A cyclic AMP receptor protein mutant that constitutively activates an Escherichia coli promoter disrupted by an IS5 insertion

Previously an Escherichia coli mutant that had acquired the ability to grow on propanediol as the sole carbon and energy source was isolated. This phe notype is the result of the constitutive expression of the fucO gene (in th e fucAO operon), which encodes one of the enzymes in the fucose metabolic p athway. The mutant was found to bear an IS5 insertion in the intergenic reg ulatory region between the divergently oriented fucAO and fucPIK operons, T hough expression of the fucAO operon was constitutive, the fucPIK operon be came noninducible such that the mutant could no longer grow on fucose. A fu cose-positive revertant which was found to contain a suppressor mutation in the crp gene was selected. Here we identify this crp mutation, which resul ts in a single amino acid substitution (K52N) that has been proposed previo usly to uncover a cryptic activating region in the cyclic AMP receptor prot ein (CRP), We show that the mutant CRP constitutively activates transcripti on from both the ISS-disrupted and the wild-type fucPIK promoters, and we i dentify the CRP-binding site that is required for this activity. Our result s show that the fucPIK promoter, a complex promoter which ordinarily depend s on both CRP and the fucose-specific regulator FucR for its activation, ca n be activated in the absence of FucR by a mutant CRP that uses three, rath er than two, activating regions to contact RNA polymerase. For the ISS-disr upted promoter, which retains a single CRP-binding site, the additional act ivating region of the mutant CRP evidently compensates for the lack of upst ream regulatory sequences.