FIG. 5.
Identification of a KIX domain mutant that binds a KID helix αB mutant. (A) Schematic of the reporter system used for the carbenicillin selection. The promoter region containing the λcI binding site (OR2) as well as the α and cI fusion proteins (here indicated as α-X and cI-Y) are identical to those used in the original reporter system (Fig. 1). In this case, however, the bla gene is inserted directly downstream of the promoter region. If the protein domains X and Y interact, the bacteria harboring these two fusion proteins will express the bla gene at a higher level and will be resistant to higher levels of carbenicillin. Because the lacZ gene is expressed cocistronically with the bla gene, these bacteria will also contain increased levels of β-Gal activity. (B) PKA-induced KID-KIX interaction confers carbenicillin resistance on host E. coli. The bacterial reporter strain US3F′3.1 was cotransformed with an expression vector encoding the wild-type α-KID fusion protein and a second vector encoding the wild-type cI-KIX fusion protein either together with PKA (solid squares) or without PKA (open squares). The graph shows the number of bacterial colonies remaining on an LB agar plate at different concentrations of carbenicillin (in the presence of 100 μM IPTG). (C) A library of cI-KIX expression vectors containing the randomly mutagenized KIX domain was transformed into a pooled mix of bacterial reporter strain US3F′3.1 cells containing 1 of 11 different α-KID mutants bearing substitutions in helix αB (see Materials and Methods). The cI-KIX expression vector also encoded PKA. These bacteria were then plated on carbenicillin-containing plates, and 24 colonies that grew were picked as potential positives. Plasmid DNA was then isolated from 14 of these 24 colonies and used to retransform strain US3F′3.1, and the KID-KIX interaction was assessed by β-Gal assay. Also included as controls in the β-Gal assay were US3F′3.1 bacteria transformed with both wild-type α-KID and wild-type cI-KIX in either the presence (+) or absence (−) of PKA.