PG isolation and analysis by UPLC.

Ah65 was subcultured 1:100 into 400 ml of buffered LB medium or buffered LB medium with 270 mM Gly. Cultures were harvested at an OD₆₀₀ of approximately 2.0. The PG was isolated using the boiling SDS method (31). Muropeptides for UPLC analysis were prepared by digesting PG samples with 20 μg lysozyme at 37°C overnight. Digested PG samples were freeze-dried prior to further analysis. Supernatants were reduced by adding 150 μl of 0.5 M sodium borate (pH 9.5) and sodium borohydride to a final concentration of 10 mg/ml and incubating at room temperature (RT) for 30 min. Finally, samples were adjusted to pH 3.5 with phosphoric acid.

UPLC analyses of muropeptides were performed on an Acquity UPLC BEH C₁₈ column (130 Å, 1.7 μm, and 2.1 mm by 150 mm; Waters) and detected at 204 nm. Muropeptides were separated using a linear gradient from buffer A (phosphate buffer, 50 mM, pH 4.35) to buffer B (phosphate buffer, 50 mM, pH 4.95; methanol, 15% [vol/vol]) over a 20-min run.