Figure 4. Effect of amicoumacin A on autolysis.

(A) Effect on whole cell autolysis. Cultures grown in the absence (open circles) and the presence (closed circles) of amicoumacin A were washed and suspended in autolysis buffer to an initial OD₆₀₀ of around 1.0 and autolysis was monitored as decline in OD₆₀₀. (B) Quantitative assay of murein hydrolase activity against cell wall. An equal amount (90 µg protein) of extracellular proteins were added to cell wall purified from amicoumacin-A-untreated cells and OD₆₀₀ was monitored as described in Materials and Methods. Symbols: open circles, extracellular proteins from untreated cells; closed circles, extracellular proteins from amicoumacin-A-treated cells; open triangles, 10 mM Tris-HCl (pH 7.5). (C) Susceptibility assay of purified cell wall to murein hydrolase. Ninety µg of extracellular proteins from amicoumacin-A-untreated cells were added to cell wall purified from amicoumacin-A-treated and -untreated cells and OD₆₀₀ was monitored. Symbols: open circles, cell wall from untreated cells with 10 mM Tris-HCl (pH 7.5); closed circles, cell wall from untreated cells with extracellular proteins; open triangles, cell wall from amicoumacin-A-treated cells with 10 mM Tris-HCl (pH 7.5); closed triangles, cell wall from amicoumacin-A-treated cells with extracellular proteins. (D) Zymographic analysis of murein hydrolase activity from amicoumacin-A-treated and -untreated cells against purified S. aureus COL cell wall. Autolytic extracts were prepared and assayed by electrophoresis on an SDS-polyacrylamide gel (10%) containing 1 mg/ml purified cell wall as described in Materials and Methods. Lanes: M, prestained molecular weight markers; 1, cell-wall-associated proteins from untreated cells; 2, cell-wall-associated proteins from amicoumacin-A-treated cells; 3, extracellular proteins from untreated cells; 4, extracellular proteins from amicoumacin-A-treated cells.