Figure 2. vfr inactivation alters speB expression profile.

(A) Growth curve of indicated strains in THY broth. Samples were collected at early-exponential (EE, (A₆₀₀ ~ 0.3), mid-exponential (ME, A₆₀₀ ~ 0.6), late-exponential (LE, A₆₀₀ ~ 1.0) and stationary phase (SP, A₆₀₀ ~ 1.7) for transcript and protein analysis. Arrows indicated the sampling time points. (B) Transcript levels of speB, ropB and vfr in strain MGAS10870 measured by Taqman qRT-PCR. (C) speB transcript level in strains MGAS10870 and isogenic mutant strain Δvfr. For (B) and (C) duplicate biological replicates were grown on two separate occasions and analyzed in duplicate. Data graphed are mean ± standard deviation. (D) Representative western immunoblot analysis of SpeB in filtered growth media from strain MGAS10870 and isogenic mutant strain Δvfr. Each experiment was performed on three different occasions with essentially identical results. Growth media collected at indicated sampling points from strains MGAS10870 and Δvfr were analyzed using anti-SpeB polyclonal rabbit antibody and chemiluminescence. The bands corresponding to the mature form of SpeB are labeled (SpeBm) and indicated by an arrow. (E) Assay of SpeB protease activity by azocasein hydrolysis assay. Filtered growth media collected at indicated growth phases from strains MGAS10870 and Δvfr were assayed for secreted SpeB protease activity. Azocasein hydrolysis by SpeB was determined by measuring the absorption at A₃₆₀ nm.