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Christopher T Walsh - One of the best experts on this subject based on the ideXlab platform.
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construction of a synthetic gene for the metalloregulatory Protein MerR and analysis of regionally mutated Proteins for transcriptional regulation
Biochemistry, 1994Co-Authors: Kenneth M Comess, Lisa M Shewchuk, Kathryn M Ivanetich, Christopher T WalshAbstract:The transcriptional control Protein MerR is a metalloregulatory switch, activating transcription of a mercury resistance operon in the presence of mercuric ions and repressing transcription in their absence. We report here the construction and utilization of a synthetic MerR gene and a single-copy merT'-lacZ fusion reporter for mutagenic analysis of the MerR Protein's function. Site-directed mutagenesis of clustered acidic residues within the central region of the MerR Protein indicated that these residues are important to the Protein's ability to repress transcription. Quadruple or sextuple mutations involving residues E83 and E84 and other nearby acidic residues result in a repression-deficient (RD) phenotype. One of the mutant Proteins was purified and shown by gel shift assay to retain binding to its operator DNA with an affinity similar to wild-type Protein, suggesting that transcriptional repression does not correlate with MerR binding affinity. A small region of MerR corresponding to residues 81-92 also was mutagenized in a search for other RD mutants and for mutants displaying sufficient transcriptional activation in the absence of mercuric ion to be classified as constitutive activation (CA) mutants. In this case, oligonucleotide-directed randomization of the target region and a screening/selection protocol were employed. Sixteen different mutants with an RD phenotype were identified, as well as eight different mutants with a CA phenotype. A high frequency of S87C mutations is evident in the RD set of mutants. The CA mutants have a high incidence of S86C and A89V mutations. The CA double mutant S86C/A89V was purified and found to bind to its DNA site with an affinity similar to that of the wild-type Protein. Chemical nuclease activity assays indicate that the nonmercurated S86C/A89V CA mutant has a DNA distortion activity identical to that of mercurated wild-type MerR. A unique disulfide bond bridging this CA mutant's dimer interface was found and is proposed to constrain Protein conformation in a manner analogous to mercuric ion binding in the wild-type Protein.
Soonyong Choi - One of the best experts on this subject based on the ideXlab platform.
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the sctr of salmonella enterica serova typhimurium encoding a homologue of MerR Protein is involved in the copper responsive regulation of cuid
Fems Microbiology Letters, 2002Co-Authors: Jin Sook Kim, Mi Hwa Kim, Min Ho Joe, Sang Sun Song, In Soo Lee, Soonyong ChoiAbstract:We have identified the cuiD gene in Salmonella enterica serova Typhimurium that codes for a putative multicopper oxidase. Expression of cuiD is induced by copper ion and its promoter/operator has sequence similarity to the promoters controlled by the transcriptional regulators of the MerR family. We also identified and isolated a gene from S. enterica serova Typhimurium that encodes a 138-amino acid residue Protein, sctR, a new member of the MerR family of transcriptional regulators. Transposon-insertional disruption of sctR shows sensitivity to copper ion and no response of cuiD expression. Copper-responsive induction and copper tolerance were restored by providing sctR in trans, suggesting that SctR plays an important role in intracellular copper detoxification.
Wilfred Chen - One of the best experts on this subject based on the ideXlab platform.
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Affinity purification of plasmid DNA by temperature-triggered precipitation
Nature protocols, 2007Co-Authors: U Loi Lao, Jan Kostal, Ashok Mulchandani, Wilfred ChenAbstract:This protocol presents a new method to purify plasmid DNA using temperature-triggered precipitation. The principle is based on the specific DNA-binding affinity of a bacterial metalloregulatory (MerR) Protein to its cognate DNA sequence and the temperature responsiveness of elastin-like Protein (ELP). A bifunctional ELP-MerR fusion Protein is created to enable the precipitation of plasmid DNA, designed to contain the MerR recognition sequence, by a simple temperature trigger. The protocol covers all stages of the process from the design of ELP-MerR fusion Proteins and MerR-binding plasmids, to the isolation of plasmid DNA from Escherichia coli cultures after boiling lysis, the subsequent temperature-triggered precipitation of plasmid DNA-fusion Protein complexes and final elution of plasmid DNA by mild heating. This protocol is well suited to laboratory research-scale applications, producing plasmid DNA of better purity and similar yield as one of the most commonly used laboratory methods, standard alkaline lysis (known as the midiprep procedure). The protocol takes approximately 30 min to obtain pure plasmid DNA from cell cultures using the temperature-triggered precipitation method.
