Evidence for direct interactions between the mercuric ion transporter (MerT) and mercuric reductase (MerA) from the Tn<Emphasis Type="Italic">501</Emphasis><Emphasis Type="Italic">mer</Emphasis> operon

Mercuric ion resistance in bacteria requires transport of mercuric ions (Hg²⁺) into the cytoplasmic compartment where they are reduced to the less toxic metallic mercury (Hg⁰) by mercuric reductase (MR). The long-established model for the resistance mechanism predicts interactions between the inner membrane mercuric ion transporter, MerT, and the N-terminal domain of cytoplasmic MR, but attempts to demonstrate this interaction have thus far been unsuccessful. A recently developed bacterial two-hybrid protein interaction detection system was used to show that the N-terminal region of MR interacts with the cytoplasmic face of MerT. We also show that the cysteine residues on the cytoplasmic face of the MerT protein are required for maximal mercuric ion transport but not for the interaction with mercuric reductase.     

Sequence and analysis of a plasmid-encoded mercury resistance operon from Mycobacterium marinum identifies MerH, a new mercuric ion transporter

In this study, we report the DNA sequence and biological analysis of a mycobacterial mercury resistance operon encoding a novel Hg²⁺ transporter. MerH was found to transport mercuric ions in Escherichia coli via a pair of essential cysteine residues but only when coexpressed with the mercuric reductase.     

Pseudomonas putida Strains Which Constitutively Overexpress Mercury Resistance for Biodetoxification of Organomercurial Pollutants

Improved biocatalysts for mercury (Hg) remediation were generated by random mutagenesis of Pseudomonas putida with a minitransposon containing merTPAB, the structural genes specifying organomercury resistance. Subsequent selection for derivatives exhibiting elevated resistance levels to phenylmercury allowed the isolation of strains that constitutively express merTPAB at high levels, conferring the ability to cleave Hg from an organic moiety and reduce the freed Hg(II) to the less toxic elemental form, Hg⁰, at greater rates. Constitutive overexpression of merTPAB had no apparent effect on culture growth rates, even when Hg(II) was initially present at otherwise toxic concentrations. These properties were also combined with benzene and toluene catabolism, allowing detoxification of the metal component of phenyl mercuric acetate, as well as degradation of its aromatic moiety.     

Tn1 generated mutants in the mercuric ion reductase of the Inc P plasmid, R702

Summary Physiological, biochemical and genetic aspects of resistance to inorganic mercury compounds were examined in a group of mercury sensitive derivatives generated in the Inc P plasmid, R702, by Tn1 insertion. Strains carrying each of these insertion mutations had no detectable mercuric ion reductase, were more sensitive to mercuric ion than a plasmidless strain, and exhibited inducible uptake of Hg²⁺. These characteristics indicate that the mutants are altered in the Hg(II) reductase. This hypothesis was supported by complementation and recombination analysis with known point and deletion mutations in the mer operon of the Inc FII plasmid, R100. Such experiments showed that the eight insertions studied had occurred in four distinct regions of the Hg(II) reductase structural gene (merA). Complementation data also demonstrated that the regulatory protein determined by the R702 plasmid has no effect on the expression of the micro-constitutive Hg(II) reductase activity expressed by merR mutants of R100.     

Structural characterization of intramolecular Hg(2+) transfer between flexibly linked domains of mercuric ion reductase

The enzyme mercuric ion reductase MerA is the central component of bacterial mercury resistance encoded by the mer operon. Many MerA proteins possess metallochaperone-like N-terminal domains (NmerA) that can transfer Hg²⁺ to the catalytic core domain (Core) for reduction to Hg⁰. These domains are tethered to the homodimeric Core by ∼ 30-residue linkers that are susceptible to proteolysis, the latter of which has prevented characterization of the interactions of NmerA and the Core in the full-length protein. Here, we report purification of homogeneous full-length MerA from the Tn21 mer operon using a fusion protein construct and combine small-angle X-ray scattering and small-angle neutron scattering with molecular dynamics simulation to characterize the structures of full-length wild-type and mutant MerA proteins that mimic the system before and during handoff of Hg²⁺ from NmerA to the Core. The radii of gyration, distance distribution functions, and Kratky plots derived from the small-angle X-ray scattering data are consistent with full-length MerA adopting elongated conformations as a result of flexibility in the linkers to the NmerA domains. The scattering profiles are best reproduced using an ensemble of linker conformations. This flexible attachment of NmerA may facilitate fast and efficient removal of Hg²⁺ from diverse protein substrates. Using a specific mutant of MerA allowed the formation of a metal-mediated interaction between NmerA and the Core and the determination of the position and relative orientation of NmerA to the Core during Hg²⁺ handoff.     

