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. 2005 Jul;187(14):4830–4843. doi: 10.1128/JB.187.14.4830-4843.2005

FIG. 3.

FIG. 3.

PrgY/TraB functional assays in pCF10 and pAD1 backgrounds. (A) prgY or traB was cloned into pMSP3545S and coexpressed with the indicated plasmid in E. faecalis strain OG1RF. The clumpy phenotype is represented by strains harboring the pMSP3545S vector control (bottom) and indicates constitutive induction by endogenous pheromone. Cultures were grown overnight with shaking in THB with 25 ng nisin/ml for induction of protein expression. (B) Western blot assay showing cross-reactivity of the TraB protein from pPD1 (P) and pAD1 (A) with the PrgY polyclonal antibody. The TraB proteins were expressed from pMSP3545S in strain OG1RF(pCF389). Extracts from OG1RF(pCF389/pMSP3545S-1) expressing wild-type prgY (+) or from strain OG1RF(pCF389/pMSP3545S) carrying the vector (−) were run for comparison. The arrow at 43 kDa corresponds to the expected size of PrgY, and the band below is a nonspecific cross-reacting band. Overnight cultures were diluted 1:5 in fresh medium plus 25 ng nisin/ml and grown 2.5 h before harvesting.