Analysis of Peptidoglycan Composition

The peptidoglycan isolated from all four strains (wild-type, ΔltgA, ΔltgA^ltgA and ΔltgA^ltgAΔ30) was incubated for 16 hr in the presence of 10 µg of mutanolysin in 12.5 mM sodium phosphate buffer (pH 5.6) at 37°C (total reaction volume 150 µl). The reaction was stopped by boiling the samples for 3 min, and the supernatant containing the soluble muropeptides was collected after centrifugation at 16,000 ×g for 10 min. The supernatant was analyzed by reversed-phase HPLC using a Hypersil GOLD aQ column (5 μm particle size, 150 × 4.6 mm, flow rate 0.5 ml at 52°C, Thermo Fisher Scientific) with a mobile phase of H₂O-0.05% trifluoroacetic acid and a 25% acetonitrile gradient over 130 min. Muropeptides of interest were collected and identified by mass spectrometry as previously described (Williams et al., 2017 (link); Williams et al., 2018 (link)).