The merR regulatory gene in Thiobacillus ferrooxidans is spaced apart from the mer structural genes

Two distinct merR genes, which regulate expression of the mercuric ion resistance gene (mer), of Thiobacillus ferrooxidans strain E-15 have been cloned, sequenced and termed merR1 and merR2. As a result of gene walking around two merR genes, it was found that these two genes were quite close in distance. The nucleotide sequence of the region (5,001 base pairs; PstI-EcoRI fragment) containing the merR genes was determined. Between the two merR genes, there were five potential open reading frames (ORFs). Two of these were identified as merC genes, and the other three as ORFs 1 to 3. ORFs 1 to 3 show significant homology to merA, tnsA from transposon Tn7, and merA, respectively. Both merR genes consist of a 408 bp ORF coding for 135 amino acids. Their gene products, MerR1 and MerR2, differed at three amino acid positions, and shared 56-57% and 32-38% identity with the MerRs from other Gram-negative and Gram-positive bacteria, respectively. Competitive primer extension analysis revealed that both regulatory genes were expressed in the host cells. These merR genes were located more than 6 kb from either end of the mer structural genes (merC-merA). This is the first example of merR being separated from the mer structural genes. The two merC genes, each of which coded for a 140-amino-acid protein, appeared to be functionally active because Escherichia coli cells carrying these merC genes on plasmid vectors showed hypersensitivity to HgCl2. However, ORFs 1 and 3, which were homologous to merA, seemed to be inactive both structurally and enzymatically. The gene arrangement in this region took on a mirror image, with the truncated tnsA as the symmetrical centre. It is suggested that the Tn7-like factor may have participated in gene duplication events of the mer region, and in its chromosomal integration.     

Effects of organic solvents on the high performance liquid chromatographic analysis of the cyanobacterial toxin cylindrospermopsin and its recovery from environmental eutrophic waters by solid phase extraction

An Escherichia coli strain was generated by fusion of a merA-deleted broad-spectrum mer operon from Pseudomonas K-62 with a bacterial polyphosphate kinase gene (ppk) from Klebsiella aerogenes in vector pUC119. A large amount of the ppk-specified polyphosphate was identified in the mercury-induced bacterium with the fusion plasmid designated pMKB18 but not in the cells without mercury induction. These results suggest that the synthesis of polyphosphate as well as the expression of the mer genes is mercury-inducible and regulated by merR. The E. coli strain with pMKB18 was more resistant to both Hg2+ and C6H5Hg+ than its isogenic strain with cloning vector pUC119. The recombinant strain accumulated more mercury from Hg2+- and C6H5Hg+-contaminated medium. Hg2+ transported into the cytoplasm appeared to be bound by chelation with the polyphosphate produced by the recombinant cells. The transported phenylmercury was degraded to Hg2+ before the chelation since polyphosphate did not directly chelate with C6H5Hg+. These results indicate that polyphosphate is capable of reducing the cytotoxicity of the transported Hg2+ probably via chelation between polyphosphate and Hg2+.     

Polypeptides specified by the mercuric resistance (mer) operon of plasmid R100

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.     

Deletions in the r-determinant mer region of plasmid R100-1 selected for loss of mercury hypersensitivy.

A mutant of plasmid R100-1, which conferred cellular hypersensitivity to Hg2+ because of the insertion of Tn801 (TnA) into the gene determining synthesis of mercuric reductase enzyme, allowed further mutational events to be selected which resulted in either reversion to Hg2+ resistance (characteristic plasmid R100-1) or sensitivity at a level characteristic of plasmidless strains. Restriction endonuclease EcoRI and BamHI analysis showed that reversion to resistance resulted from loss of TnA from the R100-mer:Tn801 plasmid, whereas the change from hypersensitivity to sensitivity to Hg2+ usually resulted from deletion of part or all of Tn801 plus plasmid deoxyribonucleic acid sequences corresponding to the operator-proximal end of the mer operon.     

Cloning a promoter that puts the expression of tetracycline resistance under the control of the regulatory elements of the mer operon

We have sub-cloned from the Eco RI-H fragments of the IncFII plasmid R100 a 260-bp EcoRI fragment, using the promoter-cloning vehicle, pBRH4, (The Inc FII plasmid codes for the mer operon, and pBRH4 expresses tetracycline resistance only when the deleted tet promoter has been replaced by another sequence that can serve as a promotor). With the 260-bp fragment inserted, the derivative plasmid, pFB4, directs the expression of tetracycline resistance only if there is a second plasmid in the strain that carries the merR-positive regulatory element. Under these conditions, the level of tetracycline resistance is directly proportional to the concentration of Hg2+ present in the medium. The 260-bp fragment also allows low-level constitutive expression of tet resistance when transactivated with merR mutants that have a "micro-constitutive" phenotype. The 260-bp mer promoter fragment contains a single HincII site; there is also but one HincII site in the EcoRI-H fragment of R100 from which the promoter fragment was derived. Restriction analysis of purified Eco RI-H DNA shows that the single HincII site is at 550 bp from the "right"terminus of the IS1b element, which is also present in the EcoRI-H fragment. Because of its biological activity and its location within the "H" fragment, this promoter is very likely a promoter for the structural genes of the operon.     

