FIG 8.

Signaling through GraS contributes to S. aureus USA300 production of secreted proteases during growth at acidic pH. (A) SDS-PAGE profile of secreted proteins (top) and zymogram for detection of SspA serine protease (bottom) in culture supernatants of USA300 and isogenic ΔgraS or protease-deficient Δpro mutants after growth for 20 h in TSB or TSB (pH 5.5). Arrows indicate the positions of proteins that represent the signature protein profile of the staphylococcal proteolytic cascade, including the precursor of glycerol ester hydrolase proGeh, mature Geh lipase, mature SspA serine protease, and SspB cysteine protease. (B) Same as described for panel A, with strains USA300 and ΔgraS complemented with pgraS or pgraS3^D>G. Expression from the P_xyl/tetO promoter of pACL2073 was induced using 120 ng/ml aTc. For SDS-PAGE profiles of secreted proteins, TCA-precipitated protein equivalent to 3.0 OD₆₀₀ units of culture supernatant were applied to each lane, while for zymogram analyses, a volume of culture supernatant equivalent to 0.075 OD₆₀₀ unit was applied to each lane.