FIG 6.

The nuclease activity of EssD is dispensable for secretion. (A) S. aureus strain USA300 (lanes 1) and the isogenic essD::erm (lanes 2) or essD^L546P (lanes 3) variant were grown at 37°C in TSB supplemented with serum. Cultures were spun to separate the medium (MD) from intact cells that were subsequently lysed with lysostaphin to release all cellular content (cell). Proteins were TCA precipitated, suspended in sample buffer prior to separation by SDS-PAGE, and transferred to PVDF membranes for immunoblot analyses with anti-EssD^N, anti-EsxC, anti-L6, or anti-HLA polyclonal sera. Numbers to the left indicate the mobility of molecular mass markers. (B) Cultures of S. aureus strain USA300 and the isogenic essD::erm, essD^L546P, ΔessB, and ΔessB/pessB variants were grown at 37°C in TSB supplemented with serum and appropriate antibiotics. The supernatant of these cultures was used to assay for nuclease activity using plasmid DNA. Sterile TSB was used as a control (sterile). Following incubation, reaction products were separated on an agarose gel and visualized with a UV transilluminator for image acquisition. Lane M, molecular mass markers.