Cysteine and histidine residues are involved in Tn MerE methylmercury transport.

Bacterial resistance to mercury compounds (mercurials) is mediated by proteins encoded by mercury resistance () operons. Six variants with site-directed mutations were constructed to investigate the roles of the cysteine and histidine residues in MerE protein during mercurial transport. By comparison of mercurial uptake by the cell with intact and/or variant MerE, we showed that the cysteine pair in the first transmembrane domain was critical for the transport of both Hg(II) and CH Hg(I).

A Novel Role of MerC in Methylmercury Transport and Phytoremediation of Methylmercury Contamination.

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 CHHg(I) and the uptake of CHHg(I) by these cells was higher than that by cells of the isogenic strain.

Increase methylmercury accumulation in Arabidopsis thaliana expressing bacterial broad-spectrum mercury transporter MerE.

The bacterial merE gene derived from the Tn21 mer operon encodes a broad-spectrum mercury transporter that governs the transport of methylmercury and mercuric ions across bacterial cytoplasmic membranes, and this gene is a potential molecular tool for improving the efficiency of methylmercury phytoremediation. A transgenic Arabidopsis engineered to express MerE was constructed and the impact of expression of MerE on methylmercury accumulation was evaluated. The subcellular localization of transiently expressed GFP-tagged MerE was examined in Arabidopsis suspension-cultured cells.

Role of MerC, MerE, MerF, MerT, and/or MerP in resistance to mercurials and the transport of mercurials in Escherichia coli.

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.

Mercurial-resistance determinants in Pseudomonas strain K-62 plasmid pMR68.

We report the complete nucleotide sequence of plasmid pMR68, isolated from Pseudomonas strain K-62, two plasmids contribute to broad-spectrum mercury resistance and that the mer operon from one of them (pMR26) has been previously characterized. The plasmid was 71,020 bp in length and contained 75 coding regions. Three mer gene clusters were identified.

Expression of the bacterial heavy metal transporter MerC fused with a plant SNARE, SYP121, in Arabidopsis thaliana increases cadmium accumulation and tolerance.

The bacterial merC gene from the Tn21-encoded mer operon is a potential molecular tool for improving the efficiency of metal phytoremediation. Arabidopsis SNARE molecules, including SYP111, SYP121, and AtVAM3 (SYP22), were attached to the C-terminus of MerC to target the protein to various organelles. The subcellular localization of transiently expressed GFP-fused MerC-SYP111, MerC-SYP121, and MerC-AtVAM3 was examined in Arabidopsis suspension-cultured cells.

[Application of mercury-resistant genes in bioremediation of mercurials in environments].

Mercury and its organic compounds, especially methylmercury are extremely hazardous pollutants that have been released into the environment in substantial quantities by natural events and anthropogenic activities. Due to the acute toxicity of these contaminants, there is an urgent need to develop an effective and affordable technology to remove them from the environments. Recently, attempts have been made to utilize bacterial mer operon-mediated reduction and volatilization of mercurials for environmental remediation of mercury pollution.

Development of a luminescence-based biosensor for detection of methylmercury.

A recombinant whole-cell bacterial sensor for highly selective and sensitive detection of the bioavailable methylmercury in the environment was constructed. The biosensor carries luciferase gene, luxAB, from Vibrio harveyi as a reporter under the control of the mercury inducible regulatory part of mer-operon from Pseudomonas K-62 plasmid pMR26. In addition, a merB gene encoding organomercurial lyase which cleaves the C-Hg bond of methylmercury to give Hg(2+ )was coexpressed in the sensor.

Engineering expression of the heavy metal transporter MerC in Saccharomyces cerevisiae for increased cadmium accumulation.

The merC gene from the Tn21-encoded mer operon has potential uses as a molecular tool for bioremediation. It was overexpressed as the fusion proteins MerC-Sso1p or MerC-Vam3p in Saccharomyces cerevisiae. Green fluorescent protein (GFP)-MerC-Sso1p fusion proteins located primarily in the plasma membrane, although some protein was detected in the endoplasmic reticulum.

Genetic engineering of transgenic tobacco for enhanced uptake and bioaccumulation of mercury.

To further enhance the efficiency and potential of plants for phytoremediation of mercury pollution, a genetically engineered tobacco to simultaneously express mercury transporter, mercury transporter (MerT) and mercury chelator, polyphosphate (polyP) was constructed by integrating bacterial merT gene in polyphosphate kinase gene (ppk)-transgenic tobacco, and its ability to phytoremediate mercury was evaluated. Integration of merT gene into ppk-transgenic tobacco did not significantly affect the mercury resistant phenotypes and polyP production. Transgenic expression of MerT in ppk-transgenic tobacco resulted in accelerated and enhanced mercury uptake into tobacco.

The MerE protein encoded by transposon Tn21 is a broad mercury transporter in Escherichia coli.

