FIG. 3.

Biological activities associated with legiobactin. (A) 10⁴ CFU of wild-type strain 130b were inoculated into non-iron-supplemented BCYE agar (leftmost plate) or non-iron-supplemented BCYE agar containing 600 μM DIP (four rightmost plates), and a center well was filled, as indicated, with 50 μl of either deferrated CDM, 10 mM FeCl₃, or the <3-kDa fraction from a CAS-negative (i.e., 194 μM DFX equivalents) or CAS-positive (i.e., 1,100 μM DFX equivalents) supernatant of strain 130b. After 21 days of incubation at room temperature, the growth of the bacteria was recorded. (B) We plated 10⁵ CFU of feoB mutant bacteria onto the surface of standard (i.e., iron-supplemented) BCYE agar (leftmost plate) or non-iron-supplemented BCYE agar (four rightmost plates), and a center well was filled, as indicated above, with either deferrated CDM, FeCl₃, or a CAS-negative or CAS-positive supernatant fraction. After 5 days of incubation at 37°C, the growth of the bacteria was recorded. The results presented for each type of bioassay are representative of at least three independent experiments.