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

Abstract

Previously an Escherichia coli mutant that had acquired the ability to grow on propanediol as the sole carbon and energy source was isolated. This phenotype is the result of the constitutive expression of the fucO gene (in the fucAO operon), which encodes one of the enzymes in the fucose metabolic pathway. The mutant was found to bear an IS5 insertion in the intergenic regulatory region between the divergently oriented fucAO and fucPIK operons. Though expression of the fucAO operon was constitutive, the fucPIK operon became noninducible such that the mutant could no longer grow on fucose. A fucose-positive revertant which was found to contain a suppressor mutation in the crp gene was selected. Here we identify this crp mutation, which results in a single amino acid substitution (K52N) that has been proposed previously to uncover a cryptic activating region in the cyclic AMP receptor protein (CRP). We show that the mutant CRP constitutively activates transcription from both the IS5-disrupted and the wild-type fucPIK promoters, and we identify the CRP-binding site that is required for this activity. Our results show that the fucPIK promoter, a complex promoter which ordinarily depends on both CRP and the fucose-specific regulator FucR for its activation, can be activated in the absence of FucR by a mutant CRP that uses three, rather than two, activating regions to contact RNA polymerase. For the IS5-disrupted promoter, which retains a single CRP-binding site, the additional activating region of the mutant CRP evidently compensates for the lack of upstream regulatory sequences.

FIG. 1

The divergent fuc operons. Shown schematically is the intergenic regulatory region between the fucAO and fucPIK operons. The bent arrow indicates the start point of transcription from the fucPIK promoter (+1). Potential CRP-binding sites are indicated by the rectangles. The sequence of CRP site 1 (hatched rectangle) is shown, as are the three base pair substitutions that were introduced to inactivate this CRP-binding site (vertical arrows). The site of the 1.3-kb IS5 insertion is also indicated (inverted triangle).

FIG. 2

CRP-binding sites associated with wild-type (WT) and IS5-disrupted promoters. Shown are the results of a DNase I protection experiment performed with purified CRP. The reaction mixtures loaded in lanes 1 and 4 contained no CRP, that loaded in lane 2 contained 320 nM CRP dimer, and those loaded in lanes 3 and 5 contained 64 nM CRP dimer. CRP site 1 is present on both templates, whereas CRP site 2 is present on the wild-type template only (see text).

FIG. 3

Interactions between CRP and RNAP at a class I and a class II promoter. When bound at a class I promoter, CRP uses AR1 (small black semicircle) in the promoter-proximal subunit to contact the α-CTD. When bound at a class II promoter, wild-type CRP makes two contacts with RNAP by using AR1 in the promoter-distal subunit to contact the α-CTD and AR2 (small black rectangle) in the promoter-proximal subunit to contact the α-NTD. The CRP^K52N mutant possesses a third activating region, designated AR3 (small black triangle), that has been proposed to contact the ς subunit (see text).