Irreversible kinetics of β-N-acetyl-d-glucosaminidase from prawn (Penaeus vannamei) inactivated by mercuric ion

Cysteine residues in prawn (Penaeus vannamei) β-N-acetyl-d-glucosaminidase (NAGase, EC 3.2.1.52) have been modified by p-chloromercuribenzoate (PCMB). The results show that sulfhydryl group is essential for the activity of the enzyme. Inactivation kinetics of the enzyme by mercuric chloride (HgCl₂) has been studied using the kinetic method of the substrate reaction during inactivation of enzyme previously described by Tsou. The kinetic results show that the inactivation of the enzyme is an irreversible reaction. The microscopic rate constants for the reaction of Hg²⁺ with free enzyme and with the enzyme-substrate complex are determined. Comparison of these rate constants indicates that the presence of substrate offers marked protection of this enzyme against inactivation by Hg²⁺. The above results suggest that the cysteine residue is essential for activity.     

Protein Method for Investigating Mercuric Reductase Gene Expression in Aquatic Environments

A colorimetric assay for NADPH-dependent, mercuric ion-specific oxidoreductase activity was developed to facilitate the investigation of mercuric reductase gene expression in polluted aquatic ecosystems. Protein molecules extracted directly from unseeded freshwater and samples seeded with Pseudomonas aeruginosa PU21(Rip64) were quantitatively assayed for mercuric reductase activity in microtiter plates by stoichiometric coupling of mercuric ion reduction to a colorimetric redox chain through NADPH oxidation. Residual NADPH was determined by titration with phenazine methosulfate-catalyzed reduction of methyl thiazolyl tetrazolium to produce visible formazan. Spectrophotometric determination of formazan concentration showed a positive correlation with the amount of NADPH remaining in the reaction mixture (r² = 0.99). Mercuric reductase activity in the protein extracts was inversely related to the amount of NADPH remaining and to the amount of formazan produced. A qualitative nitrocellulose membrane-based version of the method was also developed, where regions of mercuric reductase activity remained colorless against a stained-membrane background. The assay detected induced mercuric reductase activity from 10² CFU, and up to threefold signal intensity was detected in seeded freshwater samples amended with mercury compared to that in mercury-free samples. The efficiency of extraction of bacterial proteins from the freshwater samples was (97 ± 2)% over the range of population densities investigated (10² to 10⁸ CFU/ml). The method was validated by detection of enzyme activity in protein extracts of water samples from a polluted site harboring naturally occurring mercury-resistant bacteria. The new method is proposed as a supplement to the repertoire of molecular techniques available for assessing specific gene expression in heterogeneous microbial communities impacted by mercury pollution.     

The relationships of Hg(II) volatilization from a freshwater pond to the abundance ofmer genes in the gene pool of the indigenous microbial community

The role of biological activities in the reduction and volatilization of Hg(II) from a polluted pond was investigated. Elemental mercury was evolved from pond water immediately following spiking with²⁰³Hg(NO₃)₂, whereas an acclimation period of 36 hours was required in control samples collected from a nearby, unpolluted river before onset of volatilization. Genes encoding the bacterial mercuric reductase enzyme (mer genes) were abundant in DNA fractions extracted from biomass of the pond microbial community, but not in samples extracted from control communities. Thus, evolution of Hg⁰ was probably due to activities mediated by the bacterial mercuric reductase. Of four characterizedmer operons, the system encoded by transposon 501 (mer(Tn501)) dominated and likely contributed to the majority of the observed Hg(II) volatilization. Thus,mer-mediated reduction and volatilization could be used to reduce Hg(II) concentrations in polluted waters, in turn decreasing rates of methylmercury formation by limiting substrate availability.     

