2. Autolysis assays were performed as described previously (Singh et al., 2008). Briefly, wild-type and the lytM mutant cultures of S. aureus were grown to an OD600 nm of 0.7 at 37 °C in PYK medium (0.5% Bacto peptone, 0.5% yeast extract, 0.3% K2HPO4, pH 7.2). After one wash with cold water (8500 g, 4 °C, 15 min), cells were suspended in 0.05 M Tris-HCl buffer, pH 7.2, containing 0.05% Triton X-100 to an OD600 nm of 1.0. selleck products Cell suspension was incubated in flasks at 37 °C with shaking (125 r.p.m.) and autolysis was determined by measuring decline in the turbidity spectrophotometrically at 600 nm every 30 min. Autolysis was also analyzed using a zymographic procedure
as described previously (Singh et al., 2008). The total autolysins were extracted after bead beating bacterial cells in 0.25 M phosphate buffer (pH 7.2) using a BioSpec Mini-Beadbeater after growth in PYK to an OD600 nm=0.7. Purified His6–LytM, extracts from E. coli cells overexpressing selleck chemicals llc His6–LytM and an S. aureus bead-beated cell-free extract was analyzed for the presence of autolysins in a zymographic method using autoclaved S. aureus 8325-4 cells as described previously (Singh et al., 2008). To construct a mutation, lytM upstream and downstream flanking regions were PCR amplified and sandwiched with a tetracycline resistance cassette in plasmid
pTZ18R. This construct was used to replace the wild-type lytM gene in the S. aureus chromosome by double homologous recombination. This mutant represents a deletion of 706 nt of the 966 nt lytM gene. In PCR assays, primers P9 and P10 amplified an ∼1.0 kb lytM region when the genomic DNA from the wild-type S. aureus was used as the template (Fig. 1, lane 1) as compared with an ∼2.5 kb amplicon when genomic DNA from the lytM mutant strain was used as a template (Fig. 1, lane 2). The mutation in the lytM gene was also confirmed by Southern blot analysis (data not shown). The deletion of LytM was investigated for any impact on the growth of S. aureus in TSB or in modified TSB to
impose stresses such as acidic stress (pH 5.5), alkaline stress (pH 9.0) or salt stress (TSB added with additional 1.5 M NaCl). No growth defect was observed whether the lytM mutants used were in S. aureus strain SH1000 or 8325-4 (data not shown). Surprisingly, the presence of oxacillin led to increased Clostridium perfringens alpha toxin lysis of mid-log-phase lytM mutant cells compared with a culture of wild-type S. aureus 8325-4 cells under identical conditions (Fig. 2). To verify whether it was indeed the lack of a functional LytM that is responsible for oxacillin-induced lysis, the mutant was complemented with the lytM gene under its own promoter in trans on plasmid pCU1. As evident in Fig. 2, the level of resistance to oxacillin-induced lysis was restored in the complemented strain. Expression of lytM was monitored using the lytM promoter–lacZ fusion in S. aureus SH1000.