Figure 2.

Induced overproduction of staphylococcal proteins in B. subtilis 168 and PG10. The spaRK genes were introduced in the amyE locus of B. subtilis 168 and PG10 to allow subtilin-inducible expression of reporter proteins with the aid of the spaS promoter on plasmids pRAG3::chp (SP_xynA), pRAG1::scn (SP_amyQ), pRAG3::isaA (SP_xynA) and pRAG3::nuc (SP_xynA). Protein expression was induced with subtilin and culture samples were collected 2 h post induction. (a) The cellular (cells) and extracellular (medium) levels of the expressed staphylococcal proteins were assessed by Western blotting with an anti-his6 antibody to detect CHIPS, the monoclonal antibody 6D4 against SCIN, or the monoclonal antibody 1D9 against IsaA. (b) The subcellular localization of SCIN produced in B. subtilis 168 was assessed by fractionation. As a negative control, noninduced cells were used. TrxA and EfeM were used as markers for cytoplasmic and membrane-bound proteins, respectively. (c) CHIPS, SCIN, IsaA, and Nuc were produced and secreted by the miniBacillus strain PG10. Precursor forms of CHIPS, SCIN, IsaA and Nuc, and full-size TrxA and EfeM are marked with filled arrow heads; mature forms of CHIPS, SCIN, IsaA, and Nuc are marked with open arrow heads.