Identification of three merB genes and characterization of a broad-spectrum mercury resistance module encoded by a class II transposon of Bacillus megaterium strain MB1.

The complete structure of a broad-spectrum mercury resistance module was shown by sequencing the Gram-positive bacterial transposon TnMERI1 of Bacillus megaterium MB1. The regions encoding organomercury resistance were identified. Upstream of a previously identified organomercurial lyase merB (merB1) region of TnMERI1, a second merR (merR2) and a second merB gene (merB2) were found. These genes constitute a second operon (mer operon 2) following a promoter/operator (P(merR2)) region. A third organomercurial lyase gene (merB3) was found immediately upstream of the mer operon (mer operon 1) followed by a promoter/operator (P(merB3)) region homologous to that of the mer operon 1 (P(merR1)-merR1-merE-like-merT-merP-merA). The complete genetic structure of the mercury resistance module is organized as P(merB3)-merB3-P(merR1)-merR1-merE-like-merT+ ++ -merP-merA-P(merR2)-merR2 -merB2-merB1. The subcloning analysis of these three merB genes showed distinct substrate specificity as different organomercury lyase genes.     

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

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, 15.1, 14 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, Tn501 [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).     

Overexpression and DNA-binding properties of the mer-encoded regulatory protein from plasmid NR1 (Tn21).

In plasmid NR1 the expression of genes involved in mercury resistance (Tn21) is regulated by the trans-acting product of the merR gene. An in vivo T7 RNA polymerase-promoter overexpression system was used to detect a protein of approximately 16,000 daltons encoded by the merR reading frame. Overexpressed MerR constituted about 5% of labeled proteins. An in vitro MerR-mer-op (mer-op is the mer operator and promoter region) gel electrophoresis binding assay established that the binding site for MerR was located between the putative -35 and -10 sequences of the promoter for the mer structural genes. A nonsense mutation in the carboxyl half of MerR resulted in the loss of biological function and the loss of in vitro mer-op binding properties.     

Simultaneous detection and removal of organomercurial compounds by using the genetic expression system of an organomercury lyase from the transposon Tn<Emphasis Type="Italic">MERI1</Emphasis>

Using a newly identified organomercury lyase gene (merB3) expression system from Tn MERI1, the mercury resistance transposon first found in Gram-positive bacteria, a dual-purpose system to detect and remove organomercurial contamination was developed. A plasmid was constructed by fusing the promoterless luxAB genes as bioluminescence reporter genes downstream of the merB3 gene and its operator/promoter region. Another plasmid, encoding mer operon genes from merR1 to merA, was also constructed to generate an expression regulatory protein, MerR1, and a mercury reductase enzyme, MerA. These two plasmids were transformed into Escherichia coli cells to produce a biological system that can detect and remove environmental organomercury contamination. Organomercurial compounds, such as neurotoxic methylmercury at nanomolar levels, were detected using the biomonitoring system within a few minutes and were removed during the next few hours.     

The meroperon of a mercury-resistant Pseudoalteromonas haloplanktis strain isolated from Minamata Bay, Japan

A mer operon of mercury-resistant Pseudoalteromonas haloplanktis strain M1, isolated from sea water of Minamata Bay, was cloned and analyzed. The mer genes were located in the chromosome and organized as merR-merT-merP-merC-merA-merD, the same order as that in Tn21. However, the orientation of the merR gene is the same as that of other mer genes (opposite direction to Tn21), and merR was cotranscribed with other mer genes, a pattern that has not been previously seen with mer determinants from other Gram-negative bacteria. Furthermore, the amino acid similarities of the corresponding mer gene products between those from strain M1 and Tn21 were unusually low.     

Effect of gene amplification on mercuric ion reduction activity of Escherichia coli.

The mercury resistance (mer) operon of plasmid R100 was cloned onto various plasmid vectors to study the effect of mer gene amplification on the rate of Hg2+ reduction by Escherichia coli cells. The plasmids were maintained at copy numbers ranging from 3 to 140 copies per cell. The overall Hg2+ reduction rate of intact cells increased only 2.4-fold for the 47-fold gene amplification. In contrast, the rate of the cytoplasmic reduction reaction, measured in permeabilized cells, increased linearly with increasing gene copy number, resulting in a 6.8-fold overall amplification. RNA hybridizations indicated that mRNA of the cytoplasmic mercuric reductase (merA gene product) increased 11-fold with the 47-fold gene amplification, while mRNA of the transport protein (merT gene product) increased only 5.4-fold. Radiolabeled proteins produced in maxicells were used to correlate the expression levels of the mer polypeptides with the measured reduction rates. The results indicated that, with increasing gene copy number, there was an approximately 5-fold increase in the merA gene product compared with a 2.5-fold increase in the merT gene product. These data demonstrate a parallel increase of Hg2+ reduction activity and transport protein expression in intact cells with plasmids with different copy numbers. In contrast, the expression level of the mercuric reductase gene underwent higher amplification than that of the transport genes at both the RNA and protein levels as plasmid copy number increased.