K. S. Pustovoit - One of the best experts on this subject based on the ideXlab platform.
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Antirestriction activity of the mercury resistance nonconjugative transposon Tn5053 is controlled by the protease ClpXP
Russian Journal of Genetics, 2014Co-Authors: G. B. Zavilgelsky, V. Yu. Kotova, O. E. Melkina, K. S. PustovoitAbstract:When transformed into Escherichia coli K12 strains, the mercury resistance transposon Tn 5053 exhibits high antirestriction activity against the Eco KI type I restriction and modification system. The products of the genes MerR and ardD contribute to the antirestriction activity of Tn 5053 . The MerR gene encodes the MerR Protein, the transcription regulator of the mer operon genes. The ardD gene is responsible for ArdD Protein synthesis and is located within the tni operon. In the following study, it was demonstrated that the antirestriction activity of the transposon Tn 5053 is absent in E. coli K12 strains with the mutant genes clpX , clpP , and recA . The antirestriction effect of Tn 5053 is not enhanced by 2-aminopurine. The Tn 5053 antirestriction activity is not altered in E. coli K12 with the mutant dam gene; however, it is decreased in the E. coli K12 mutD . It is assumed that the activities of the MerR and ArdD Proteins lead to the formation of a significant amount of unmodified DNA in the bacterial cell, causing the SOS-dependent reduction of the Eco KI (R_2M_2S) enzyme activity associated with ClpXP-induced proteolysis of the R-subunit.
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Antirestriction activity of the mercury resistance nonconjugative transposon Tn5053 is controlled by the protease ClpXP
Russian Journal of Genetics, 2014Co-Authors: G. B. Zavilgelsky, V. Yu. Kotova, O. E. Melkina, K. S. PustovoitAbstract:When transformed into Escherichia coli K12 strains, the mercury resistance transposon Tn 5053 exhibits high antirestriction activity against the Eco KI type I restriction and modification system. The products of the genes MerR and ardD contribute to the antirestriction activity of Tn 5053 . The MerR gene encodes the MerR Protein, the transcription regulator of the mer operon genes. The ardD gene is responsible for ArdD Protein synthesis and is located within the tni operon. In the following study, it was demonstrated that the antirestriction activity of the transposon Tn 5053 is absent in E. coli K12 strains with the mutant genes clpX , clpP , and recA . The antirestriction effect of Tn 5053 is not enhanced by 2-aminopurine. The Tn 5053 antirestriction activity is not altered in E. coli K12 with the mutant dam gene; however, it is decreased in the E. coli K12 mutD . It is assumed that the activities of the MerR and ArdD Proteins lead to the formation of a significant amount of unmodified DNA in the bacterial cell, causing the SOS-dependent reduction of the Eco KI (R_2M_2S) enzyme activity associated with ClpXP-induced proteolysis of the R-subunit.
Kenneth M Comess - One of the best experts on this subject based on the ideXlab platform.
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construction of a synthetic gene for the metalloregulatory Protein MerR and analysis of regionally mutated Proteins for transcriptional regulation
Biochemistry, 1994Co-Authors: Kenneth M Comess, Lisa M Shewchuk, Kathryn M Ivanetich, Christopher T WalshAbstract:The transcriptional control Protein MerR is a metalloregulatory switch, activating transcription of a mercury resistance operon in the presence of mercuric ions and repressing transcription in their absence. We report here the construction and utilization of a synthetic MerR gene and a single-copy merT'-lacZ fusion reporter for mutagenic analysis of the MerR Protein's function. Site-directed mutagenesis of clustered acidic residues within the central region of the MerR Protein indicated that these residues are important to the Protein's ability to repress transcription. Quadruple or sextuple mutations involving residues E83 and E84 and other nearby acidic residues result in a repression-deficient (RD) phenotype. One of the mutant Proteins was purified and shown by gel shift assay to retain binding to its operator DNA with an affinity similar to wild-type Protein, suggesting that transcriptional repression does not correlate with MerR binding affinity. A small region of MerR corresponding to residues 81-92 also was mutagenized in a search for other RD mutants and for mutants displaying sufficient transcriptional activation in the absence of mercuric ion to be classified as constitutive activation (CA) mutants. In this case, oligonucleotide-directed randomization of the target region and a screening/selection protocol were employed. Sixteen different mutants with an RD phenotype were identified, as well as eight different mutants with a CA phenotype. A high frequency of S87C mutations is evident in the RD set of mutants. The CA mutants have a high incidence of S86C and A89V mutations. The CA double mutant S86C/A89V was purified and found to bind to its DNA site with an affinity similar to that of the wild-type Protein. Chemical nuclease activity assays indicate that the nonmercurated S86C/A89V CA mutant has a DNA distortion activity identical to that of mercurated wild-type MerR. A unique disulfide bond bridging this CA mutant's dimer interface was found and is proposed to constrain Protein conformation in a manner analogous to mercuric ion binding in the wild-type Protein.