FIG. 5.

Bacterial two-hybrid assay: characterization of σ²⁸_Ct substitution that affects its interaction with the β-flap. (A) Diagram showing how interaction of the σ²⁸_Ct region 4 (in pBRα-σ²⁸_Ct) with the β-flap (in pACλcI-β-flap_Ct or pACλcI-β-flap) activates transcription from the test promoter plac O_R2-62 located on the chromosome in KS1. (B) Effects of substitution in the σ moiety of the α-σ²⁸_Ct chimera on transcription from plac O_R2-62 in the presence of the chimera λcI-β-flap_Ct. (C) Effects of substitutions in the σ moiety of the α-σ²⁸_Ct chimera on transcription from plac O_R2-62 in the presence of the chimera λcI-β-flap. KS1 cells harboring two compatible plasmids, which direct the synthesis of α-σ²⁸_Ct or derivatives and λcI-β-flap_Ct or pACλcI-β-flap as indicated, were grown in the presence of carbencillin, chloramphenicol, and kanamycin. Protein expression was induced by treatment with different concentrations of IPTG for 1 hour when cultures reached an optical density at 600 nm of 0.3. β-Galactosidase activity was measured in duplicate on at least three independent occasions. Values shown are the averages from one experiment.