FIG 6.

Induced overproduction of the staphylococcal antigen IsaA in the parental strain 168 and midiBacillus. To allow induction of IsaA expression from plasmid pRAG3::isaA with subtilin, the spaRK genes were introduced in the amyE locus of the midiBacillus and 168 strains. Culture samples were collected 2 h after induction with subtilin. At the same time, samples were withdrawn from parallel noninduced cultures. Cells were separated from the growth medium by centrifugation, and proteins in the respective fractions were analyzed by LDS-PAGE and Western blotting with the IsaA-specific monoclonal antibody 1D9. The cytoplasmic marker protein for cell lysis TrxA was detected with a specific polyclonal antibody. The positions of precursor and mature forms of IsaA and the TrxA protein are marked with arrowheads. Of note, precursor and mature forms of IsaA are distinguished based on the fact that the designated precursor form is exclusively identified in the cell fraction, has a lower mobility on LDS-PAGE, and shows a deduced M_w difference with the secreted mature protein that is consistent with the presence of a signal peptide.