Reduction of Hg2+ with reduced mammalian cytochrome c by cytochrome c oxidase purified from a mercury-resistant acidithiobacillus ferrooxidans strain, MON-1

Acidithiobacillus ferrooxidans AP19-3, ATCC 23270, and MON-1 are mercury-sensitive, moderately mercury-resistant, and highly mercury-resistant strains respectively. It is known that 2,3,5,6-tetramethyl-p-phenylendiamine (TMPD) and reduced cytochrome c are used as electron donors specific for cytochrome c oxidase. Resting cells of strain MON-1 had TMPD oxidase activity and volatilized metal mercury with TMPD as an electron donor. Cytochrome c oxidase purified from strain MON-1 reduced mercuric ions to metalic mercury with reduced mammalian cytochrome c as well as TMPD. These mercury volatilization activities with reduced cytochrome c and TMPD were completely inhibited by 1 mM NaCN. These results indicate that cytochrome c oxidase is involved in mercury reduction in A. ferrooxidans cells. The cytochrome c oxidase activities of strains AP19-3 and ATCC 23270 were completely inhibited by 1 muM and 5 muM of mercuric chloride respectively. In contrast, the activity of strain MON-1 was inhibited 33% by 5 muM, and 70% by 10 muM of mercuric chloride, suggesting that the levels of mercury resistance in A. ferrooxidans strains correspond well with the levels of mercury resistance of cytochrome c oxidase.     

Bacterial removal of mercury from sewage

Mercury-resistant bacteria, which are able to reduce mercuric ion (Hg(2+)) to metallic mercury (Hg(0)), were examined for their ability to remove mercury from waste-water aerobically. Growth studies in artificial medium indicated that mercury increases the lag phase, but does not effect the growth rate of these bacteria. Further studies demonstrated that growth was minimal during a phase of rapid mercury removal, after which growth resumed. Small but significant amounts of carbohydrates are required for the mercuric ion reduction. Prolonged periods of bacterial growth under nonsterile conditions was accomplished without the loss of the mercuric reducing ability of the culture. A continuous culture of the resistant organism was maintained on raw sewage for two weeks, during which time relatively high concentrations of mercury (70 mg/L) were removed from the sewage at a rate of 2.5 mg/L h and at efficiencies exceeding 98%.     

Uptake of metallic mercury and mercuric ion by human erythrocytes

Uptake of metallic mercury (Hg degrees) and mercuric ion (Hg2+) by erythrocytes was studied by incubating erythrocytes with various concentrations of radioactive metallic mercury and mercuric ion in phosphate-buffered saline (pH 6.8) or plasma at 25 degrees C for 30 min. Radioactivity taken up in the cytosol (endsome) and stroma were determined with a gamma scintillation counter. The radioactivity ratio of the mercury recovered in the cytosol fraction to metallic mercury incubated in the saline was significantly higher than the ratio of that to mercuric ion. Similar findings were observed in erythrocytes incubated with metallic mercury and mercuric ion in plasma, although the recovered radioactivity of mercury in the cytosol of erythrocytes incubated with metallic mercury or mercuric ion in plasma was less than that incubated in phosphate-buffered saline. Thus, erythrocytes incubated with metallic mercury took up a larger amount of mercury than those incubated with mercuric ion. Discussion is made on these findings.     

Overexpression of a single membrane component from the Bacillus mer operon enhanced mercury resistance in an Escherichia coli host

Overexpression of a mercuric ion binding protein, MerP, from the mercury resistance operon genes of Gram-positive bacterial strain Bacillus megaterium MB1 and from Gram-negative bacterial strain Pseudomonas aeruginosa K-62 was found to enhance the mercury resistance level of Escherichia coli host cells, even though they share only 27.3% identity. Immunoblot analysis showed that MerP (BMerP) from Bacillus could be expressed on the membrane fraction of E. coli cells. Treated with 10 microM Hg2+, a recombinant strain harboring the BMerP gene significantly improved, showing a 27% increase in mercuric ion adsorption capacity, 16% better than that of a Pseudomonas merP gene (PMerP)-harboring strain. While multiple heavy metals co-existed, the mercuric ion adsorption capacity of the BMerP-harboring E. coli was not affected while that of the PMerP-harboring strain decreased. These results suggest that BMerP can act as a bio-adsorbent compartmentalizing the toxic mercuric ion on the cell membrane and enhancing resistance.     

Cytochemical localization of mercury in Saccharomyces cerevisiae treated with mercuric chloride

We describe a cytochemical method for localizing mercury at the electron microscopic level in the yeast Saccharomyces cerevisiae. After addition of a lethal concentration of mercuric chloride to growing yeast cells, mercury was associated with the cell wall and cytoplasmic membrane. Little or no mercury was present in the cytoplasm. Electron probe X-ray microanalysis (EPMA) confirmed that the cytochemical reaction, visualized as mercury-silver complexes, was localized in dense bodies consisting of a core of mercury sulfide polymers surrounded by a shell of silver atoms.     

