Cyclic AMP Receptor Protein and RhaR Synergistically Activate Transcription from the
            <scp>l</scp>
            -Rhamnose-Responsive
            <i>rhaSR</i>
            Promoter in
            <i>Escherichia coli</i>

Wickstrum, Jason R.; Santangelo, Thomas J.; Egan, Susan M.

doi:10.1128/jb.187.19.6708-6718.2005

Cited by 22 publications

(23 citation statements)

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“…(Gallegos et al, 1997). Some members of this family are known to be L-rhamnose-responsive transcription activators, regulating L-rhamnose catabolic genes (Wickstrum et al, 2005). Thus, araC is a potential transcriptional regulator of the rha operon in L. plantarum.…”

Section: Selection Of Bacteria With A-l-rhamnosidase Activitymentioning

confidence: 99%

Physiological and biochemical characterization of the two α-l-rhamnosidases of Lactobacillus plantarum NCC245

et al. 2009

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This work is believed to be the first report on the physiological and biochemical characterization of a-L-rhamnosidases in lactic acid bacteria. A total of 216 strains representing 37 species and eight genera of food-grade bacteria were screened for a-L-rhamnosidase activity. The majority of positive bacteria (25 out of 35) were Lactobacillus plantarum strains, and activity of the L. plantarum strain NCC245 was examined in more detail. The analysis of a-L-rhamnosidase activity under different growth conditions revealed dual regulation of the enzyme activity, involving carbon catabolite repression and induction: the enzyme activity was downregulated by glucose and upregulated by L-rhamnose. The expression of the two a-L-rhamnosidase genes rhaB1 and rhaB2 and two predicted permease genes rhaP1 and rhaP2, identified in a probable operon rhaP2B2P1B1, was repressed by glucose and induced by L-rhamnose, showing regulation at the transcriptional level. The two a-L-rhamnosidase genes were overexpressed and purified from Escherichia coli. RhaB1 activity was maximal at 50 6C and at neutral pH and RhaB2 maximal activity was detected at 60 6C and at pH 5, with high residual activity at 70 6C. Both enzymes showed a preference for the a-1,6 linkage of L-rhamnose to b-D-glucose, hesperidin and rutin being their best substrates, but, surprisingly, no activity was detected towards the a-1,2 linkage in naringin under the tested conditions. In conclusion, we identified and characterized the strain L. plantarum NCC245 and its two a-L-rhamnosidase enzymes, which might be applied for improvement of bioavailability of health-beneficial polyphenols, such as hesperidin, in humans.

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Section: Selection Of Bacteria With A-l-rhamnosidase Activitymentioning

confidence: 99%

Physiological and biochemical characterization of the two α-l-rhamnosidases of Lactobacillus plantarum NCC245

et al. 2009

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“…As observed for the MhpR regulator, CRP also regulates expression of other transcription factors in E. coli such as MelR (Webster et al, 1988), MalT (Chapon & Kolb, 1983), RhaS (Wickstrum et al, 2005), MaoB (Yamashita et al, 1996), HcaR (Turlin et al, 2001), GalS (Weickert & Adhya, 1993), RpoH (Kallipolitis & Valentin-Hansen, 1998), BlgG (Gulati & Mahadevan, 2000), PrpR (Lee et al, 2005) and Fis (Nasser et al, 2001). …”

Section: Discussionmentioning

confidence: 99%

“…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).…”

mentioning

confidence: 99%

“…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). The mechanism of CRP activation of these promoters is different, the Pr promoter of mhpR and the promoter of melR being class II CRP-dependent promoters (Webster et al, 1988;Samarasinghe et al, 2008) whereas the promoter of malT, and the P SR and P prpR promoters behave as class I CRP-dependent promoters (Chapon & Kolb, 1983;Wickstrum et al, 2005;Lee et al, 2005).…”

mentioning

confidence: 99%

“…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). The mechanism of CRP activation of these promoters is different, the Pr promoter of mhpR and the promoter of melR being class II CRP-dependent promoters (Webster et al, 1988;Samarasinghe et al, 2008) whereas the promoter of malT, and the P SR and P prpR promoters behave as class I CRP-dependent promoters (Chapon & Kolb, 1983;Wickstrum et al, 2005;Lee et al, 2005). The consensus nucleotide sequence for the cAMP-CRP binding site (Busby & Ebright, 1999) is compared to the Pr promoter sequence from nt "51 to "30 and the known cAMP-CRP binding sites at pgalP1 and pmelR, which are located from "52 to "31 with respect to its transcription start point.…”

mentioning

confidence: 99%

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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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Allosteric regulation within the highly interconnected structural scaffold of AraC/XylS homologs tolerates a wide range of amino acid changes

et al. 2021

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To create bacterial transcription “circuits” for biotechnology, one approach is to recombine natural transcription factors, promoters, and operators. Additional novel functions can be engineered from existing transcription factors such as the E. coli AraC transcriptional activator, for which binding to DNA is modulated by binding L‐arabinose. Here, we engineered chimeric AraC/XylS transcription activators that recognized ara DNA binding sites and responded to varied effector ligands. The first step, identifying domain boundaries in the natural homologs, was challenging because (i) no full‐length, dimeric structures were available and (ii) extremely low sequence identities (≤10%) among homologs precluded traditional assemblies of sequence alignments. Thus, to identify domains, we built and aligned structural models of the natural proteins. The designed chimeric activators were assessed for function, which was then further improved by random mutagenesis. Several mutational variants were identified for an XylS•AraC chimera that responded to benzoate; two enhanced activation to near that of wild‐type AraC. For an RhaR•AraC chimera, a variant with five additional substitutions enabled transcriptional activation in response to rhamnose. These five changes were dispersed across the protein structure, and combinatorial experiments testing subsets of substitutions showed significant non‐additivity. Combined, the structure modeling and epistasis suggest that the common AraC/XylS structural scaffold is highly interconnected, with complex intra‐protein and inter‐domain communication pathways enabling allosteric regulation. At the same time, the observed epistasis and the low sequence identities of the natural homologs suggest that the structural scaffold and function of transcriptional regulation are nevertheless highly accommodating of amino acid changes.

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Cyclic AMP Receptor Protein and RhaR Synergistically Activate Transcription from the l -Rhamnose-Responsive rhaSR Promoter in Escherichia coli

Cited by 22 publications

References 40 publications

Physiological and biochemical characterization of the two α-l-rhamnosidases of Lactobacillus plantarum NCC245

Physiological and biochemical characterization of the two α-l-rhamnosidases of Lactobacillus plantarum NCC245

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

Allosteric regulation within the highly interconnected structural scaffold of AraC/XylS homologs tolerates a wide range of amino acid changes

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