3-Hydroxyphenylpropionate and phenylpropionate are synergistic activators of the MhpR transcriptional regulator from Escherichia coli

Abstract

The degradation of the aromatic compound phenylpropionate (PP) in Escherichia coli K-12 requires the activation of two different catabolic pathways coded by the hca and the mhp gene clusters involved in the mineralization of PP and 3-hydroxyphenylpropionate (3HPP), respectively. The compound 3-(2,3-dihydroxyphenyl)propionate (DHPP) is a common intermediate of both pathways which must be cleaved by the MhpB dioxygenase before entering into the primary cell metabolism. Therefore, the degradation of PP has to be controlled by both its specific regulator (HcaR) but also by the MhpR regulator of the mhp cluster. We have demonstrated that 3HPP and DHPP are the true and best activators of MhpR, whereas PP only induces no response. However, in vivo and in vitro transcription experiments have demonstrated that PP activates the MhpR regulator synergistically with the true inducers, representing the first case of such a peculiar synergistic effect described for a bacterial regulator. The three compounds enhanced the interaction of MhpR with its DNA operator in electrophoretic mobility shift assays. Inducer binding to MhpR is detected by circular dichroism and fluorescence spectroscopies. Fluorescence quenching measurements have revealed that the true inducers (3HPP and DHPP) and PP bind with similar affinities and independently to MhpR. This type of dual-metabolite synergy provides great potential for a rapid modulation of gene expression and represents an important feature of transcriptional control. The mhp regulatory system is an example of the high complexity achievable in prokaryotes.

Phenylpropanoic and phenylpropenoic acids and their hydroxylated derivatives are widely distributed in the environment, arising from digestion of aromatic amino acids or as breakdown products of lignin and other plant-derived phenylpropanoids and flavonoids. The bacterial catabolism of these aromatic compounds plays a key role in recycling of such carbon sources in the ecosystem (1, 2). Most Escherichia coli strains are able to degrade these compounds via a meta-fission pathway (3). A scheme of the biochemical pathway for the catabolism of 3-hydroxyphenylpropionate (3HPP)2 and 3-hydoxycinnamate (3HCI) in E. coli K-12 is shown in Fig. 1B. The first step is catalyzed by the MhpA hydroxylase, which inserts one atom of molecular oxygen at the position 2 of the phenyl ring of 3HPP to give 3-(2,3-dihydroxyphenyl)propionic acid (DHPP). This intermediate is then converted to succinate, pyruvate, and acetyl-CoA through the action of a meta-cleavage hydrolytic route whose enzymes are encoded by the mhp cluster located at minute 8.0 of the genome (Fig. 1A), being the first hydroxyphenylpropionate degradation pathway described both at the biochemical and genetic levels (3⇓⇓–6). The mhp cluster is arranged as follows: (i) six catabolic genes encoding the initial monooxygenase (mhpA), the extradiol dioxygenase (mhpB), and the hydrolytic meta-cleavage enzymes (mhpCDFE); (ii) a gene (mhpT) that encodes a potential transporter; (iii) a regulatory gene (mhpR) which is adjacent to the catabolic genes but transcribed in the opposite direction (5). Promoters Pr and Pa control the expression of the divergently transcribed mhpR regulatory gene and mhp catabolic genes, respectively (Fig. 1A).

Remarkably, the catabolism of 3HPP is connected with degradation of phenylpropionic acid (PP) through the common intermediate DHPP (6). The hca cluster encoding the enzymes responsible for the early steps of PP catabolism is located at minute 57.5 of the genome and contains (i) five genes encoding PP-dioxygenase (hcaEFCD; formerly named as hcaA1A2CD) and PP-dihydrodiol dehydrogenase (hcaB), (ii) a regulatory gene (hcaR), and (iii) a gene (hcaT) that might encode a transporter. The genes hcaR and hcaT are transcribed in the opposite direction from the other genes of cluster (Fig. 1A). The first biochemical step of PP degradation is catalyzed by a PP dioxygenase (HcaA1A2CD), which adds oxygen atoms to positions 2 and 3 of the PP phenyl ring and is subsequently oxidized by the HcaB dehydrogenase to give DHPP (Fig. 1B). Therefore, this compound links the catabolism of PP and 3HPP in E. coli.

As mentioned above, the 3HPP and PP catabolic pathways are regulated by two different regulatory proteins, MhpR and HcaR, respectively. Expression of mhp catabolic genes depends on the transcriptional activator MhpR belonging to the IclR family of transcriptional regulators (Fig. 1A) comprising more than 500 members identified from bacterial and Archaea genomes. IclR regulators typically have an N-terminal helix-turn-helix DNA binding motif and are linked by a long helix to the effector binding domain (the best defining trait of the family) located at the C-terminal domain. Sequence analysis of IclR regulators revealed a very low conservation of the amino acids residues involved in the effector binding, reflecting the chemical diversity of effector molecules recognized by the members of this family. HcaR belongs to the LysR family of transcriptional regulators and positively controls the neighboring genes, hcaA1A2CBD, in the presence of PP and negatively controls its own expression (Fig. 1A) (7, 8). MhpR behaves as a 3HPP-dependent activator of the Pa catabolic promoter by binding to its specific operator sequence centered at position −58 with respect to the transcription start site in Pa promoter. In contrast to HcaR, MhpR does not autoregulate its own expression (9). Expression of Pa promoter is also influenced by the cAMP receptor protein (CRP), which allows expression of the mhp catabolic genes when the preferred carbon source (glucose) is not available but 3HPP is present in the medium (Fig. 1A). MhpR shows a synergistic transcription activation mechanism with CRP (9).

Although transcriptional regulators often respond to one molecule which alters their binding to the promoter region, it has been described that in some particular occasions multiple effectors can regulate gene expression (10⇓–12). The combined effect of these compounds on transcriptional control by a single regulator is in general poorly understood. Such combined effects could play a relevant role in those cases where two or more pathways share common intermediates and must be synchronically regulated, as appears to be the case for the mhp and hca pathways. In this work we have used different in vivo and in vitro experimental approaches to describe the synergistic activation of MhpR in response to different metabolites