Search

MerC, encoded by merC in the transposon Tn21 mer operon, is a heavy metal transporter with potential applications for phytoremediation of heavy metals such as mercuric ion and cadmium. In this study, we demonstrate that MerC also acts as a transporter for methylmercury. When MerC was expressed in Escherichia coli XL1-Blue, cells became hypersensitive to CH3Hg(I) and the uptake of CH3Hg(I) by these cells was higher than that by cells of the isogenic strain. Moreover, transgenic Arabidopsis plants expressing bacterial MerC or MerC fused to plant soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) accumulated CH3Hg(I) effectively and their growth was comparable to the wild-type plants. These results demonstrate that when the bacterium-derived merC gene is ectopically introduced in genetically modified plants, MerC expression in the transgenic plants promotes the transport and sequestration of methylmercury. Thus, our results show that the expression of merC in Arabidopsis results in transgenic plants that could be used for the phytoremediation and elimination of toxic methylmercury from the environment.

The characteristics of bacteria take up mercury into cells via membrane potential-dependent sequence-divergent members of the mercuric ion (Mer) superfamily, i.e., a periplasmic mercuric ion scavenging protein (MerP) and one or more inner membrane-spanning proteins (MerC, MerE, MerF, and MerT), which transport mercuric ions into the cytoplasm, have been applied in engineering of bioreactor used for mercurial bioremediation. We engineered bacteria to express MerC, MerE, MerF, or MerT with or without MerP to clarify their individual role and potential in transport of mercurial. By immunoblot analysis using specific polyclonal antibody, the proteins encoded by merC, merE, merF, merT or merP, were certainly expressed and identified in the membrane fraction. Bacteria expressing MerC, MerE, MerF or MerT in the absence of MerP transported significantly more C6H5Hg(I) and Hg(II) across bacterial membrane than their isogenic strain. In vivo expression of MerP in the presence of all the transporters did not cause apparent difference to the C6H5Hg(I) transport, but gives an apparently higher Hg(II) transport than that did by MerE, MerF or MerT but not by MerC. Among the four transporters studied, MerC showed more potential to transport Hg(II) across bacterial membrane than MerE, MerF and MerT. Together these findings, we demonstrated for the first time that in addition to MerE and MerT, MerF and MerC are broad-spectrum mercury transporters that mediate both Hg(II) and phenylmercury transport into cells. Our results suggested that MerC is the most efficient tool for designing mercurial bioremediation systems, because MerC is sufficient for mercurial transport into cells.

The feasibility of transgenic tobacco, engineered to express bacterial polyphosphate (polyP), for phytoremediation of mercury pollution was evaluated. T3 progeny of the transgenic tobacco produced a large amount of polyP in leaves and showed a relatively high resistant phenotype to Hg2+ than its wild-type progenitors. These results suggest that the integrated ppk gene, encoding polyphosphate kinase (PPK), a key enzyme for polyP biosynthesis, is stably conserved in tobacco genome, and translated to active PPK which catalyzed biosynthesis of polyP, and suggest that polyP is capable of reducing the cytotoxicity of Hg2+, probably via chelation formation with polyP. The transgenic tobacco expressing polyP accumulated significantly more mercury than its wild-type progenitors from Hg2+-containing agar medium and simulated soils without taxing the tobacco plants suggesting that the transported Hg2+ was accumulated as a less toxic Hg-polyP complex in the tobacco tissues. Based on the results obtained in the present study, the polyP-mediated accumulation of mercury from mercurial-contaminated soils may provide an ecologically compatible approach for phytoremediation of mercury pollution.

To prevent environmental mercury poisoning incidents, an effective technology for treating mercury-polluted environments is urgent. Recently, with advances in biotechnology, bioremediation utilizing microorganisms to remove mercurials from contaminated sites has become one of the most rapidly developing fields of environmental restoration. A number of bioremediation strategies, including biotransformation, biosorption, and bioprecipitation of mercurials, have been developed to treat mercurial-polluted environments and mercury-containing waste. To construct bacteria that are capable of specifically accumulating mercury, we have genetically engineered Escherichia coli to express a mercury transport system and organomercurial lyase enzyme simultaneously, and overexpress polyphosphate, a strong chelator of essential divalent metals. The mercury accumulation system was designed so that overexpressed polyphosphate would serve as a mercury accumulator; the mercury transport system would make the bacterial cell specifically accumulate mercury; and the intracellular accumulation process would allow the bioaccumulation system to be less sensitive to ambient conditions. The applicability of the new engineered bacteria in the environmental remediation of mercurials is evaluated and discussed in this review.

A new whole-cell bacterial sensor for the detection of low concentration of mercury in environment was constructed by gene fusion between a mercury resistance (mer) operon from pMR26 of Pseudomonas strain K-62 and a promoterless luxAB gene from Vibrio harveyi. The luminescence-based biosensor was evaluated for the selectivity and sensitivity of the detection of mercury. Cadmium, lead, chromium and zinc ions did not interfere with the assay even at same concentration compared to Hg2+. Methylmercury, phenylmercury and mercuric sulfide also did not affect the biosensor. These results reveal that the specificity of the construct is restricted to bioavailable Hg2+. The sensitivity of the biosensor was improved by decreasing the cell density in the bioassay in addition to genetically expressing an Hg2+ transport system which was expected to increase the amount of mer operon-inducing mercury in the cytoplasm. In optimized assay conditions, the lowest detectable concentration of Hg2+ was 2 pM with 1 ml sample. This detection limit is enough to detect this compound in many contaminated and some pristine environmental samples.

