CadC-Mediated Activation of the &lt;i&gt;cadBA&lt;/i&gt; Promoter in &lt;i&gt;Escherichia coli&lt;/i&gt;

Küper, Christoph; Jung, Kirsten

doi:10.1159/000090346

Cited by 81 publications

(109 citation statements)

References 71 publications

(45 reference statements)

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“…Expression of BcrR in E. faecalis and E. coli-To study membrane-bound DNA-binding proteins, DNA binding assays have been either performed with inverted membrane vesicles that may contain other potentially contaminating proteins (10,35), soluble variants of the protein (36,37), in the presence of detergent (38), or by reconstitution of purified protein into liposomes (39). The level of BcrR expression in native membranes of E. faecalis was too low for EMSAs with bcrABD promoter DNA (data not shown).…”

Section: Resultsmentioning

confidence: 99%

“…These include a low ratio of protein-to-lipid in the proteoliposomes, BcrR His orientated in the opposite direction with the DNA-binding domain on the inside of the proteoliposomes, and potential clumping of proteoliposomes preventing BcrR His from accessing target DNA. Previously described EMSAs with proteoliposomes have also required large quantities of protein to DNA for similar reasons (39).…”

Section: Sds-page Analysismentioning

confidence: 99%

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Molecular Analysis of BcrR, a Membrane-bound Bacitracin Sensor and DNA-binding Protein from Enterococcus faecalis

Gauntlett

Gebhard

Keis

et al. 2008

Journal of Biological Chemistry

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BcrR has been identified as a novel regulatory protein of high level bacitracin resistance encoded by the bcrABD operon in Enterococcus faecalis. The N-terminal domain of BcrR has similarity to the helix-turn-helix motif of DNAbinding proteins, and topological modeling predicts that the C-terminal domain contains four transmembrane ␣-helices. These data have led to the hypothesis that BcrR functions as both a membrane-bound sensor and transducer of bacitracin availability to regulate bcrABD expression. To characterize the bcrABD promoter and identify the promoter elements to which BcrR binds, a series of bcrA-lacZ fusions were constructed. A 69-bp region was identified that was essential for bacitracin-dependent bcrA-lacZ expression. Mutations that targeted this region were used to identify two inverted repeat sequences, each with the sequence 5-GACA(N) 7 TGTC-3, on the bcrABD promoter that were required for bcrA-lacZ expression. To study BcrR binding to this region, we overproduced BcrR with a C-terminal hexa-histidine tag in Escherichia coli membranes, extracted the protein with n-dodecyl-␤-D-maltoside, and subsequently purified it via Ni 2؉ -nitrilotriacetic acid and gel filtration chromatography to apparent homogeneity. Purified BcrR was reconstituted into liposomes, and BcrR binding to bcrABD promoter DNA was analyzed using electrophoretic mobility shift assays. Both inverted repeat sequences were required for BcrR binding, both in the presence and absence of bacitracin. These data demonstrate that membrane-bound BcrR binds specifically to the bcrABD promoter, irrespective of bacitracin concentration. We therefore propose that bacitracin-dependent induction of bcrABD expression by BcrR occurs after DNA binding.Bacitracin is a polypeptide antibiotic that functions by binding to and sequestering undecaprenyl pyrophosphate (UPP) 4 (1). Because UPP acts as a carrier of peptidoglycan monomeric units across the cell membrane (2), the antibacterial nature of bacitracin results from its ability to block cell wall synthesis. High level bacitracin resistance in Enterococcus faecalis is encoded by the bcrABD operon that is under the control of a putative membrane-bound DNA-binding protein, BcrR (3). In the presence of bacitracin, transcription of the bcrABD operon leads to the production of BcrA and BcrB, which are proposed to act as an ABC exporter of bacitracin, thus conferring high level bacitracin resistance (minimum inhibitory concentration Ն 256 g/ml) to the cell (3). BcrD is proposed to function as an undecaprenyl pyrophosphate phosphatase that functions to increase the amount of undecaprenyl available to the cell (3). It has been demonstrated that bcrR, which is transcribed constitutively, is essential for high level bacitracin resistance in E. faecalis and that transcription of bcrABD is abolished in the absence of bcrR (3). The expression of bcrABD was found to be inducible with increasing concentrations of bacitracin (3). The N-terminal domain (amino acid residues 5-61) of BcrR has homology to the Xre famil...

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Section: Resultsmentioning

confidence: 99%

Section: Sds-page Analysismentioning

confidence: 99%

Molecular Analysis of BcrR, a Membrane-bound Bacitracin Sensor and DNA-binding Protein from Enterococcus faecalis

Gauntlett

Gebhard

Keis

et al. 2008

Journal of Biological Chemistry

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“…Its altogether 11 a-helices (nos. [4][5][6][7][8][9][10][11][12][13][14] are organized as a bundle of five antiparallel a-helical pairs, whereby helix 13 comprises just one turn. These pairs are twisted in a clockwise direction, resulting in a spiral bundle such that the C-terminus of helix 4 is situated close to the N-terminal end of helix 12.…”

