A second positive regulatory function in the mer (mercury resistance) operon

doi:10.1016/0378-1119(83)90225-1

Gene

Volume 25, Issues 2–3, November 1983, Pages 209-221

https://doi.org/10.1016/0378-1119(83)90225-1 Get rights and content

Abstract

Transpositional mutagenesis of the mer operon of the IncFII plasmid, R100, has revealed a second, trans-acting positive regulatory function. Mutants in this function do not synthesize any of the three small mer operon peptides and have no inducible Hg(II) uptake activity. This second regulatory function is part of complementation group B and so depends upon the activity of the previously described trans-acting positive regulatory function merR. All mutants in this new function map in the amino-terminal 20 kDal of the Hg(II) reductase, suggesting either that this enzyme is also a regulatory protein or that there is a distinct protein whose reading frame is superimposed on that of the Hg(II) reductase. While we have only seen the five previously described mer operon peptides of 69, 66, and 12 (13) kDal encoded in minicells by single-copy plasmids, we have observed two new HgCl₂-inducible polypeptides of approx. 20 kDal in minicells carrying a multicopy derivative of the mer operon of R100. Sequence data for the Hg(II) reductase region of the related mer operon of the transposon, Tn 501 [Brown, N.L., Ford, S.J., Pridmore, R.D. and Fritzinger, D.C., Biochemistry 22 (1983) 4089–4095], shows a second reading frame very rich in cysteine and arginine which overlaps the amino-terminal 20 kDal of the Hg(II) reductase structural gene. We believe that this reading frame is the structural gene for this new regulatory function and propose the name merC (for control).

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Cited by (11)