To investigate the individual role of MerT and MerP encoded by Pseudomonas K-62 pMR26 in the transport of phenylmercury, a series of mutants with a specific point mutation in merT and/or genetic deletion in merP were constructed and transformed into Escherichia coil XL-1-Blue. Transport of phenylmercury across the cytoplasmic membrane of E. coli mediated by MerT and MerP was inhibited by NaCN and by cold temperatures. Deletion of merP reduced, but did not completely abolish the C6H5Hg+-hyperuptake and -hypersensitive phenotypes suggesting that transport of phenylmercury into the cytoplasm of E. coli is still occurring. Mutations of the vicinal cysteine residues (Cys24 and Cys25) in the first transmembrane region of MerT to serine caused complete loss of Hg2+-hyperuptake and -hypersensitivity, whereas the mutations did not affect the C6H5Hg+-hyperuptake and -hypersensitive phenotypes. In addition, no additive effect on the C6H5Hg+-hyperuptake and -hypersensitive phenotypes was found, when mutations of the two cysteines in MerT to serine were further introduced in the merP-deleted mutants. These results clearly demonstrated that the vicinal cysteine residues of MerT are not involved in the transport of C6H5Hg+, but indeed are involved in the transport of Hg2+ as previously reported.

The inducibility by organomercury of the broad-spectrum mer operon on pMRA17 cloned from Pseudomonas K-62 plasmid pMR26 was assessed in the absence of a functional merB gene. The mer polypeptides encoded by the mer genes on pMRA17 were almost identified in maxicell induced not only by Hg2+ but also by C6H5Hg+ or CH3Hg+. Maxicell with pMRD103, a merB-deletion plasmid constructed from pMRA17, also produced the corresponding mer polypeptides when maxicell was induced by Hg2+, whereas no mer polypeptides were detected in the maxicell induced by C6H5Hg+ or CH3Hg+. These results suggest that merB is needed for induction of the pMRA17 mer operon expression by organomercurials. Next, to test the inducibility of pMRA17 mer operon expression from its own promoter, a promoterless lacZ was fused with the mer operon, where merB was deleted in plasmid pB43merlacZ. Only Hg2+, but not C6H5Hg+ or CH3Hg+, can activate β-galactosidase expression in bacteria with pB43merlacZ. These results not only imply that the pMRA17 MerR is a narrow-spectrum regulator that did not recognize organomercury as a direct inducer, but also confirms that merB is required for induction of the pMRA17 mer operon expression by organomercurials.

pMRB01 cloned from Pseudomonas K-62 plasmid pMR26 conferred bacterial hypersensitivity to organomercurials. DNA sequence analysis of a 2.3-kb SacI-Aor51HI fragment encompassing the whole region required for expression of the hypersensitive phenotype, revealed three open reading frames. The DNA sequence of these frames had 82.5%, 99.2% and 97.0% homology with the pDU1358 merR, merB and merD, respectively. The pMRB01 mer operon differs from the already known mer operon by the absence of the merT, merP and merA genes in this plasmid. An inverted repeat-like sequence upstream from the predicted merR was observed suggesting that this defective mer operon could be part of a transposon-like structure. Induction experiments and maxicell analysis of the mer-polypeptide showed that the lyase enzyme encoded by pMRB01 merB gene is mercurial-inducible and regulated by the transacting product of the merR gene. These results suggest that the hypersensitivity to organomercurials resulted from the expression of lyase activity encoded by the defective mer operon in the absence of reductase activity. The lyase enzyme encoded by pMRB01 merB catalyzes the protonolysis of the C-Hg bond of both arylmercury and alkylmercury compounds.

To investigate the role of aromatic amino acids of MerT protein in phenylmercury transport, two merT variants (pMRTm1P and pMRTm2P) with specific site-directed mutations were constructed and examined their effects on C6H5Hg+-transport. Substitution of Phe-36 and Trp-40 residues located on the periplasmic loop of MerT in turn with Ala and Val, respectively, did not affect the Hg2+-uptake, but caused a significant reduction in the C6H5Hg+-uptake by bacterial cells with intact merT gene. Introduction of specific mutations changing Phe-108, 114, 115 to Ala, and Tyr-110, 116 to Ser in the C-terminal region of the third transmembrane of MerT also caused large reduction in the uptake of C6H5Hg+, but had no effect on the Hg2+-uptake. In addition, both mutations caused a significant reduction in the hypersensitivity to C6H5Hg+, but without affecting the Hg2+-hypersensitive phenotype. Together these results suggest that the aromatic amino acids of MerT protein may play an important role in the transport of C6H5Hg+ across the cell membrane.

The merB-merA-deleted plasmid pMRD141 which contains the intact merT-merP genes of pMRA17 conferred bacterial hypersensitivity not only to Hg2+ but also to C6H5Hg+. The bacterium with pMRD141 took up significantly more C6H5Hg+ than its isogenic strain with the cloning vector Bluescript II. The hypersensitivity to C6H5Hg+ seems to be based on hyperaccumulation of toxic C6H5Hg+ in the absence of detoxifying enzymes encoded by merB and merA. Our results show that bacterial transport of C6H5Hg+ into the cytoplasm is regulated by merT-merP genes.