Regulation of the mhp Cluster Responsible for 3-(3-Hydroxyphenyl)propionic Acid Degradation in Escherichia coli

Abstract

The mhp gene cluster from Escherichia coli constitutes a model system to study bacterial degradation of 3-(3-hydroxyphenyl)propionic acid (3HPP). In this work the regulation of the inducible mhp catabolic genes has been studied by genetic and biochemical approaches. The Pr and Pa promoters, which control the expression of the divergently transcribed mhpR regulatory gene and mhp catabolic genes, respectively, show a peculiar arrangement leading to transcripts that are complementary at their 5'-ends. By using Pr-lacZ and Pa-lacZ translational fusions and gel retardation assays, we have shown that the mhpR gene product behaves as a 3HPP-dependent activator of the Pa promoter, being the expression from Pr constitutive and MhpR-independent. DNase I footprinting experiments and mutational analysis mapped an MhpR-protected region, centered at position -58 with respect to the Pa transcription start site, which is indispensable for MhpR binding and in vivo activation of the Pa promoter. Superimposed in the specific MhpR-mediated regulation of the Pa promoter, we have observed a strict catabolite repression control carried out by the cAMP receptor protein (CRP) that allows expression of the mhp catabolic genes when the preferred carbon source (glucose) is not available and 3HPP is present in the medium. Gel retardation assays revealed that the specific activator, MhpR, is essential for the binding of the second activator, CRP, to the Pa promoter. Such peculiar synergistic transcription activation has not yet been observed in other aromatic catabolic pathways, and the MhpR activator becomes the first member of the IclR family of transcriptional regulators that is indispensable for recruiting CRP to the target promoter.