensus on its primary enzymatic activity. Using synthetic Lipid II, we show here that PBP4 preferentially acts as a transpeptidase (TP) <i>in vitro</i>. Moreover, it is the PBP primarily responsible for incorporating exogenous d-amino acids into cellular PG, implying that it also has TP activity <i>in vivo</i>. Notably, PBP4 efficiently exchanges d-amino acids not only into PG polymers but also into the PG monomers Lipid I and Lipid II. This is the first demonstration that any TP domain of a PBP can activate the PG monomer building blocks. Exploiting the promiscuous TP activity of PBP4, we developed a simple, highly sensitive assay to detect cellular pools of lipid-linked PG precursors, which are of notoriously low abundance. This method, which addresses a longstanding problem, is useful for assessing how genetic and pharmacological perturbations affect precursor levels, and may facilitate studies to elucidate antibiotic mechanism of action