Transcription analyses Prewarmed LB broth was inoculated with an overnight selleck culture to an OD600 0.05 and incubated at 37°C. Cells were harvested at OD600 0.2, 0.5, 1, 3 and 6, centrifuged for 5 min at 20’000 g and 4°C. Cells were immediately snap frozen in liquid nitrogen and stored at – 80°C. Total RNA was extracted as described in [60].

Seven μg RNA was separated in a 1.5% agarose gel containing 20 mM guanidine thiocyanate in 1× TBE [61]. RNA was transferred onto a positively charged nylon membrane (Roche) using the downward capillary transfer method. The blots were hybridized with specific digoxigenin (DIG)-labeled DNA probes (Roche). Primers used are listed in Additional file 2 Table S1. Analyses of subcellular protein fractions Cells were sampled as described for transcription analyses and culture supernatant was collected as described for zymographic analysis. Cells were fractionated basically according to Schneewind et al. [38]. Briefly, cells GSK3326595 mw were digested in SMM buffer supplemented with each 72 μg/ml lysostaphin and lysozyme, 36 μg/ml DNase and 2 mM PMSF. Protoplasts AR-13324 order were

separated from the cell wall containing supernatant by centrifugation for 4 min at 16’000 g. Protoplasts were resuspended in membrane buffer (0.1 M NaCl, 0.1 M Tris-HCl, 0.01 MgCl2 pH 7.5) and lysed by three cycles of freezing in liquid nitrogen/thawing at 20°C. Cell membranes were separated from the cytoplasm by centrifugation for 30 min at 20’000 g and 4°C. Membrane pellets were Cell press solubilized in

buffer B (25 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM MgCl2, 30% glycerol) supplemented with 1% Triton X-100 and 0.5% N-lauroylsarcosine, by gently mixing end-over-end at 4°C. Where necessary, protein fractions were concentrated with Amicon Ultra-15, -4 or -0.5 centrifugal filter units (MWCO 10 kDa, Millipore). Cell fractions were kept at – 20°C. Five μg of protein was separated by SDS-10% PAGEs and either stained with Coomassie Imperial™ Protein Stain (Thermo Scientific) or blotted onto a PVDF-membrane (Immobilon-P, Millipore). For detection of SpA, membranes were blocked with 5% milk powder in PBS and then incubated with goat anti-human IgA conjugated with horseradish peroxidase (HRP, Sigma-Aldrich), 1:10’000 in 0.5% milk powder/PBS, 0.05% Tween 20 (AppliChem). After washing three times with PBS pH 7.4, HRP was detected with SuperSignal West Pico Chemiluminescent substrate (Thermo Scientific). PBP2a was detected as described in [28]. For detection of PBP4, membranes were blocked with 5% milk powder in PBS. Membranes were pre-incubated with 40 μg/ml human IgG in 0.5% milk powder/PBS. Rabbit anti-PBP4 antibodies (1:2000, [62]) and 0.05% Tween 20 were then added. After incubation for 1 h, membranes were washed three times with PBS before addition of goat anti-rabbit IgG-HRP (Jackson ImmunoResearch), 1:10’000 in 0.5% milk powder/PBS/0.05% Tween 20. After washing three times with PBS, HRP was detected as described for SpA.