MerF is a mercury transport protein: Different structures but a common mechanism for mercuric ion transporters?
2000, FEBS Letters
Mercury resistance determinants are widespread in Gram-negative bacteria, but vary in the number and identity of genes present. We have shown that the merF gene from plasmid pMER327/419 encodes a 8.7 kDa mercury transport protein, by determining in vivo mercury volatilisation when MerF is expressed in the presence of mercuric reductase. We have confirmed that MerC of Tn21 is also a mercuric ion transporter. We have been able to detect interaction of the periplasmic protein MerP only with the MerT transporter, and not with MerF or MerC. Hydropathy analysis led to the prediction of models for MerT, MerC and MerF having three, four and two transmembrane regions respectively. In all three cases one pair of cysteine residues is predicted to be within the inner membrane with a second pair of cysteine residues on the cytoplasmic face, and the second helix contains a proline and at least one charged residue. The mechanisms of mercuric ion transport may be similar in these transporters even though their structures in the membrane differ.
Synthesis and degradation of the mRNA of the Tn21 mer operon
1992, Journal of Molecular Biology
The mercury resistance locus encoded by Tn21 on the monocopy IncFII plasmid R100 (mer^Tn21) consists of a metal-responsive activator/repressor, merR, which controls initiation of a polycistronic message that includes genes for the uptake (merTPC) and reduction (merA) of Hg²⁺ and merD, which may also play a minor regulatory role. Comparison of the relative abundance of the 5′ and 3′ ends of the merTPCAD transcript revealed a strong transcriptional gradient in the operon, consistent with previous observations of lower relative abundance of the more promoter-distal gene products. In vivo mRNA degradation rates varied only slightly for the different genes: however, the rates of mRNA synthesis varied considerably from the beginning to the end of the operon. Specifically, mRNA corresponding to the promoter-proximal genes, merTPC, achieved a maximum in vivo synthesis rate between 60 and 120 seconds after induction; this rate was maintained for approximately ten minutes. In contrast, the synthesis rates of mRNA corresponding to the promoter-distal genes merA and merD, were initially fivefold lower than the rates of the promoter-proximal genes for the first five minutes after induction, and then rose gradually to approximately 50% of the merTPC synthesis rates. These data suggested that early after induction only 20% of the transcripts initiating at merT proceed beyond merC. At later times after induction approximately 50% of the transcripts proceed beyond merC. Nuclease end mapping did not reveal any discrete termination events in the merPCA region, thus, premature termination may occur at many sites.
Polypeptides specified by the mercuric resistance (mer) operon of plasmid R100
1986, Gene
Overlapping deletion mutations were constructed in chimaeric plasmids carrying the mer operon of plasmid R100. Polypeptides specified by the mutant plasmids in Escherichia coli minicells correlated with the mer genes as follows: merT, 17- and 16-kDa polypeptides; merP, 9.8- and 9.5-kDa polypeptides ; merC, a 14-kDa polypeptide; merA, 65- and 62-kDa polypeptides. The products of the merR and merD genes were not identified. The revised nomenclature of the mer genes is explained.
Versatile mercury-resistant cloning and expression vectors
1985, Gene
Cloning vectors have been constructed employing two diverse replicons, IncQ and P15A. Both vectors confer resistance to kanamycin (Km) and mercuric ions (Hg²⁺). One of these vectors, pDG105, is a broad-host-range, nonconjugative, oligocopy IncQ plasmid, which is capable of transforming Escherichia coli, Acinetobacter calcoaceticus, and Pseudomonas putida. The second vector, pDG106, is a narrow-host-range, multicopy cloning vector compatible with pBR322. Both vectors contain unique cloning sites in the Km-resistance gene for HindIII, SmaI, and XhoI, as well as unique EcoRI and ScaI sites in the mer operon. Cloning into the EcoRI site in the mer operon results in the mercury “supersensitive” phenotype, easily detectable by replica plating. Insertion of the galK gene into the EcoRI site in the mer operon results in Hg²⁺ -inducible galactokinase activity, demonstrating the application of these plasmids as regulated expression vectors.
Mercuric reductase structural genes from plasmid R100 and transposon Tn501: functional domains of the enzyme
1985, Gene
The nucleotide sequence for the 2240 bp of plasmid R100 following the merC gene of the mercuric resistance operon has been determined and compared with the homologous sequence of transposon Tn507. The sequences following merC and preceding the next structural gene merA are unrelated between R100 and Tn501 and differ in length, with 72 bp in Tn501 and 509 bp in R100. The R100 sequence has a potential open reading frame (ORF) for a 140 amino acid polypeptide with a reasonable translational start signal preceding it. The merA genes contain 1686 (Tn5O1) and 1695 (R100) bp respectively. When optimally aligned, the merA sequences differ in 18% of their positions. These differences were clustered in specific regions. In addition, there was one nucleotide triplet in the Tn501 sequence which has no counterpart in the R100 sequence and one dodecylnucleotide sequence in the R100 sequence without counterpart in Tn501. Thus the predicted merA polypeptide of Tn501 contains 561 amino acids and the R100 counterpart contains 564 amino acids. Comparison of the R100 mercuric reductase sequences with that for human glutathione reductase [Krauth-Siegel et al.: Eur. J. Biochem. 121 (1982) 259–267], for which there is a 2 Å resolution electron density map [Thieme et al. : J. Mol. Biol. 152 (1981) 763–782] shows a strong homology, with 26% identical amino acids and many conservative substitutions. This homology allows the conclusion that the active site of these enzymes and the contact positions for flavin adenine dinucleotide (FAD) and NADPH are highly conserved, while the amino- and carboxyl-terminal sequences differ.
Physical and genetic map of the organomercury resistance (Omr) and inorganic mercury resistance (Hgr) loci of the IncM plasmid R831b
1984, Gene
Tn7 insertion mutagenesis has been used to facilitate the generation of a physical (restriction endonuclease) and genetic map of the IncM plasmid, R831b. The only selectable phenotypes carried by this 90-kb conjugative plasmid are resistances to inorganic mercury [Hg (II)] and to organomercury compounds. Mutants in the Hgr locus of R831b complemented previously described mutants in the mer operon of the IncFII plasmid R100, indicating functional homology of the locus in each of these different plasmids. However, the R83 Ib Hgr locus is not notably similar in restriction site pattern to either the mer operon of R100 or the mercury resistance transposon, Tn501. Although the enzymes they encode are co-ordinately regulated, the Omr locus of R831b maps approx. 13.5 kb away from the Hgr locus. Three insertions which affect neither phenotype lie between the Hgr and Omr loci; thus, the loci are separated both physically and genetically. One mutant was obtained which tentatively identifies the position of the Tra locus of R831b as adjacent to the Hgr locus.

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A second positive regulatory function in the mer (mercury resistance) operon

Abstract

Gene

Cell

J. Biol. Chem.

Cell

J. Biol. Chem.

Linkage map of Escherichia coli K-12, edition 7

Characterization of Tn 501, a transposon determining resistance to mercuric ions

Mol. Gen. Genet.

Polarity of Tn 5 insertion mutations in Escherichia coli

J. Bacteriol.

DNA sequence of a gene from the Pseudomonas transposon Tn 501 encoding mercuric reductase

Biochemistry

Mercury and organomercurial resistance determined by plasmids in Pseudomonas

J. Bacteriol.

Genetic and molecular characterization of Tn 21, a multiple resistance transposon from 11100.1

J. Bacteriol.

Properties of lambda transducing bacteriophages carrying R100 plasmid DNA: mercury resistance genes

J. Bacteriol.

Transposon A-generated mutations in the mercuric resistance genes of plasmid 11100-1

J. Bacteriol.

Mercuric reductase: purification and characterization of a transposon-encoded flavoprotein containing a redox-active disulfide

J. Biol. Chem.

Mercuric reductase; homology to glutathione reductase and lipoamidedehydrogenase. Iodoacetamide alkylation and the sequence of the active site peptide

Biochemistry

Regional preference of insertion of Tn 501 and Tn 802 into RP1 and its derivatives

Mol. Gen. Genet.

Polypeptides encoded by the mer operon

J. Bacteriol.