Sulfate-Reducing Bacteria: Principal Methylators of Mercury in Anoxic Estuarine Sediment

Substrate-electron acceptor combinations and specific metabolic inhibitors were applied to anoxic saltmarsh sediment spiked with mercuric ions (Hg²⁺) in an effort to identify, by a direct approach, the microorganisms responsible for the synthesis of hazardous monomethylmercury. 2-Bromoethane sulfonate (30 mM), a specific inhibitor of methanogens, increased monomethylmercury synthesis, whereas sodium molybdate (20 mM), a specific inhibitor of sulfate reducers, decreased Hg²⁺ methylation by more than 95%. Anaerobic enrichment and isolation procedures yielded a Desulfovibrio desulfuricans culture that vigorously methylated Hg²⁺ in culture solution and also in samples of presterilized sediment. The Hg²⁺ methylation activity of sulfate reducers is fully expressed only when sulfate is limiting and fermentable organic substrates are available. To date, sulfate reducers have not been suspected of Hg²⁺ methylation. Identification of these bacteria as the principal methylators of Hg²⁺ in anoxic sediments raises questions about the environmental relevance of previous pure culture-based methylation work.     

Mercury detoxification and natural product synthesis: the work of Christopher T. Walsh

Mercuric Reductase. Purification and Characterization of a Transposon-encoded Flavoprotein Containing an Oxidation-Reduction-active Disulfide (Fox, B., and Walsh, C. T. (1982) J. Biol. Chem. 257, 2498–2503)Cloning, Overproduction, and Characterization of the Escherichia coli Holo-acyl Carrier Protein Synthase (Lambalot, R. H., and Walsh, C. T. (1995) J. Biol. Chem. 270, 24658–24661)Christopher Thomas Walsh was born in 1944 in Boston, Massachusetts. He attended Harvard University, where he did undergraduate research with E. O. Wilson, publishing a first author paper on the composition of fire ant trail substance in Nature (1). He earned his A.B. in biology in 1965. Walsh then went to Rockefeller University to work with Leonard B. Spector, publishing six first author papers and earning a Ph.D. in 1970 with a dissertation titled “The Mechanism of Action of the Citrate Cleavage Enzyme.”Open in a separate windowChristopher T. WalshWalsh did a 2-year postdoctoral fellowship with Robert H. Abeles at Brandeis University before joining the faculty of the Massachusetts Institute of Technology (MIT) in 1972 as an assistant professor. He eventually became the Karl Taylor Compton Professor and chairman of the chemistry department there.Walsh''s initial research at MIT centered on studies of a class of enzyme inhibitors called “suicide substrates,” compounds that were not toxic to cells but resembled normal metabolites so closely that they underwent metabolic transformation to form products that were inhibitory. Walsh also started to explore novel chemical transformations in biology, which led to his elucidation of the process by which bacteria detoxify mercury-containing molecules in the environment by cleaving carbon-mercury bonds and then reducing the mercuric salt to elemental mercury. An enzyme that is central to this process is a flavoprotein called mercuric reductase. The enzyme catalyzes two-electron reduction of mercuric ions to elemental mercury using NADPH as an electron donor. The elemental mercury is volatile and is thus nonenzymatically removed from the environment.In the first Journal of Biological Chemistry (JBC) Classic reprinted here, Walsh and Barbara Fox describe the purification of mercuric reductase from Pseudomonas aeruginosa. To their surprise, they discovered that the enzyme had a high degree of similarity to lipoamide dehydrogenase and glutathione reductase, flavoenzymes that catalyze the transfer of electrons between pyridine nucleotides and disulfides. This paper initiated a series of studies investigating how the inorganic Hg²⁺ substrate is bound to two pairs of thiols, one in the active site and one as an exit site, and how electrons flow from NADPH through the FAD to the bound Hg²⁺.In 1987, Walsh moved to Harvard Medical School to learn more biology and medicine and to become chairman of the department of biological chemistry and molecular pharmacology. He continued to study biocatalysts and began exploring antibiotic and antitumor agents as well. One of his first major findings at Harvard explained the mechanism by which resistance develops to the antibiotic vancomycin (2), work that provided the foundation to create new antibiotics.Walsh also is widely recognized for spurring a renaissance in natural product biosynthesis. This started with his investigation of holo-acyl carrier protein synthase (ACPS), a phosphopantetheinyltransferase (PPTase) that transfers the 4′-phosphopantetheine (4′-PP) moiety from coenzyme A to Ser-36 of acyl carrier protein (ACP) in E. coli. Walsh and Ralph H. Lambalot purified ACPS to near homogeneity by exploiting the fact that ACPS could be refolded and reconstituted after elution from an apo-ACP affinity column under denaturing conditions. As reported in the second JBC Classic reprinted here, Walsh and Lambalot used N-terminal sequencing of ACPS to determine that dpj, an essential gene of previously unknown function, was the structural gene for ACPS. These studies led to the identification of other PPTase genes and enzymes involved in the conversion of apo forms of acyl and peptidyl carrier proteins in polyketide and nonribosomal peptide synthases/synthetases. This, in turn, allowed posttranslational activation of these multimodular enzymes when heterologously expressed in E. coli, which started Walsh on a 10-year, 200-paper focus on the characterization of the many enzymatic steps in assembly line biosynthesis of natural products.Currently, Walsh is the Hamilton Kuhn Professor of Biological Chemistry and Molecular Pharmacology at Harvard Medical School. He also was president of the Dana Farber Cancer Institute from 1992 to 1995. Walsh has received many honors and awards for his contributions to science. These include the Eli Lilly Award in Biochemistry (1979), the American Chemical Society (ACS) Arthur C. Cope Scholar Award in Organic Chemistry (1998), the ACS Repligen Award for Chemistry of Life Processes (1999), the ACS Alfred Bader Award for Bioorganic Chemistry (2003), the American Society for Microbiology Promega Biotechnology Research Award (2004), the American Society for Biochemistry and Molecular Biology Fritz Lipmann Award (2005), the ACS Murray Goodman Award (2007), and the Stanford University School of Medicine Pauling Medal and Lecture (2010). Walsh also was elected to the American Academy of Arts and Sciences (1988), the National Academy of Sciences (1989), and the American Philosophical Society (2003). He served on the JBC editorial board from 1978 to1980.¹     

