B. subtilis strain 168 (trpC2) was precultured at 30 ℃ for 24 h in NG medium supplemented with 50 mcg of tryptophan per ml, and then 0.1 ml of the resulting culture was inoculated into 10 ml of fresh NG medium, followed by incubation under shaking at 30℃. The titers of the heat-resistant spores were determined by heating the cultures for 10 min at 80 ℃ and then plating them onto an NG agar plate.

The production of bacilysocin was completely blocked when the ytpA gene, which encodes a protein homologous to lysophospholipase, was disrupted, but blockage of the ytpA gene did not significantly affect growth. Sporulation was also impaired, with a 10-fold reduction in heat-resistant spore titers being detected. Since the ytpA disruptant actually lacked phospholipase activity, we propose that the YtpA protein functions as an enzyme for the biosynthesis of bacilysocin.