Bacillus subtilis 168 Cell Culture

Cell suspensions of Bacillus subtilis 168 were prepared essentially as previously described (Nickels et al., 2017b (link)). Strain BKE32840 (ΔyusL) was obtained from the Bacillus Genetic Stock Center (The Ohio State University, Columbus, OH, United States). The culture medium used for producing samples was M9 minimal medium containing 0.4% (w/v) glucose and supplemented with 50 mg/L of L-tryptophan (Harwood and Cutting, 1990 ). Solid media were prepared by the addition of 1.5% Noble Agar (Difco). Erythromycin was added to 0.5 μg/mL for routine maintenance of BKE32840. Broth cultures were incubated at 37°C with shaking at 250 rpm. FA feeding experiments included cultures supplemented with 8 mg/L each of a15:0 and n16:0 from 25 mg/mL stock solutions in ethanol, along with 10 g/L of FA-free BSA. Cerulenin (Alfa Aesar) was added to a final concentration of 50 μg/mL. Non-fed cultures were grown in M9 minimal medium with 10 g/L BSA but without FAs or Cerulenin. Cells were harvested at mid-log phase (∼0.8 OD₆₀₀) by centrifugation at 6,000 × g for 15 min and washed three times in sterile phosphate buffered saline (pH 7.4).