, 2011). Luria–Bertani (LB) broth was used as the basic culture medium. Cells were precultured at 37 °C overnight with shaking (180 r.p.m.; BR-15: TAITEC, Tokyo, Japan). This culture (50 μL) was inoculated into 5 mL of LB at 37 °C with shaking (180 r.p.m.; BR-15). Logarithmic-phase cells were collected at an OD600 of 0.3. Cells from an overnight culture were harvested 15 h after inoculation from a glycerol
stock. To inhibit transcription/translation, cells were treated check details with 100 μg mL−1 rifampicin and 100 μg mL−1 chloramphenicol for 60 min prior to harvesting. Cells equivalent to 8 × 108 colony-forming units (CFU) were collected at the logarithmic or stationary phase, washed with PBS and suspended in high osmotic or acid solutions. The high osmotic solutions were 4 M NaCl, 4 M KCl and 20% raffinose. The acid solutions were 10 mM HCl (pH 2.0) and citrate-phosphate buffer (pH 2.6, 4.6 or 6.6) supplemented with 100 mM NaCl and 10 mM KCl. After incubation for 5, 15 or 60 min, the cells were washed with PBS and collected for subsequent viability testing (CFU counting) and thin-layer chromatography (TLC). For heat- or cold-shock treatment, cultures containing 8 × 108 CFU CH5424802 supplier were directly shifted to the
appropriate temperature. Lipid extraction and TLC were carried out as described previously (Tsai et al., 2011). Cells equivalent to 8 × 108 CFU were washed with PBS and resuspended in 200 μL of 2% NaCl. Lysostaphin was added to the cell suspension Cell Penetrating Peptide (final concentration 0.1 mg mL−1) and incubated at 37 °C for 3 min. The lysed cell suspension was then subjected to chloroform–methanol extraction. Lipids were dissolved in chloroform–methanol (2 : 1;
v/v), applied to silica TLC plates (Silica gel 60; Merck, Darmstadt, Germany) and developed with chloroform–methanol–acetic acid (65 : 25 : 10; v/v/v). The TLC plates were sprayed with 100 mg mL−1 CuSO4 containing 8% phosphoric acid and heated at 180 °C to detect phospholipids. A digital image was obtained by a scanner, and the signal intensities were quantified using Image J software (version 1.44p; NIH). The number of CL synthase genes varies among bacterial species (Supporting Information, Table S1). Staphylococcal cls1 (SA1155) and cls2 (SA1891) share higher levels of similarity with each other than with cls genes from other species. They were grouped with Bacillus subtilis cls (BSU36590) and Listeria monocytogenes lmo2503, but not with B. subtilis ywjE (BSU37190) and ywiE (BSU37240) or L. monocytogenes lmo0008 (Fig. S1). This indicates that the two staphylococcal cls genes were not acquired by horizontal gene transfer from different species. We found a single insertion/deletion (INDEL) site in the N-terminal region of Cls (Fig. S1). The INDEL in Cls2 is considered to be the ancestral type because it is shared with the Cls of other bacterial species.