penicillin - binding 
 proteins ( pbps ) are involved in the synthesis 
 and remodeling of bacterial peptidoglycan ( pg ) . 
 staphylococcus 
 aureus expresses four pbps . 
 genetic studies in s. 
 aureus 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 ) in vitro . moreover , it is the pbp primarily responsible for incorporating 
 exogenous d - amino acids into cellular pg , implying that it 
 also has tp activity in vivo . 
 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 .