epidermidis, as described elsewhere [24–26].
SE1457ΔsaeRS, SE1457, and SE1457saec cells were diluted in TSB containing 1 M NaCl, grown to mid-exponential phase (OD600 = ~0.6-0.8), washed twice in cold sterile Idasanutlin distilled water, resuspended in the same volume of 0.05 M Tris-HCl containing 0.05% Triton X-100 (pH 7.2), and incubated at 30°C. OD600 was measured every 30 min. The Triton X-100-induced autolysis rate was calculated as follows: Ra = OD0-ODt/OD0. Zymogram The murein hydrolase activities
of SE1457, SE1457ΔsaeRS, SE1457saec, and SE1457ΔatlE were detected by zymographic analysis as described elsewhere [26, 27]. Extracts from lysostaphin- learn more and SDS-treated S. epidermidis (Ex-Lys and Ex-SDS, Nirogacestat molecular weight respectively) and the concentrated supernatants of the bacterial culture (Ex-Sup) were used to analyze the murein hydrolase activities of each strain. Ex-Lys were obtained by treating S. epidermidis cells with 30 μg/mL of lysostaphin for 2 h at 37°C and subsequently centrifuged at 8,000 g for 30 min. Ex-SDS
were obtained by treating S. epidermidis cells in 100 μL of 100 mM phosphate buffer containing 4% SDS at 37°C for 30 min and centrifuged (10,000 g) for 10 min. Ex-Sup were acquired by concentrating fantofarone supernatants of overnight S. epidermidis cultures to 10% initial volume using a centrifugal filter device (Millipore, Billerica, MA). S. epidermidis cell extracts were separated on a SDS-PAGE gel (10% acrylamide, pH 8.8) containing 0.2% (wt/vol) lyophilized Micrococcus luteus (M. luteus) or S. epidermidis cells. After electrophoresis, the gels were washed four times with distilled water for 30 min at room temperature, incubated in 25 mM Tris-HCl containing 1% Triton X-100 (pH 8.0) at 37°C for 6 h, and then stained with methylene blue. Quantification of eDNA Extracellular DNA isolation from biofilms was performed as described by Rice et al. [7, 19, 28]. Briefly, SE1457, SE1457ΔsaeRS, and SE1457saec biofilms (grown for 24 h) were chilled at 4°C for 1 h and treated with 1.0 μL of 0.5 M EDTA.