Figure 1.

The null Fis mutation decreased the antimicrobial activity of D. zeae strain EC1. (A) Quantitative analysis of zeamine yield of wild-type strain EC1 and its derivative strains. The antimicrobial activity bioassay plates were prepared by pouring 15 mL of LB agar medium into the 120 × 120 mm plates, and then overlaid with 20 mL of 1% agarose containing 1.0 × 10⁸ cells of fresh E. coli harboring pBBR1MCS4 plasmid. Wells of 5 mm in diameter were punched after solidification. Overnight bacterial cultures were grown in LS5 medium to OD₆₀₀ at around 1.4, which was centrifuged twice at 12,000 rpm for 10 min, and 20 μl of the supernatants were added into the wells. The plates were incubated at 37 °C for 10 h. The antimicrobial activity was determined by measuring the radius of the visible clear zone surrounding the well. The concentration of zeamines was determined by this formula: zeamines (unit) = 0.5484e^0.886x (R² = 0.9957), X is the radius in millimeters of the inhibition zone surrounding the well. (B) Plate assay of the antimicrobial activities of wild-type strain EC1 and its derivative strains. The photograph was taken after 12 h of incubation at 37 °C. Final results of fis mutant were normalized to that of the wild-type EC1, which was set to a value of 100%, for easy comparison. Experiments were repeated at least three times in triplicates. Symbol: ***P < 0.0001 (Student’s t-test).