The Escherichia coli C homoprotocatechuate degradative operon: hpc gene order, direction of transcription and control of expression

Abstract

Homoprotocatechuate (HPC; 3,4-dihydroxyphenylacetate) is catabolized to Krebs cycle intermediates via extradiol (meta-) cleavage and the necessary enzymes are chromosomally encoded in a variety of bacteria. Based on an analysis of the cloned pathway genes, the Escherichia coli C hpc gene cluster was thought to be arranged in two gene blocks transcribed from a central, divergent, operator/promoter region, which was negatively regulated by the Hpc repressor. By a variety of techniques including expression of cloned hpc genes in pUC18/19 vectors, unidirectional deletion subcloning, hybridization studies and nucleotide sequencing it has now been shown that the hpc pathway structural genes are transcribed in one direction. These experiments have also indicated that a decarboxylase and an isomerase of the pathway are encoded by a single gene (hpcE) and have established the exact structural gene order as hpcRphpcECBDGH. The position of the putative regulatory gene, hpcR, is upstream of the first structural gene (hpcE) for the Hpc pathway enzymes. The deduced open reading frame for the Hpc repressor specifies a protein of 148 amino acids with a subunit molecular weight of 17 kDa. The region between hpcR and the first gene for the pathway enzymes has a sequence similar to that for catabolite activator protein (CAP) binding. This region is immediately upstream of a promoter for the pathway structural genes, which has been identified by transcript mapping.

Molecular characterization of the 4-hydroxyphenylacetate catabolic pathway of Escherichia coli W: engineering a mobile aromatic degradative cluster

“…It was surprising that the 5-oxo-pent-3-ene-1,2,5-tricarboxylic acid decarboxylase and HHDD isomerase activities of E. coli C were not found associated in the earliest biochemical analyses of HpcE (25). In fact, this finding allowed the isolation of the unstable product HHDD (enol form), which was further used to assay the isomerase and hydratase activities of HpcE and HpcG, respectively (17,20,25,42,43). Hence, we believe that there are not conclusive data to assume that 2-oxo-hept-3-ene-1,7-dioic acid is the substrate of HpcG hydratase (HpaH in E. coli W), and we propose that the transformation of HHDD into 2,4-dihydroxy-hept-2-ene-1,7-dioic …”

“…The hpaR gene encodes a protein of 17,235 Da, identical to the putative regulatory hpcR gene of the HPC meta-cleavage pathway of E. coli C. HpcR has been proposed to function as a repressor and appears to be unrelated to any other regulator (43).…”

“…The HPC degradative operon of E. coli C has been partially cloned (25,43), and some of its products have been characterized (16-18, 40-42, 44, 48). In addition, we have previously demonstrated that the first step in the 4-HPA degradation in E. coli W, i.e., the formation of HPC, is catalyzed by a twocomponent aromatic hydroxylase (38,39).…”

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

“…CRP Mediates a Superimposed Level of Regulation on the Pa Promoter-It is known that some aromatic catabolic pathways in E. coli, such as the paa, hca, hpa, and mao-encoded pathways responsible for the catabolism of phenylacetate, phenylpropionate, 4-hydroxyphenylacetate, and 2-phenylethylamine, respectively, are repressed by the presence of glucose in the culture medium (26,27,(42)(43)(44)(45). To check whether the mhp cluster is also under catabolite repression control, E. coli AFM-CRAL cells were grown in glycerol-or glucose-containing minimal medium in the presence of 3HPP.…”

“…The Pg promoter of the hpa cluster for 3,4-dihydroxyphenylacetic acid degradation and the Pa and Pz promoters of the paa cluster for phenylacetic acid degradation in E. coli can also be considered class III CRP-dependent promoters because they require both CRP and the integration host factor protein as transcriptional activators (27,44). However, all these promoters show different architectures, and whereas the Pa promoter of the mhp cluster requires a pathway-specific activator (MhpR), promoters from the hpa and paa clusters are controlled by the cognate pathway-specific HpaR and PaaX repressors, respectively (27,42,44). This suggests that the molecular mechanisms of transcriptional regulation of the aromatic catabolic clusters in E. coli are highly diverse, and they constitute a useful model system to unravel complex regulatory circuits that involve specific and overimposed levels of regulation.…”