Figure 5.

Identification of the EfCIV583 inducer. (a) Affinity chromatography of p1 EF0309 using His6-Rpr_EfCIV583. E. coli strain expressing the EF0309/His6-Rpr_EfCIV583 pair was IPTG (isopropyl β-d-1-thiogalactopyranoside)-induced and, after disruption of the cells, the expressed proteins were applied to a Ni2+ agarose column and eluted. The presence of the different proteins was monitored in the load (lanes E), flow-through, wash and elute fractions (P) by Coomassie staining. The identity of the purified proteins was determined by in-gel enzymatic digestion and mass fingerprinting. It is assumed that the presence of Xis in the eluate represents an Rpr–Xis complex. (b) MC (1 μg ml⁻¹) was added to a culture of E. faecalis JP11028 (EfCIV583/p1-positive) or E. faecalis JP13142 (JP11028 p1Δxis), followed by incubation at 32 °C. Samples were removed at the indicated time points and used to prepare minilysates, which were resolved on a 0.7% agarose gel, and Southern blotted with an EfCIV583 probe. (c) EfCIV583 interference with phage reproduction. Approximately 10⁸ bacteria (carrying or not the EfCIV583 element) were infected with 400 plaque forming units (PFU) of phage φ1 or phage p1 Δxis, plated on phage bottom agar and incubated 24 h at 32 °C. Plates were stained with 0.1% triphenyl tetrazolium chloride in TSB (tryptic soy broth) media and photographed. CCC, closed circular form.