Transcription activation at the <i>Escherichia coli melAB</i> promoter: the role of MelR and the cyclic AMP receptor protein

Belyaeva, Tamara A.; Wade, Joseph T.; Webster, Christine; Howard, Victoria J.; Thomas, Mark S.; Hyde, Eva I.; Busby, Stephen J. W.

doi:10.1046/j.1365-2958.2000.01849.x

Cited by 41 publications

(80 citation statements)

References 29 publications

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“…Taking our results together with previous data (Torres et al, 2003) we have demonstrated that the glucose effect on mhp expression is exerted at the level of both catabolic and regulatory promoters. This simultaneous CRP control is not unique to the mhp pathway, since it has been also described for the genes for propionate catabolism in E. coli and Salmonella enterica (Lee et al, 2005) and for some regulons from E. coli such as those for L-rhamnose (Holcroft & Egan, 2000;Wickstrum et al, 2005), melibiose (Webster et al, 1988;Belyaeva et al, 2000) and maltose (Chapon & Kolb, 1983;Richet & Sogaard-Andersen, 1994;Richet, 2000). While the expression of the mhpR, melR and malT regulators depends only on the presence of CRP (Webster et al, 1988;Chapon & Kolb, 1983), the expression of rhaSR and prpR depends on the presence of CRP and the transcription activators RhaR and PrpR, respectively (Tobin & Schleif, 1990a, b;Wickstrum et al, 2005;Lee et al, 2005).…”

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

2011

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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...

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

2011

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“…Recombinant plasmids were propagated in strain WAM131 (11), an mel ϩ strain derived from GM-1 (14). ␤-Galactosidase levels in these cells were measured using the method of Miller (15): cells * This work was supported by grants from the Ministry of Education, Science and Culture of Japan and from the United Kingdom Biotechnology and Biological Sciences Research Council.…”

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confidence: 99%

“…1). First, using PCR, we amplified the KK81 fragment that carries the entire melAB promoter with all four MelR binding sites (sites 1Ј, 1, 2, and 2Ј) that are known to be essential for optimal melAB promoter activity (10,11). Second, we amplified the shorter JK14 fragment that carries the melAB transcript start site and only the MelR binding sites 2 and 2Ј.…”

Section: In Vivo Assays With the W3133-2 Mutant Melabmentioning

confidence: 99%

“…In preliminary experiments, we checked the activity of the melAB promoter carried by these fragments. To do this, the different EcoRIHindIII fragments were cloned into the broad host range lac fusion vector, pRW50, and the resulting plasmids were transformed into WAM131, a ⌬lac mel ϩ strain derived from GM-1 (11,14). Expression of the promoter melAB::lacZ fusions carried by the pRW50 recombinants was measured in transformants grown either with or without the inducer, melibiose.…”

Section: In Vivo Assays With the W3133-2 Mutant Melabmentioning

confidence: 99%

“…1). Recent studies (11) have shown that the occupation of site 2Ј by MelR is the most crucial for the activation of the melAB promoter, but that, of the four target sites, site 2Ј binds MelR most weakly. We noted that the base changes in the melAB promoter in the W3133-2 mutant changed the DNA sequence of MelR binding site 2Ј such that it corresponded better to the tighter binding site 1Ј, site 1, and site 2.…”

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Mutations That Increase the Activity of the Promoter of theEscherichia coli Melibiose Operon Improve the Binding of MelR, a Transcription Activator Triggered by Melibiose

Tamai¹,

Belyaeva²,

Busby³

et al. 2000

Journal of Biological Chemistry

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MelR is an Escherichia coli transcription factor that activates expression of the melAB operon in response to the presence of melibiose in the environment. MelR stimulates transcription initiation at the melAB promoter by binding to four sites centered at positions ؊120.5, ؊100.5, ؊62.5, and ؊42.5 upstream of the transcript start point. In a previous study, we described a spontaneous mutant that exhibited increased melAB expression. Sequence analysis showed that this mutant carries five consecutive base changes at positions ؊49, ؊50, ؊51, ؊52, and ؊53 upstream of the melAB transcript start. Here we show that these changes improve MelR binding to the target site centered at position ؊42.5 at the melAB promoter and that this improvement is responsible for increased promoter activity. Thus, the activity of the melAB promoter is fixed by the occupation by MelR of a DNA site that overlaps the ؊35 hexamer: MelR appears to be a typical class II-type transcription activator.

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Transcriptional regulation of the moe (molybdate metabolism) operon of Escherichia coli

Hasona

Self

Shanmugam

2001

Archives of Microbiology

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Regulation of transcription of the Escherichia coli moe operon, which codes for proteins connecting molybdate metabolism, molybdopterin synthesis, and apomolybdoenzyme synthesis, was investigated. Expression of the moe operon was independent of genes coding for molybdate transport and Mo-cofactor biosynthesis. Expression of moeA-lacZ increased during anaerobic growth (2.5-fold over the aerobic value) and in the presence of nitrate and trimethylamine N-oxide (3.5- and 1.5-fold, respectively). The nitrate-dependent increase in moe expression required the NarL protein, while the anaerobiosis-dependent increase in moeA-lacZ expression required Arc proteins. ArcA-phosphate and not ArcA bound to the DNA upstream of moe, shifted the electrophoretic mobility of moe promoter DNA, and protected the DNA from DNase I hydrolysis. Nitrate-independent transcription of moeA-lacZ was repressed by the FNR protein, which also protected moe operator DNA from DNase I hydrolysis. These results show that ArcA-phosphate and FNR have opposite effects on the transcriptional regulation of the moe operon, and the combined action of the two redox regulators modulate the level of Mo-cofactor in the cell. Apparently, the control of synthesis of Mo-cofactor and the apomolybdoenzymes nitrate reductase and trimethylamine N-oxide reductase are coupled at the level of the moe operon.

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Transcription activation at the Escherichia coli melAB promoter: the role of MelR and the cyclic AMP receptor protein

Cited by 41 publications

References 29 publications

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

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

Mutations That Increase the Activity of the Promoter of theEscherichia coli Melibiose Operon Improve the Binding of MelR, a Transcription Activator Triggered by Melibiose

Transcriptional regulation of the moe (molybdate metabolism) operon of Escherichia coli

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