Remazol brilliant blue

Peptidoglycan was purified from NTHI wild-type strain 176 by the method of Uehara et al. (15 (link)). One liter of exponential-phase culture was centrifuged, the pellet was resuspended in 20 ml of phosphate-buffered saline (PBS), boiled with 80 ml of 5% SDS for 30 min, and left overnight at room temperature (RT). Samples were ultracentrifuged for 1 h at 52,000 × g at RT and then washed with water several times to remove SDS. The peptidoglycan pellet was resuspended in 1 ml of PBS and incubated with 200 µg/ml amylase (catalog no. A6380; Sigma) overnight at 37°C. The sample was pelleted by ultracentrifugation at 200,000 × g for 15 min, washed with water three times, and resuspended in 1 ml of water. The dye release assay was performed staining purified peptidoglycan with remazol brilliant blue (catalog no. R8001; Sigma) (14 (link)) and then incubating it with 4 µM purified LytM proteins or mutanolysin (positive control) at 37°C for different incubation times. Dye release was quantified measuring the absorbance at 595 nm. Peptidoglycan incubated with PBS alone was used as a negative control (blank) and subtracted from all values.