The cls1 mutant did not differ from the parental strain in its growth rate, survival at stationary phase, total CL accumulation, or L-form generation. However, our data indicate that the synthesis of CL by Cls1 helps the cls2

mutant to survive prolonged incubation under high-salt conditions (Figure 5E), suggesting that Cls1 has a specific function under stress conditions if Cls2 is unavailable. Future studies should examine the functional characteristics of these two CL synthases, including possible differences in their subcellular localizations. Conclusions Improved lipid extraction and molecular genetic analyses showed that both cls1 and this website cls2 participate in CL accumulation. The cls2 gene Capmatinib cost appears to serve a housekeeping function, while cls1 is active under stress conditions. Staphylococcus aureus can grow under conditions of high salinity without CL, but CL is required to survive prolonged high

salinity stress and to generate L-form variants. This CL-dependent survival helps to explain the success of S. aureus as a human pathogen and skin/mucus membrane commensal. Edoxaban Methods Bacterial strains and culture conditions The S. aureus strains used in this study are shown in Table 1. Luria-Bertani (LB) broth was the basic culture medium, and its NaCl content was modified as indicated. Cells were pre-cultured aerobically at 37°C overnight with shaking (180 rpm; BR-15; TAITEC, Tokyo, Japan).

Culture inoculate (200 μl) was added to 40 ml of LB containing 0.1% NaCl or 15% NaCl in a 200 ml Erlenmeyer flask and incubated at 37°C with shaking (230 rpm; BR-23UM; TAITEC). To achieve the 25% NaCl culture condition, 0.4 ml of an overnight culture was mixed with 2 ml of LB containing 30% NaCl, and the culture was incubated at 37°C with shaking (180 rpm; BR-15; TAITEC). When necessary, the pH was adjusted to 7.0 or 4.8, and the cells were harvested at exponential phase before any change in pH. The growth rate was measured spectrophotometrically as optical density at 600 nm (OD600). Anaerobic growth was C646 carried out at 37°C without shaking. Mutant isolation procedures used tryptic soy broth (TSB) or brain heart infusion (BHI) medium. Table 1 Bacterial strains, plasmids, and primers used in this study Strain or plasmid Relevant characteristics Source/reference S.