Preparation of a DNA gene probe for detection of mercury resistance genes in gram-negative bacterial communities

A DNA gene probe was prepared to study genetic change mechanisms responsible for adaptation to mercury in natural bacterial communities. The probe was constructed from a 2.6-kilobase NcoI-EcoRI DNA restriction fragment which spans the majority of the mercury resistance operon (mer) in the R-factor R100. The range of specificity of this gene probe was defined by hybridization to the DNA of a wide variety of mercury-resistant bacteria previously shown to possess the mercuric reductase enzyme. All of the tested gram-negative bacteria had DNA sequences homologous to the mer probe, whereas no such homologies were detected in DNA of the gram-positive strains. Thus, the mer probe can be utilized to study gene flow processes in gram-negative bacterial communities.     

Preparation of a DNA gene probe for detection of mercury resistance genes in gram-negative bacterial communities.

A DNA gene probe was prepared to study genetic change mechanisms responsible for adaptation to mercury in natural bacterial communities. The probe was constructed from a 2.6-kilobase NcoI-EcoRI DNA restriction fragment which spans the majority of the mercury resistance operon (mer) in the R-factor R100. The range of specificity of this gene probe was defined by hybridization to the DNA of a wide variety of mercury-resistant bacteria previously shown to possess the mercuric reductase enzyme. All of the tested gram-negative bacteria had DNA sequences homologous to the mer probe, whereas no such homologies were detected in DNA of the gram-positive strains. Thus, the mer probe can be utilized to study gene flow processes in gram-negative bacterial communities.     

Development of a biological mercury removal-recovery system

A mercury removal-recovery system was developed for collection of elemental mercury volatilized by biological mercuric ion reduction. Using the mercury removal-recovery system, removal of mercuric chloride from mercury-containing buffer without nutrients by resting cells of mercury-resistant bacterium, Pseudomonas putida PpY101/pSR134 was tested. Optimum temperature, pH, thiol compounds and cell concentration on removal of mercuric chloride were determined, and 92 to 98% of 40 mg Hg l^–1 was recovered in 24 h. The efficiency of mercuric chloride removal from river water and seawater was as high as that observed when using a buffered solution.     

Levels of metallic mercury and mercuric ion in the venous and arterial bloods of normal and acatalasemic mice following exposure to mercury vapor

Levels of metallic mercury and mercuric ion in the arterial and venous bloods of normal and acatalasemic mice exposed to metallic mercury vapor in vitro and in vivo were investigated. Mercury uptake in venous blood from air saturated with mercury vapor with or without hydrogen peroxide in vitro was determined. Level of mercuric ion in venous blood of normal mice was significantly higher than that of acatalasemic mice. By contrast, metallic mercury in venous blood of acatalasemic mice was elevated relative to level in normal mice. Metallic mercury level in red blood cells and plasma was also significantly higher in acatalasemic mice. The ratio of metallic mercury to total mercury (Hg degrees + Hg2+) in the arterial and venous bloods of acatalasemic mice exposed to metallic mercury vapor was increased relative to normal mice. This ratio in red blood cells and plasma in the venous bloods of acatalasemic mice in vivo was also significantly higher than those of normal mice. The significance of metallic mercury in plasma for distribution of mercury in organs is discussed.     

Overexpression of a Single Membrane Component from the Bacillus mer Operon Enhanced Mercury Resistance in an Escherichia coli Host

Overexpression of a mercuric ion binding protein, MerP, from the mercury resistance operon genes of Gram-positive bacterial strain Bacillus megaterium MB1 and from Gram-negative bacterial strain Pseudomonas aeruginosa K-62 was found to enhance the mercury resistance level of Escherichia coli host cells, even though they share only 27.3% identity. Immunoblot analysis showed that MerP (BMerP) from Bacillus could be expressed on the membrane fraction of E. coli cells. Treated with 10 μM Hg²⁺, a recombinant strain harboring the BMerP gene significantly improved, showing a 27% increase in mercuric ion adsorption capacity, 16% better than that of a Pseudomonas merP gene (PMerP)-harboring strain. While multiple heavy metals co-existed, the mercuric ion adsorption capacity of the BMerP-harboring E. coli was not affected while that of the PMerP-harboring strain decreased. These results suggest that BMerP can act as a bio-adsorbent compartmentalizing the toxic mercuric ion on the cell membrane and enhancing resistance.     

Reduction of mercuric ion in vitro by superoxide anion

The reduction rate of mercuric ion to metallic mercury by a superoxide anion produced by a xanthine-xanthine oxidase system increased with an increased concentration of xanthine oxidase in the presence of enough xanthine. The reduction rate of mercuric ion by a superoxide anion in the presence of nitroblue tetrazolium (NBT) was proportional to the concentration of NBT. The result suggests that NBT was reduced to diformazan by a superoxide anion produced by a xanthine-xanthine oxidase system and that mercuric ion will be reduced to metallic mercury by diformazan. The reduction rate of mercuric ion was also indicative that a superoxide anion produced by an NADH-phenazine methosulfate (PMS) system increased with an increased concentration of PMS.     

