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.     

Deletion mutant analysis of the Staphylococcus aureus plasmid pI258 mercury-resistance determinant

Deletion mutant analysis of the mercury-resistant determinant (mer operon) from the Staphylococcus aureus plasmid pI258 was used to verify the location of the merA and merB genes and to show the existence of mercuric ion transport gene(s). ORF5 was confirmed to be a transport gene and has an amino acid product sequence homologous to the merT gene products from several gram-negative bacteria and a Bacillus species. Deletion analysis established that inactivation of merA on a broad-spectrum mer resistance determinant resulted in a mercury-hypersensitive phenotype. Gene dosage had no apparent effect on the level of resistance conferred by the intact mer operon or on the expression of an inducible phenotype, except that when the intact pI258 mer operon was on a high copy number plasmid, uninduced cells possessed a volatilization rate that was at most only 3.5-fold less than that observed for induced cells. There was no need for mercury ion transport proteins for full resistance when the mer operon was expressed in a high copy number plasmid.     

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.     

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.     

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.     

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).     

Operon mer: bacterial resistance to mercury and potential for bioremediation of contaminated environments

Mercury is present in the environment as a result of natural processes and from anthropogenic sources. The amount of mercury mobilized and released into the biosphere has increased since the beginning of the industrial age. Generally, mercury accumulates upwards through aquatic food chains, so that organisms at higher trophic levels have higher mercury concentrations. Some bacteria are able to resist heavy metal contamination through chemical transformation by reduction, oxidation, methylation and demethylation. One of the best understood biological systems for detoxifying organometallic or inorganic compounds involves the mer operon. The mer determinants, RTPCDAB, in these bacteria are often located in plasmids or transposons and can also be found in chromosomes. There are two classes of mercury resistance: narrow-spectrum specifies resistance to inorganic mercury, while broad-spectrum includes resistance to organomercurials, encoded by the gene merB. The regulatory gene merR is transcribed from a promoter that is divergently oriented from the promoter for the other mer genes. MerR regulates the expression of the structural genes of the operon in both a positive and a negative fashion. Resistance is due to Hg2+ being taken up into the cell and delivered to the NADPH-dependent flavoenzyme mercuric reductase, which catalyzes the two-electron reduction of Hg2+ to volatile, low-toxicity Hg0. The potential for bioremediation applications of the microbial mer operon has been long recognized; consequently, Escherichia coli and other wild and genetically engineered organisms for the bioremediation of Hg2+-contaminated environments have been assayed by several laboratories.     

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.     

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+.