Section: Resultsmentioning

confidence: 99%

“…12 The DNA-binding sites were identified and the interaction between purified CadC and the promoter region was demonstrated in vitro. 13 Expression of the cadBA operon is activated at low external pH and concomitantly available lysine. Recently, it was shown that CadC is not a direct sensor of the external lysine concentration.…”

Section: Introductionmentioning

confidence: 99%

Crystal structure of the sensory domain of Escherichia coli CadC, a member of the ToxR‐like protein family

et al. 2011

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The membrane-integral transcriptional activator CadC comprises sensory and transcriptional regulatory functions within one polypeptide chain. Its C-terminal periplasmic domain, CadC pd , is responsible for sensing of environmental pH as well as for binding of the feedback inhibitor cadaverine. Here we describe the crystal structure of CadC pd (residues 188-512) solved at a resolution of 1.8 Å via multiple wavelength anomalous dispersion (MAD) using a ReCl 6 22 derivative. CadC pd reveals a novel fold comprising two subdomains: an N-terminal subdomain dominated by a b-sheet in contact with three a-helices and a C-terminal subdomain formed by an eleven-membered a-helical bundle, which is oriented almost perpendicular to the helices in the first subdomain. Further to the native protein, crystal structures were also solved for its variants D471N and D471E, which show functionally different behavior in pH sensing. Interestingly, in the heavy metal derivative of CadC pd used for MAD phasing a ReCl 6 22 ion was found in a cavity located between the two subdomains. Amino acid side chains that coordinate this complex ion are conserved in CadC homologues from various bacterial species, suggesting a function of the cavity in the binding of cadaverine, which was supported by docking studies. Notably, CadC pd forms a homodimer in solution, which can be explained by an extended, albeit rather polar interface between two symmetry-related monomers in the crystal structure. The occurrence of several acidic residues in this region suggests protonation-dependent changes in the mode of dimerization, which could eventually trigger transcriptional activation by CadC in the bacterial cytoplasm.

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“…The cadBA operon encodes lysine decarboxlase (CadA) and lysine-cadaverine antiporter (CadB). The expression of the cadBA operon is dependent upon the transcriptional activator CadC (8,9,11,12,16,22). CadA has a pH optimum of 5.7 and converts lysine into cadaverine and CO 2 (19).…”

Section: Smentioning

confidence: 99%

Transcriptional Regulation of stj Fimbrial Operon in Salmonella Typhimurium

Deniz;AKÇELİK¹

2012

Ankara Üniversitesi Veteriner Fakültesi Dergisi

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Summary:The stj fimbrial operon harbors five genes, stjABCDE. The operon plays role in the persistence and virulence of S.Typhimurium in host systems. In this study, in vitro activation of the stj operon was achieved by transposon (TPOP) mutagenesis. The transposon insertion site was located within the cadC gene promoter in the stj operon-activated mutant strain MDN20. Comparative analysis of two-dimensional gel electrophoresis protein patterns of the wild type strain (S. Typhimurium LT2) and mutant strain MDN20 revealed that the CadC and StjB proteins were produced by mutant strain MDN20 but not by the wild type strain. These results were also confirmed by Maldi-TOF analysis. These findings indicate that cadC gene is a transcriptional activator of stj fimbrial operon in S. Typhimurium.Key words: S. Typhimurium, stj operon, cadC, transcriptional activator.Salmonella Typhimurium'da stj fimbriyal operonunun transkripsiyonel regülasyonu.Özet: Salmonella Typhimurium'da konakçı sistemde kalıcılığı ve virulensliğinde rol oynayan stj fimbriyal operonu, stjABCDE olmak üzere beş gen içermektedir. Bu çalışmada, stj fimbriyal operonunun in vitro koşullarda aktivasyonu transpozon (TPOP) mutasyonu yolu ile gerçekleştirildi. stj operonu aktive edilen mutant suşta (MDN20), traspozonun cadC geni promotoruna girdiği belirlendi. Doğal suş (S. Typhimurium LT2) ve MDN20 mutant suşunda gerçekleştirilen iki yönlü jel elektroforezi çalışmaları sonucunda; CadC ve StjB proteinlerinin doğal suşta üretilmediği, ancak MDN20 mutant suşunda üretildiği saptandı. Bu sonuçlar ayrıca Maldi-TOF analizleri ile de doğrulandı. Bu bulgular, S. Typhimurium'da cadC geninin, stj operonunun transkripsiyonel aktivatörü olduğunu gösterdi. Anahtar sözcükler: S. Typhimurium, stj operonu, cadC, transkripsiyonel aktivatör.

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CadC-Mediated Activation of the <i>cadBA</i> Promoter in <i>Escherichia coli</i>

Cited by 81 publications

References 71 publications

Molecular Analysis of BcrR, a Membrane-bound Bacitracin Sensor and DNA-binding Protein from Enterococcus faecalis

Molecular Analysis of BcrR, a Membrane-bound Bacitracin Sensor and DNA-binding Protein from Enterococcus faecalis

Crystal structure of the sensory domain of Escherichia coli CadC, a member of the ToxR‐like protein family

Transcriptional Regulation of stj Fimbrial Operon in Salmonella Typhimurium

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