Purification and functional characterization of MerD. A coregulator of the mercury resistance operon in gram-negative bacteria.
Mercury resistance operons (mer) from transposons Tn21, Tn501, and plasmid pDU1358 are highly homologous and inducible with Hg2+. The regulatory gene merR is transcribed from one promoter, which is divergently oriented from the promoter for the other mer genes. MerR, the product of the regulatory gene, negatively regulates its own expression as well as the expression of the other genes. MerR activates transcription of the operon in the presence of inducing concentrations of Hg2+. The most promoter distal gene, merD, which is cotranscribed with the structural genes, down regulates the mer operon. A frame-shift mutation in merD, created by deletion of 3 bp and an insertion of a 16 bp sequence upstream of the major inverted repeats present at the 3' end of the merD sequence, resulted in increased synthesis of the structural gene transcript and higher level of resistance to Hg2+ by a factor of about 2. MerD protein was over-produced using a T7 expression system. The overproduced protein was present in the pellet fraction, when cell lysates were centrifuged at a low speed. Approximately 80% pure MerD protein was recovered from the pellet fraction by extracting with a buffer solution containing 5 M urea. The purified protein migrated as a 13,500 molecular weight protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the N-terminal amino acid sequence corresponded to that deduced from the DNA sequence of merD. MerD bound specifically with the mer promoter sequence. DNase I footprinting experiments identified a common mer operator sequence for MerR and MerD.
Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago 60612.
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Mukhopadhyay
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Yu
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Nucifora
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TK
Misra
D001426:Bacterial Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004352:Drug Resistance, Microbial / Q000235:genetics; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D005798:Genes, Bacterial; D005809:Genes, Regulator; D008628:Mercury / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009154:Mutation; D009875:Operator Regions, Genetic; D009876:Operon; D011401:Promoter Regions, Genetic; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C051124:MerR protein, Bacteria; D008628:Mercury