Purification and characterization of a novel organometallic receptor protein regulating the expression of the broad spectrum mercury-resistant operon of plasmid pDU1358.

Abstract

The narrow spectrum mercury-resistant (mer) operons of transposons Tn21 and Tn501 are inducible by inorganic mercury salts. The major regulatory gene merR is transcribed divergently from the other mer genes, which are cotranscribed. The MerR protein represses its own expression, as well as the expression of the other mer genes in the absence of the inducers. The synthesis of the polycistronic mer message is stimulated by MerR in the presence of the inducers. The MerRBS protein encoded by the broad spectrum mer operon of plasmid pDU1358 was characterized as a novel organomercurial receptor, distinguishing it from the narrow spectrum MerRNS proteins, described above. Several organomercurial compounds directly effected cellular activation of the mer operon transcription via the receptor protein MerRBS, but not by MerRNS. The merR gene from pDU1358 was cloned under the tac promoter, and the overexpressed MerRBS protein was soluble in buffer solutions containing 0.5 M NaCl at pH 7.5, but precipitated when NaCl concentration was reduced to 0.1 M (MerRBS concentrations at or above 0.1 mg/ml). MerRBS was purified to near homogeneity by selective precipitation and solubilization by varying the salt concentration in buffer solutions, followed by Sephadex G-75 column chromatography. Both MerRBS and Tn21-encoded MerRNS bound with DNA fragments containing the pDU1358 mer operator sequence with comparable affinities. In vitro run-off transcription studies revealed that MerRBS activated mer operon expression in the presence of Hg2+ or phenylmercuric acetate. Phenylmercuric acetate did not induce mer operon expression when the MerRNS was used in the assay.