In order to clarify the physiological role of the merE gene of transposon Tn21, a pE4 plasmid that contained the merR gene of plasmid pMR26 from Pseudomonas strain K-62, and the merE gene of Tn21 from the Shigella flexneri plasmid NR1 (R100) was constructed. Bacteria with plasmid pE4 (merR-o/p-merE) were more hypersensitive to CH(3)Hg(I) and Hg(II), and took up significantly more CH(3)Hg(I) and Hg(II), than the isogenic strain. The MerE protein encoded by pE4 was localized in the membrane cell fraction, but not in the soluble fraction.

Engineering expression of polyphosphate confers cadmium resistance in tobacco.

The feasibility of transgenic tobacco, genetically engineered to express bacterial polyphosphate (polyP) for phytoremediation of cadmium pollution was examined. The transgenic tobacco showed more resistance to Cd2+ and accumulated more Cd2+ than its wild-type progenitors. These results suggest that polyP has abilities to reduce Cd2+ toxicity, probably via a chelation mechanism, and to accumulate cadmium in the transgenic tobacco.

Hepatic changes in adenine nucleotide levels and adenosine 3'-monophosphate forming enzyme in streptozotocin-induced diabetic mice.

To elucidate the pathophysiological significance of adenosine 3'-monophosphate (3'-AMP) forming enzyme in mice, the effect of streptozotocin (STZ) on the enzyme activities and adenine nucleotide levels in the ICR mice (4-week-old) liver was examined. After 2 weeks, treatment with a single dosage of STZ (100, 150 or 200 mg/kg i.p.

Electrochemical behaviors of sulfhydryl compounds in the presence of elemental mercury.

In the expression of bioaccumulated elemental mercury (Hg 0) toxicity, the first Hg 0 oxidation step is crucial. Therefore, to clarify the mechanism underlying the interactions of sulfhydryl (SH) compounds and Hg 0 in the present study, we analyzed the oxidation of reduced glutathione (GSH) and L-cysteine (Cys) in the presence of Hg 0 in aqueous solution by cyclic voltammetry (CV). Production of Hg2+ in the reaction mixture was found to increase along with a decrease in free SH residues.

Accumulation of mercury in transgenic tobacco expressing bacterial polyphosphate.

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.

Engineering expression of bacterial polyphosphate kinase in tobacco for mercury remediation.

To develop the potential of plants to sequester and accumulate mercurials from the contaminated sites, we engineered a tobacco (Nicotiana tabacum) plant to express a bacterial ppk gene, encoding polyphosphate kinase (PPK), under control of a plant promoter. The designated plant expression plasmid pPKT116 that contains the entire coding region of ppk was used for Agrobacterium-mediated gene transfer into tobacco plants. A large number of independent transgenic tobacco plants were obtained, in some of which the ppk gene was stably integrated in the plant genome and substantially translated to the expected PPK protein in the transgenic tobacco.

Enhancement of cellular adenosine triphosphate levels in PC12 cells by 2,5-dideoxyadenosine, a P-site inhibitor of adenylate cyclase.

To elucidate the biological significance of the P-site inhibitor of adenylate cyclase, the effect of 2,5-dideoxyadenosine (DDA) on cellular levels of adenine compounds in PC12 cells was studied. The addition of DDA and deoxyadenosine (deoxyAdo), P-site inhibitors of adenylate cyclase, as well as the addition of adenosine (Ado) to the incubation medium containing glucose as the sole nutrient significantly enhanced cellular ATP levels in PC12 cells. N6-Methyladenosine and N6-cyclohexyladenosine did not augment the ATP levels.

Effect of iron lactate overloading on adenine nucleotide levels and adenosine 3'-monophosphate forming enzyme in rat liver and spleen.

To elucidate the pathophysiological significance of adenosine 3'-monophosphate (3'-AMP) forming enzyme in rats, the effect of iron lactate overloading on the enzyme activities and adenine nucleotide levels in the liver and spleen was examined. Sprague-Dawley rats were fed a diet supplemented with 0%, 0.625% or 5.

A screening system of prodrugs selective for MAO-A or MAO-B.

We synthesized several prodrugs of glycine and gamma-aminobutyric acid. In order to establish a screening system from the prodrugs of selective activity to MAO-A or MAO-B, we examined purification conditions such as solubilization with Triton X-100, precipitation with ammonium sulfate, gel filtration and anion exchange chromatography. MAO-B was purified from various tissues such as guinea pig brain, kidney and spleen.

Polyphosphate produced in recombinant Escherichia coli confers mercury resistance.

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.

Enhancement of cellular adenosine triphosphate levels in PC12 cells by extracellular adenosine.

To elucidate the biological significance of extracellular adenine compounds, the effects of adenosine (Ado) on cellular levels of adenine compounds, especially adenosine triphosphate (ATP), in PC12 cells were studied. Ado and inosine but not adenosine 5'-monophosphate, adenosine 5'-diphosphate, ATP, guanosine, cytosine, thymidine, and uridine, significantly enhanced cellular ATP levels in PC12 cells in time- and dose-dependent manners. Various P1 receptor agonists of Ado did not enhance the ATP level.