FIG 2.

Bacterial two-hybrid assays confirm the interaction of adenylate cyclase and MshH with native EIIA^Glc and EIIA^Glc point mutants mimicking the phosphorylated and unphosphorylated states. (A and B) Interaction of EIIA^Glc fused to the N terminus of the DNA-binding protein Zif (EIIA^Glc-Zif) with adenylate cyclase (AC-ω) (A) or MshH (MshH-ω) (B) fused to the ω subunit of RNA polymerase in an E. coli strain encoding the lacZ gene preceded by a Zif binding site. (C and D) A similar experiment was performed to assess the interactions of adenylate cyclase (AC-ω) (C) or MshH (MshH-ω) (D) with EIIA^Glc point mutants in which an alanine (EIIA^GlcH91A-Zif) or an aspartate (EIIA^GlcH91D-Zif) was substituted for histidine 91 to mimic the unphosphorylated and phosphorylated states of EIIA^Glc, respectively. Controls, which are traced in gray for each experiment, include (i) a vector encoding Zif alone (Zif) combined with a vector encoding the protein of interest fused to ω, (ii) a vector encoding native EIIA^Glc or point mutants fused to Zif combined with a vector encoding ω alone (ω), and (iii) vectors encoding Zif and ω alone. Two experimental replicates were included in each trial, and several trials were performed. A representative trial is shown here.