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Dual Bait-Compatible Bacterial Two-Hybrid System 293Fig. 1. The bacterial two-hybrid system. (A) Schematic showing the componentsof the bacterial two-hybrid system. A first protein, the bait, is fused to a DBD providedby λcI that in turn binds to a specific DNA-binding site positioned near a weak promoter.A second protein, the prey, is fused to the α-subunit of the E. coli RNAP. Interactionbetween bait and prey mediates the recruitment of RNAP to the weak promoter,thereby activating transcription of a downstream reporter gene(s). (B) Expression ofthe yeast HIS3 gene following bait–prey interaction can be selected for E. coli strainslacking a functional hisB gene (KJ1567 or Bacteriomatch). The bacterial aadA geneencodes for streptomycin and spectinomycin antibiotic resistance, and provides a secondselection. (C) The LacZ gene in the strain AG58(RP28) encodes β-galactosidase,which can be assayed either qualitatively on a plate-based assay, or quantitatively, asdiscussed in the text.