An NADH:Quinone Oxidoreductase Active during Biodegradation by the Brown-Rot Basidiomycete Gloeophyllum trabeum

The brown-rot basidiomycete Gloeophyllum trabeum uses a quinone redox cycle to generate extracellular Fenton reagent, a key component of the biodegradative system expressed by this highly destructive wood decay fungus. The hitherto uncharacterized quinone reductase that drives this cycle is a potential target for inhibitors of wood decay. We have identified the major quinone reductase expressed by G. trabeum under conditions that elicit high levels of quinone redox cycling. The enzyme comprises two identical 22-kDa subunits, each with one molecule of flavin mononucleotide. It is specific for NADH as the reductant and uses the quinones produced by G. trabeum (2,5-dimethoxy-1,4-benzoquinone and 4,5-dimethoxy-1,2-benzoquinone) as electron acceptors. The affinity of the reductase for these quinones is so high that precise kinetic parameters were not obtainable, but it is clear that k_cat/K_m for the quinones is greater than 10⁸ M⁻¹ s⁻¹. The reductase is encoded by a gene with substantial similarity to NAD(P)H:quinone reductase genes from other fungi. The G. trabeum quinone reductase may function in quinone detoxification, a role often proposed for these enzymes, but we hypothesize that the fungus has recruited it to drive extracellular oxyradical production.     

Community Analysis of a Mercury Hot Spring Supports Occurrence of Domain-Specific Forms of Mercuric Reductase

