FIG 3.
Analysis of PltB-ArtB interactions. (A) Solvation energies of the interface calculated between monomers in the binding subunit are color coded to represent the interaction: PltB-PltB are shown in blue, ArtB-ArtB in red, and PltB-ArtB in purple. IA, interface area in Angstroms (Å); ΔG, solvation free energy gain upon formation of the interface (kcal/M). Negative ΔG values correspond to hydrophobic interfaces, and therefore a positive protein affinity. (B) Schematic of the method used to screen two-hybrid system clones. In the example shown, DNA fragments encoding the PltB polypeptide or C-terminal oligomerization region, as predicted from TT structure, were cloned at the 5′ or 3′ ends of regions encoding adenylate cyclase domain T18 or T25. All T18 or T25 clones were pooled into “banks” that were cotransformed into the cAMP-null (ΔcyaA) E. coli BTH101. Screening was performed by growing the transformants on LB agar supplemented with IPTG and X-Gal. Plasmid DNA was isolated from clones with positive interactions and was retransformed into E. coli BTH101 to exclude constitutively active CAP* mutants. Clones with confirmed interactions were submitted for Sanger sequencing to determine the identity of the interacting domains. (C) E. coli BTH101 two-hybrid system strains grown on LB agar supplemented with IPTG and X-Gal. Clones that showed positive interactions appear blue. Colored boxes show the fusion of either the interacting domain (_ID) or the full-length protein (_Full) for PltB, PltA, CdtB, and ArtB to either the CyaA-25 (shown in teal) or CyaA-18 subunits (shown in mauve) of the two-hybrid system. (D) Detection of interactions of ArtB and PltB in S. Javiana ΔcyaA harboring two-hybrid system plasmids. S. Javiana cells were grown for 24 h in M63 minimal medium with maltose as the sole carbon source, and absorbance was measured at 600 nm. Error bars indicate the standard deviations of the means from three independent experiments. Constructs with the C terminus are denoted with “ct.” *, P < 0.05 versus the negative control before (blue) and after (pink) Dunnett’s test for multiple comparisons adjustment; the PltBT25-ArtBT18 interaction was only marginally significant after multiple comparison corrections (corrected P value = 0.0973).