Escherichia coli mhpR gene expression is regulated by catabolite repression mediated by the cAMP–CRP complex

Abstract

The expression of the mhp genes involved in the degradation of the aromatic compound 3-(3-hydroxyphenyl)propionic acid (3HPP) in Escherichia coli is dependent on the MhpR transcriptional activator at the Pa promoter. This catabolic promoter is also subject to catabolic repression in the presence of glucose mediated by the cAMP-CRP complex. The Pr promoter drives the MhpR-independent expression of the regulatory gene. In vivo and in vitro experiments have shown that transcription from the Pr promoter is downregulated by the addition of glucose and this catabolic repression is also mediated by the cAMP-CRP complex. The activation role of the cAMP-CRP regulatory system was further investigated by DNase I footprinting assays, which showed that the cAMP-CRP complex binds to the Pr promoter sequence, protecting a region centred at position "40.5, which allowed the classification of Pr as a class II CRP-dependent promoter. Open complex formation at the Pr promoter is observed only when RNA polymerase and cAMP-CRP are present. Finally, by in vitro transcription assays we have demonstrated the absolute requirement of the cAMP-CRP complex for the activation of the Pr promoter. INTRODUCTIONThe mhp gene cluster of Escherichia coli, which encodes the 3-hydroxyphenylpropionate (3HPP) catabolic pathway, constitutes a model system for studying bacterial degradation because of its distinctive regulation. The mhp regulatory region contains the Pr and Pa promoters, which control the expression of the divergently transcribed mhpR regulatory gene and mhp catabolic genes, respectively (Ferrández et al., 1997). Expression of the mhp catabolic genes depends on the transcriptional activator MhpR, which belongs to the IclR family of transcriptional regulators. MhpR behaves as a 3HPP-dependent activator of the Pa promoter by binding to its specific operator sequence centred at position 258 with respect to the transcription start site in the Pa promoter (Fig. 1a) (Ferrández et al., 1997;Torres et al., 2003). MhpR displays a distinctive regulation mechanism in which phenylpropionic acid activates the MhpR regulator synergistically with the true inducers, representing the first case of such an unusual synergistic effect described for a bacterial regulator (Manso et al., 2009). Gene expression from the Pa promoter is also under a strict global control system mediated by the cAMP-CRP complex that allows expression of the mhp catabolic genes when glucose is not available and 3HPP is present in the medium (Torres et al., 2003). The Pa promoter contains a potential CRP-binding site centred at position 295.5 relative to the transcription start point of Pa (Fig. 1a). In vitro experiments revealed that the specific activator, MhpR, is essential for the recruitment of CRP at the Pa promoter (Torres et al., 2003). The synergistic transcription activation by the cAMP-CRP complex and the MhpR activator allowed the Pa promoter of the mhp cluster to be classified as a class III CRP-dependent promoter (Torres et al., 2003).Although the regulation of the mh...

PredCRP: predicting and analysing the regulatory roles of CRP from its binding sites in Escherichia coli

“…There are two categories of methods to identify the binding site of a specific TF. One category consists of the low-throughput methods such as the electrophoretic mobility shift assay and the DNase I footprinting assay 17 . The other category comprise high-throughput methods such as the chromatin immunoprecipitation (ChIP) assay, a sequencing-based method (ChIP-seq) 18 and elegant computational methods focusing on predicting TF-binding sites 19 – 21 .…”

“…There are two approaches to determining regulatory roles of a TF. One is a low-throughput approach such as the promoter -lacZ fusion 17 and quantitative PCR (qPCR) 22 . The other consists of a high-throughput approach such as gene expression analysis using RNA-seq 23 or microarray data.…”

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