Relevant steps of PG biosynthesis and D-amino acid dipeptide (DAAD) probes used in this study.

<p>(<b>a</b>) DAADs are taken up by bacteria where they compete with endogenous D-Ala-D-Ala (DA—DA) for incorporation into PG. De novo synthesis of DA—DA is inhibited by D-cyloserine. The pentapeptide PG subunit is then flipped across the inner membrane into the periplasm where it is transglycosylated to form glycan polymers (nascent PG) and crosslinked by penicillin binding proteins (PBPs). Transpeptidation causes cleavage of the terminal D-Ala at position 5. Because the N-terminally labeled portion of DAAD becomes the amino acid at position 4 of the pentapeptide, the label is resistant to this processing and remains on the stem peptide. (<b>b</b>) PG-labeling reagents used in this study. Clickable DAADs EDA—DA and ADA—DA. Star represents the clickable amino acid.