FIG. 5.
Decrease in peptidoglycan hydrolase activity in ΔltaS mutant cells. (A) Monitoring of autolysis of ΔltaS mutant cells. Exponentially growing cells at 30°C were harvested, suspended in 50 mM Tris-HCl (pH 7.4) buffer containing 0.05% Triton X-100, and incubated at 30°C. The OD600 was determined at the indicated times. Open circles, RN4220; filled circles, M0674/pM101 (ΔltaS). Data are means and standard deviations for at least three independent experiments. (B) Zymographic analysis of peptidoglycan hydrolases. A cell wall fraction was prepared and separated by SDS-PAGE (10%) with a gel containing 1 mg/ml heat-killed M. luteus cells. The gel was incubated in 0.2 M phosphate buffer, and visualization of the cleared zone was enhanced by staining with 0.5% methylene blue. Lanes: 1, RN4220/pKE515; 2, M0674/pM101/pKE515; 3, M0674/pM101/pSltaS.