FIG. 4.

Bacterial LLO production requires anteiso-BCFA. Abundance (A) and activity (B and C) of LLO in the supernatant of overnight bacterial cultures grown in BHI medium with or without 2MB. (A) Bacterial culture supernatant fractions were first normalized by culture optical density. Proteins were extracted from the supernatant by TCA precipitation and analyzed by SDS-PAGE and immunoblotting with an anti-LLO antibody. Recombinant LLO (rLLO) was used as a positive control. Supernatant of the Δhly mutant (Δhly) was used as a negative control. (B and C) Hemolytic assays were performed by mixing sheep red blood cells with serially diluted supernatant samples. Hemolytic activity measured by absorbance at 541 nm was also normalized by culture optical density (OD). Averages of triplicates from a single experiment are plotted, with error bars representing standard deviations. The data shown here are representative of at least 3 independent experiments.