FIG. 2.

cCF10 pheromone suppresses growth of E. faecalis cells carrying the pCF10ΔprgU plasmid. A) Serial dilution plate assay. E. faecalis overnight cultures were diluted 1:100 in fresh BHI and incubated for 1 h at 37°C in the absence (−) or presence (+) of cCF10 (10 ng ml⁻¹). Tenfold serial dilutions were inoculated onto BHI medium and assessed for growth. Strains: OG1RF with pCF10 or ΔprgU (pCF10ΔprgU) alone, or additionally with the P₂₃ vector plasmid (pDL278P₂₃) or the P₂₃::prgU expression plasmid (denoted P₂₃::U; pMB11). B) Pheromone spot assay. Overnight cultures listed at the left in Panel A were diluted 1:100 in fresh BHI and incubated for 1 h at 37°C in the absence of pheromone. Cultures were spread on BHI media, allowed to dry, and cCF10 pheromone (10 ng ml⁻¹) was added to the center of the plate. Plates were incubated overnight at 37 °C and assessed for growth. cCF10 pheromone is solubilized in DMSO, which inhibits E. faecalis growth and causes small zones of clearance independently of cCF10-induced toxicity; for example, see spot assay for OG1RF(pCF10). C) Growth curve assay. Overnight cultures were diluted 1:50 in BHI and incubated for 1 h at 37 °C. The cells were normalized to an OD₆₀₀ of 0.1 and cultures were incubated in the absence or presence of cCF10 (10 ng ml⁻¹) at 37°C. Aliquots were removed at specified time points, 10 μl of 0.5 M EDTA was added to disaggregate the cells, and the OD₆₀₀ was measured.