Extended Data Fig. 6: Crosslinked PG is not a substrate for SagB-SpdC.
S. aureus Lipid II was co-incubated with wild-type PBP2, a hydrolase, and BDL for visualization. Reactions were quenched, halved, and then one portion was treated with lysostaphin. Crosslinked peptidoglycan oligomers do not readily enter the gel matrix (lane 1) and appear as a dark smear when crosslinks are cleaved by the endopeptidase lysostaphin (lane 4). In reactions containing SagB-SpdC, a low-molecular weight ladder of bands is present without lysostaphin (lane 2) or with lysostaphin (lane 5). This shows that these products, generated by SagB-SpdC, are not crosslinked as they were not changed with lysostaphin treatment. Notably, approximately 20% of peptidoglycan peptides are crosslinked by wild-type PBP2 in vitro36. The high molecular weight signals (smears) in lanes 5 and 6 are similar to the signal in lane 4, indicating that crosslinked PG is not cleaved by either SagB-SpdC or full-length SagB.
