Sacculi was prepared from stationary-phase cells of S. aureus strain HG003 using a previously published protocol52 (link), with the following modifications. Volumes of solutions were scaled appropriately for the number of cells harvested. After 1 M HCl treatment, the pellets were washed with dH2O, flash-frozen, and lyophilized to yield purified sacculi.
A 0.5 mg/mL solution of sacculi was prepared in reaction buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% DDM, 60 μM Zn(OAc)2). The sacculi hydrolysis assay was set up in non-treated 96-well plates (Genesee Scientific). To each well, 150 μL of sacculi solution was added. Lysostaphin (dissolved in dH2O) or the purified LytH-ActH complex (stored in 50 mM HEPES, pH 7.5, 500 mM NaCl, 10% glycerol, 0.05% DDM) was mixed with the sacculi substrate. Reactions were prepared in triplicate using 0–125 nM of purified protein per reaction. The plate was covered with a lid and incubated at 25°C with shaking. Absorbance (OD600) was recorded at 20-min intervals for 20 hr using a SpectraMax Plus 384 microplate spectrophotometer (Molecular Devices). Hydrolysis of sacculi was monitored over time as a decrease in absorbance.
A 0.5 mg/mL solution of sacculi was prepared in reaction buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 0.02% DDM, 60 μM Zn(OAc)2). The sacculi hydrolysis assay was set up in non-treated 96-well plates (Genesee Scientific). To each well, 150 μL of sacculi solution was added. Lysostaphin (dissolved in dH2O) or the purified LytH-ActH complex (stored in 50 mM HEPES, pH 7.5, 500 mM NaCl, 10% glycerol, 0.05% DDM) was mixed with the sacculi substrate. Reactions were prepared in triplicate using 0–125 nM of purified protein per reaction. The plate was covered with a lid and incubated at 25°C with shaking. Absorbance (OD600) was recorded at 20-min intervals for 20 hr using a SpectraMax Plus 384 microplate spectrophotometer (Molecular Devices). Hydrolysis of sacculi was monitored over time as a decrease in absorbance.
