FIG. 2.
PrsA1 shares no apparent functional overlap with PrsA2 in in vitro assays. All assays were conducted using the wild-type strain or ΔprsA2, ΔprsA1, ΔprsA1 ΔprsA2, or ΔprsA2 plus pPL2-PprsA2-prsA1 mutant strains. (A) Measurement of LLO-associated bacterial hemolytic activity. Dilutions of bacterial culture supernatants were assessed for their ability to lyse sheep's red blood cells (RBCs) in vitro. The reciprocal of the supernatant dilution that resulted in 50% lysis of RBCs (hemolytic units) was determined in a minimum of five independent experiments, each conducted in duplicate, and the average of results is shown. (B) Phospholipase activity as determined by the incubation of bacterial strains on egg yolk agar plates followed by the observation of a zone of opacity surrounding the bacterial streak. A representative image from one of five plates is shown. (C) Plaque formation in L2 fibroblast monolayers in the presence of gentamicin. At 72 h postinfection, plaques were measured using a micrometer and the plaque size for the wild type was set to 100%. Plaque assays were repeated at least three times, with results from a minimum of 20 plaques averaged per experiment. Statistical significance was determined using one-way analysis of variance with Tukey's multiple comparison test (*, P < 0.01; ***, P < 0.0001).