FIG 2.
Effect of bacitracin treatment on pilus gene transcript levels. (A) FCT region in serotype M3 GAS. The spectinomycin resistance gene, aad9 (gray block), was used to construct in-frame deletions of the pilus backbone protein (tee3, spyM3_0100). (B) Growth curves for MGAS10870 and MGAS10870 liaSR135G in rich medium with and without bacitracin at 1.0 μg/ml. Bacitracin was added when the OD600 was 0.5, and cells were harvested 60 min after that for RNA extraction and quantitative real-time PCR. (C) Quantitative real-time PCR for determination of the transcript levels of pilus genes (cbp and tee3), pilus gene regulators (nra and msmR), and spxA2. *, P < 0.05 (t test, unpaired, Welch's correction) compared to those in parental strain MGAS10870. (D) Western immunoblotting for Tee3 in the parental invasive strain (MGAS10870), an isogenic carrier mutant, and derived pilus-negative mutants. Cells were treated with bacitracin as described in the legend to panel B, proteins were extracted, and equal amounts of purified cell wall and secreted proteins were probed with an affinity-purified antibody to Tee3 as described in Materials and Methods.