Extended Data Fig. 5: The LytH-ActH complex removes stem peptides from uncrosslinked glycan strands.
a, Chemical structure highlighting the differences between synthetic and S. aureus native Lipid II. b, PAGE autoradiographs of reactions with uncrosslinked glycan strand substrates. The glycopolymers for substrates (1) and (3) were made from synthetic Lipid II, while the glycopolymers for substrate 2 were generated from native Lipid II. For substrate (1), the radiolabel was found in the [14C]-GlcNAc residues of the glycan backbone. For substrates (2) and (3), the radiolabel was provided by a short wall teichoic acid (WTA) disaccharide branch ([14C]-LIIAWTA) attached to the hydroxyl group at the C6 position. c, Purified S. aureus sacculi were treated with lysostaphin and LytHWT-ActH. Hydrolysis of sacculi was monitored over time as a decrease in OD600. Experiments were performed in triplicate for each concentration of protein; each line represents the mean, plotted as percent of initial absorbance. d, Schematic for a PAGE assay to assess if LytH-ActH cleaves crosslinked peptidoglycan. Uncrosslinked glycan strands (substrate 2) were made from native Lipid II and radiolabeled with a short [14C]-LIIAWTA disaccharide branch (substrate 1) at the C6 position. The radiolabeled glycan strands were crosslinked by S. aureus PBP4 to generate crosslinked peptidoglycan (substrate 3). e, Peptidoglycan substrates were treated with LytHWT-ActH or lysostaphin. Reaction products were resolved by PAGE. Lysostaphin cleaves crosslinks, generating smaller species that migrate faster in the gel. Molecular weight ladders were not run on these PAGE gels. Data are representative of three independent experiments (b-c, e).