Mercury is a redox-active heavy metal that reacts with active thiols and depletes cellular antioxidants. Active resistance to the mercuric ion is a widely distributed trait among bacteria and results from the action of mercuric reductase (MerA). Protein phylogenetic analysis of MerA in bacteria indicated the occurrence of a second distinctive form of MerA among the archaea, which lacked an N-terminal metal recruitment domain and a C-terminal active tyrosine. To assess the distribution of the forms of MerA in an interacting community comprising members of both prokaryotic domains, studies were conducted at a naturally occurring mercury-rich geothermal environment. Geochemical analyses of Coso Hot Springs indicated that mercury ore (cinnabar) was present at concentrations of parts per thousand. Under high-temperature and acid conditions, cinnabar may be oxidized to the toxic form Hg²⁺, necessitating mercury resistance in resident prokaryotes. Culture-independent analysis combined with culture-based methods indicated the presence of thermophilic crenarchaeal and gram-positive bacterial taxa. Fluorescence in situ hybridization analysis provided quantitative data for community composition. DNA sequence analysis of archaeal and bacterial merA sequences derived from cultured pool isolates and from community DNA supported the hypothesis that both forms of MerA were present. Competition experiments were performed to assess the role of archaeal merA in biological fitness. An essential role for this protein was evident during growth in a mercury-contaminated environment. Despite environmental selection for mercury resistance and the proximity of community members, MerA retains the two distinct prokaryotic forms and avoids genetic homogenization.     

Skeletal muscle glycogen phosphorylase is irreversibly inhibited by mercury: Molecular,cellular and kinetic aspects

Muscle glycogen phosphorylase (GP) plays an important role in muscle functions. Mercury has toxic effects in skeletal muscle leading to muscle weakness or cramps. However, the mechanisms underlying these toxic effects are poorly understood. We report that GP is irreversibly inhibited by inorganic (Hg²⁺) and organic (CH₃Hg⁺) mercury (IC₅₀ = 380 nM and k_inact = 600 M⁻¹ s⁻¹ for Hg²⁺ and IC₅₀ = 43 μM and k_inact = 13 M⁻¹ s⁻¹ for CH₃Hg⁺) through reaction of these compounds with cysteine residues of the enzyme. Our data suggest that the irreversible inhibition of GP could represent one of the mechanisms that contribute to mercury-dependent muscle toxicity.     

Characterization of the metabolically modified heavy metal-resistant Cupriavidus metallidurans strain MSR33 generated for mercury bioremediation

Background

Mercury-polluted environments are often contaminated with other heavy metals. Therefore, bacteria with resistance to several heavy metals may be useful for bioremediation. Cupriavidus metallidurans CH34 is a model heavy metal-resistant bacterium, but possesses a low resistance to mercury compounds.

Methodology/Principal Findings

To improve inorganic and organic mercury resistance of strain CH34, the IncP-1β plasmid pTP6 that provides novel merB, merG genes and additional other mer genes was introduced into the bacterium by biparental mating. The transconjugant Cupriavidus metallidurans strain MSR33 was genetically and biochemically characterized. Strain MSR33 maintained stably the plasmid pTP6 over 70 generations under non-selective conditions. The organomercurial lyase protein MerB and the mercuric reductase MerA of strain MSR33 were synthesized in presence of Hg²⁺. The minimum inhibitory concentrations (mM) for strain MSR33 were: Hg²⁺, 0.12 and CH₃Hg⁺, 0.08. The addition of Hg²⁺ (0.04 mM) at exponential phase had not an effect on the growth rate of strain MSR33. In contrast, after Hg²⁺ addition at exponential phase the parental strain CH34 showed an immediate cessation of cell growth. During exposure to Hg²⁺ no effects in the morphology of MSR33 cells were observed, whereas CH34 cells exposed to Hg²⁺ showed a fuzzy outer membrane. Bioremediation with strain MSR33 of two mercury-contaminated aqueous solutions was evaluated. Hg²⁺ (0.10 and 0.15 mM) was completely volatilized by strain MSR33 from the polluted waters in presence of thioglycolate (5 mM) after 2 h.

Conclusions/Significance

A broad-spectrum mercury-resistant strain MSR33 was generated by incorporation of plasmid pTP6 that was directly isolated from the environment into C. metallidurans CH34. Strain MSR33 is capable to remove mercury from polluted waters. This is the first study to use an IncP-1β plasmid directly isolated from the environment, to generate a novel and stable bacterial strain useful for mercury bioremediation.     

