FIG. 2.

Inducible expression of functional PrgY in E. faecalis. (A) PrgY was expressed from the nisin-inducible vector pMSP3545S-1, whose map is depicted. (B) PrgY expression in whole-cell extracts was analyzed by Western blotting with a polyclonal anti-PrgY antibody. PrgY expression from E. faecalis strain OG1RF harboring pCF10 (lane 1) or pMSP3545S-1 induced with nisin (lane 2) or not induced with nisin (lane 3) and OG1RF harboring the pCF10-derived prgY mutant pCF389 (lane 4). The arrow at 43 kDa corresponds to the expected size of PrgY. (C) Clumping assay to assess PrgY function in trans. Strains induced for plasmid transfer express Asc10 and clump in liquid culture (see text), which causes uneven settling at the bottom of the well. This is depicted in wells 2 and 3 (compare with nonclumpy well 1). Strains shown in wells 1 to 5 are OG1RF(pCF10) (well 1), OG1RF(pCF10) induced with cCF10 (well 2), the constitutively clumpy prgY mutant OG1RF(pCF389) (see text) (well 3), and OG1RF(pCF389/pMSP3545S-1) plus nisin for expression of PrgY (well 4) or without nisin (well 5). Cultures were grown with shaking overnight in THB or in THB with synthetic cCF10 (10 ng/ml) or nisin (25 ng/ml). (D) PrgY-mediated trans-acting regulation of Asc10 expression. Asc10 expression was measured by immunoblot analysis of whole-cell extracts spotted onto a membrane and detected with a polyclonal anti-Asc10 antibody. The cCF10 concentration used to treat each sample is indicated above each sample spot, and the strain is indicated below. pMSP3545S is the vector control, and PrgY was expressed from pMSP3545S-1. All strains were treated with the indicated cCF10 concentration and 5 ng nisin/ml for 6 h following 1:10 dilution of an overnight culture into fresh medium. Cell extracts were washed twice before harvesting. An equivalent amount of protein was spotted for each sample.