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.     

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.     

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.     

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.     

Tn5037-a Tn21-like mercury transposon, detected in Thiobacillus ferrooxidans

The 6645-bp mercury resistance transposon of the chemolithotrophic bacterium Thiobacillus ferrooxidans was cloned and sequenced. This transposon, named Tn5037, belongs to the Tn21 branch of the Tn21 subgroup, many members of which have been isolated from clinical sources. Having the minimum set of the genes (merRTPA), the mercury resistance operon of Tn5037 is organized similarly to most of the Gram-negative bacteria mer operons and is closest to that of Thiobacillus 3.2. The operator-promoter region of the mer operon of Tn5037 also has the common (Tn21/Tn501-like) structure. However, its inverted, presumably MerR protein binding repeats in the operator/promoter element are two base pairs shorter than in Tn21/Tn501. In the merA region, this transposon shares 77.4, 79.1, 83.2 and 87.8% identical bases with Tn21, Tn501, T. ferrooxidance E-15, and Thiobacillus 3.2, respectively. No inducibility of the Tn5037 mer operon was detected in the in vivo experiments. The transposition system (terminal repeats plus gene tnpA) of Tn5037 was inactive in Escherichia coli K12, in contrast to its resolution system (res site plus gene tnpR). However, transposition of Tn5037 in this host was provided by the tnpA gene of Tn5036, a member of the Tn21 subgroup. Sequence analysis of the Tn5037 res site suggested its recombinant nature.     

Translation of merD in Tn21.

All four sequenced examples of the mercury resistance (mer) operon of gram-negative bacteria have a promoter-distal reading frame, merD, whose removal has little effect on the resistance phenotype and whose translation has not previously been observed. Using merD-lacZ protein fusions, we show that merD is translated. However, Hg(II)-induced merD expression, as measured by beta-galactosidase activity and immunoblotting, is 10- to 15-fold lower than that of fusions to the gene immediately preceding it, merA.