Purification and Analysis of Bacterial Peptidoglycan

Biological replicates were grown to mid-logarithmic or stationary phase in 400 mL of Luria broth. Cells were centrifuged (Avanti JXN-26 Beckman Coulter centrifuge and Beckman Coulter JA-10 rotor) at 7,000 × g for 0.5 h at 4°C, resuspended in chilled 6 mL PBS, and lysed via dropwise addition to boiling 8% sodium dodecyl sulfate (SDS). PG was further purified as previously described (84 (link)). Briefly, muropeptides were cleaved from PG by Cellosyl muramidase (Hoechst, Frankfurt am Main, Germany), reduced with sodium borohydride, and separated on a 250- by 4.6-mm 3-μm Prontosil 120-3-C₁₈ AQ reversed-phase column (Bischoff, Leonberg, Germany). The eluted muropeptides were detected by the absorbance at 205 nm. Eluted peaks were designated based on known published chromatograms (20 (link), 42 (link), 57 (link)); new peaks were analyzed by MS/MS, as previously described (20 (link)).