FIG 4.

B. subtilis subsp. natto supernatant affects the activity of cCF10. (A) Modified E. faecalis mating experiment. The SCS of B. subtilis subsp. natto was first treated separately with donor cells, recipient cells, or pheromone cCF10 at 37°C for 1 h. Next, donor cells were incubated with cCF10 at 37°C for 30 min and then mixed with recipient cells for one round of conjugation (10 min). Through this method, we were able to clarify the factor that is actually affected by the SCS of B. subtilis subsp. natto. (B) T/D ratios after separate treatment of pheromone cCF10, recipient cells, or donor cells with B. subtilis subsp. natto SCS. The y axis indicates the T/D ratio. Ctrl (control), not treated with SCS, P+N: pheromone cCF10 treated with B. subtilis subsp. natto SCS; R+N, recipient cells treated with B. subtilis subsp. natto SCS; D+N, donor cells treated with B. subtilis subsp. natto SCS. The data are presented as the means ± SDs (n = 3). Values with different uppercase letters were significantly different according to Duncan’s multiple-range tests (P < 0.05). (C) The effect of B. subtilis subsp. natto SCS on cCF10 increased over time. Donor cells were induced with cCF10 that was first treated with B. subtilis subsp. natto SCS for 0, 1, 2, and 4 h. Then, recipient cells were added and allowed one round of conjugation. The y axis indicates the T/D ratio. Ctrl (control), not treated with SCS; P+N, pheromone cCF10 treated with B. subtilis subsp. natto SCS. The data are presented as the means ± SDs (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001 compared with each negative control group (Student’s t test).