Structural and Biochemical Characterization of Free Methionine-R-sulfoxide Reductase from Neisseria meningitidis

A new family of methionine-sulfoxide reductase (Msr) was recently described. The enzyme, named fRMsr, selectively reduces the R isomer at the sulfoxide function of free methionine sulfoxide (Met-R-O). The fRMsrs belong to the GAF fold family. They represent the first GAF domain to show enzymatic activity. Two other Msr families, MsrA and MsrB, were already known. MsrA and MsrB reduce free Met-S-O and Met-R-O, respectively, but exhibit higher catalytic efficiency toward Met-O within a peptide or a protein context. The fold of the three families differs. In the present work, the crystal structure of the fRMsr from Neisseria meningitidis has been determined in complex with S-Met-R-O. Based on biochemical and kinetic data as well as genomic analyses, Cys¹¹⁸ is demonstrated to be the catalytic Cys on which a sulfenic acid is formed. All of the structural factors involved in the stereoselectivity of the l-Met-R-O binding were identified and account for why Met-S-O, DMSO, and a Met-O within a peptide are not substrates. Taking into account the structural, enzymatic, and biochemical information, a scenario of the catalysis for the reductase step is proposed. Based on the thiol content before and after Met-O reduction and the stoichiometry of Met formed per subunit of wild type and Cys-to-Ala mutants, a scenario of the recycling process of the N. meningitidis fRMsr is proposed. All of the biochemical, enzymatic, and structural properties of the N. meningitidis fRMsr are compared with those of MsrA and MsrB and are discussed in terms of the evolution of function of the GAF domain.     

Bacterial <Emphasis Type="Italic">mer</Emphasis> operon-mediated detoxification of mercurial compounds: a short review

Mercury pollution has emerged as a major problem in industrialized zones and presents a serious threat to environment and health of local communities. Effectiveness and wide distribution of mer operon by horizontal and vertical gene transfer in its various forms among large community of microbe reflect importance and compatibility of this mechanism in nature. This review specifically describes mer operon and its generic molecular mechanism with reference to the central role played by merA gene and its related gene products. The combinatorial action of merA and merB together maintains broad spectrum mercury detoxification system for substantial detoxification of mercurial compounds. Feasibility of mer operon to coexist with antibiotic resistance gene (amp ^r , kan ^r , tet ^r ) clusters enables extensive adaptation of bacterial species to adverse environment. Flexibility of the mer genes to exist as intricate part of chromosome, plasmids, transposons, and integrons enables high distribution of these genes in wider microbial gene pool. Unique ability of this system to manipulate oligodynamic property of mercurial compounds for volatilization of mercuric ions (Hg²⁺) makes it possible for a wide range of microbes to tolerate mercury-mediated toxicity.     

Expression of mercuric reductase from Bacillus megaterium MB1 in eukaryotic microalga Chlorella sp. DT: an approach for mercury phytoremediation

A eukaryotic microalga, Chlorella sp. DT, was transformed with the Bacillus megaterium strain MB1 merA gene, encoding mercuric reductase (MerA), which mediates the reduction of Hg²⁺ to volatile elemental Hg⁰. The transformed Chlorella cells were selected first by hygromycin B and then by HgCl₂. The existence of merA gene in the genomic DNA of transgenic strains was shown by polymerase chain reaction amplification, while the stable integration of merA into genomic DNA of transgenic strains was confirmed by Southern blot analysis. The ability to remove Hg²⁺ in merA transgenic strains was higher than that in the wild type. The merA transgenic strains showed higher growth rate and photosynthetic activity than the wild type did in the presence of a toxic concentration of Hg²⁺. Cultured with Hg²⁺, the expression level of superoxide dismutase in transgenic strains was lower than that in the wild type, suggesting that the transgenic strains faced a lower level of oxidative stress. All the results indicated that merA gene was successfully integrated into the genome of transgenic strains and functionally expressed to promote the removal of Hg²⁺.     

The pectic enzymes of Aspergillus niger. A mercury-activated exopolygalacturonase

1. An exopolygalacturonase was separated from a mycelial extract of Aspergillus niger with a 290-fold purification and a recovery of 8·6%. 2. The enzyme displayed its full activity only in the presence of Hg²⁺ ions; K_A for mercuric chloride was about 6×10⁻⁸m. 3. The mercury-activated enzyme progressively removed the terminal galacturonic acid residues from α-(1→4)-linked galacturonide chains and converted digalacturonic acid, trigalacturonic acid, tetragalacturonic acid and pectic acid into galacturonic acid.