CHARACTERIZATION OF THE NEGATIVE ELEMENTS INVOLVED IN SILENCING THE BGL OPERON OF ESCHERICHIA-COLI - POSSIBLE ROLES FOR DNA GYRASE, H-NS, AND CRP-CAMP IN REGULATION

The bgl operon of Escherichia coil is rendered cryptic and uninducible in wild-type cells by the presence of DNA structural elements that ne gatively regulate transcription. We have carried out a detailed analys is of the sequences implicated in negative regulation. Finestructure d eletion analysis of the upstream sequences showed the presence of at l east two elements involved in silencing the promoter. Chemical probing of genomic DNA in vivo showed that a region of dyad symmetry, present upstream of the promoter, is hypersensitive to KMnO4. The hypersensit ive region detected corresponds to the potential cruciform structure i mplicated earlier in negative regulation. Enhancement of transcription from the wild-type promoter, observed in the presence of the gyrase i nhibitor novobiocin, was absent in a mutant that carried point mutatio ns in the inverted repeat. This observation suggests that the activati on seen in a gyrase mutant is mediated by destabilization of the cruci form because of reduced supercoiling. Deletion of sequences downstream of the potential cruciform also resulted in an increase in transcript ion, indicating the presence of a second regulatory element. Measureme nt of transcription from the bgl promoter carrying the deletion, in a strain that has a mutation in the hns gene, indicated that this region is likely to be involved in binding to H-NS or a protein regulated by H-NS, which acts as a non-specific repressor. We also provide evidenc e which suggests that transcriptional activation by mutations at the c AMP receptor protein (CRP)-binding site is mediated partly by antagoni zation of the negative effect of H-NS by CRP-cAMP as a result of its i ncreased affinity for the mutant site.