FIG 8.

B. subtilis subsp. natto grows and secretes proteases under anaerobic conditions. (A) B. subtilis subsp. natto was cultured alone or together with E. faecalis donors and recipients on SGN (Schaeffer’s sporulation medium supplemented with glucose and nitrate) agar supplemented with skim milk. The amount of secreted protease was estimated from the clear zone that formed around the filter membrane. (B) Four-hour in vitro conjugation assays with exogenously added anaerobically cultured B. subtilis subsp. natto supernatant (SGN broth, 48 h) or B. subtilis subsp. natto (coculture). For bacterial coculture experiments, the cultures of B. subtilis subsp. natto were grown anaerobically in SGN broth to the early exponential phase and diluted according to the OD₆₀₀. Next, the cultures were added to E. faecalis mating cultures and allowed to grow for 4 h. The entire culture was then plated on selective medium for E. faecalis transconjugants and donors. Fresh SGN broth was added to mating cultures to serve as the negative control group. The y axis indicates the T/D ratio. The data are presented as the means ± SDs (n = 3). *, P < 0.05 compared with negative control (Duncan’s multiple-range tests).