Nucleotide sequence of a chromosomal mercury resistance determinant from a Bacillus sp. with broad-spectrum mercury resistance.

A 13.5-kilobase HindIII fragment, bearing an intact mercury resistance (mer) operon, was isolated from chromosomal DNA of broad-spectrum mercury-resistant Bacillus sp. strain RC607 by using as a probe a clone containing the mercury reductase (merA) gene. The new clone, pYW33, expressed broad-spectrum mercury resistance both in Escherichia coli and in Bacillus subtilis, but only in B. subtilis was the mercuric reductase activity inducible. Sequencing of a 1.8-kilobase mercury hypersensitivity-producing fragment revealed four open reading frames (ORFs). ORF1 may code for a regulatory protein (MerR). ORF2 and ORF4 were associated with cellular transport function and the hypersensitivity phenotype. DNA fragments encompassing the merA and the merB genes were sequenced. The predicted Bacillus sp. strain RC607 MerA (mercuric reductase) and MerB (organomercurial lyase) were similar to those predicted from Staphylococcus aureus plasmid pI258 (67 and 73% amino acid identities, respectively); however, only 40% of the amino acid residues of RC607 MerA were identical to those of the mercuric reductase from gram-negative bacteria. A 69-kilodalton polypeptide was isolated and identified as the merA gene product by examination of its amino-terminal sequence.     

Response to mercury (II) ions in Methylococcus capsulatus (Bath)

The mercury (II) ion is toxic and is usually detoxified in Bacteria by reduction to elemental mercury, which is less toxic. This is catalysed by an NAD(P)H-dependent mercuric reductase (EC 1.16.1.1). Here, we present strong evidence that Methylococcus capsulatus (Bath) - a methanotrophic member of the Gammaproteobacteria - uses this enzyme to detoxify mercury. In radiorespirometry studies, it was found that cells exposed to mercury dissimilated 100% of [(14) C]-methane provided to generate reducing equivalents to fuel mercury (II) reduction, rather than the mix of assimilation and dissimilation found in control incubations. The detoxification system is constitutively expressed with a specific activity of 352 (±18) nmol NADH oxidized min(-1) (mg protein)(-1) . Putative mercuric reductase genes were predicted in the M.?capsulatus (Bath) genome and found in mRNA microarray studies. The MerA-derived polypeptide showed high identity (>?80%) with MerA sequences from the Betaproteobacteria.     

Degradation of Organomercurials in Bacillus cereus

Degradation of phenylmercuric acetate (PMA) and p-chloromercuribenzoic acid (PCMB) by cells of a mercury resistant strain of Bacillus cereus was demonstrated. Degradation of PMA was also demonstrated with the crude extract of the B. cereus cells grown in nutrient broth containing 1 μm PMA or 10 μm HgCl₂. The crude extract seems to split the carbon-mercury bond of PMA and form benzene and the mercuric ion as degradation products. The splitting enzyme was separated from mercuric reductase which catalyzes the reduction of the mercuric ion by gel filtration on Sephadex G 100.     

Mercuric reductase enzyme from a mercury-volatilizing strain of Thiobacillus ferrooxidans.

Cell-free mercury volatilization activity (mercuric reductase) was obtained from a mercury-volatilizing Thiobacillus ferrooxidans strain, and the properties of intact-cell and cell-free activities were compared with those determined by plasmid R100 in Escherichia coli. Intact cells of T. ferrooxidans volatilized mercury at pH 2.5, whereas cells of E. coli did not. Cell-free enzyme preparations from both bacteria functioned best at or above neutral pH and not at all at pH 2.5. The T. ferrooxidans mercuric reductase was a soluble enzyme that was dependent upon added NAD(P)H. The enzyme activity was stable at 80 degrees C, required an added thiol compound, and was stimulated by EDTA. Antisera against purified mercuric reductases from transposon Tn501 and plasmid R831 (which inactivated mercuric reductases from a wide range of enteric and pseudomonad strains) did not inactivate the enzyme from T. ferrooxidans.     

A stable mercury-containing complex of the organomercurial lyase MerB: catalysis, product release, and direct transfer to MerA

Bacteria isolated from organic mercury-contaminated sites have developed a system of two enzymes that allows them to efficiently convert both ionic and organic mercury compounds to the less toxic elemental mercury. Both enzymes are encoded on the mer operon and require sulfhydryl-bound substrates. The first enzyme is an organomercurial lyase (MerB), and the second enzyme is a mercuric ion reductase (MerA). MerB catalyzes the protonolysis of the carbon-mercury bond, resulting in the formation of a reduced carbon compound and inorganic ionic mercury. Of several mercury-containing MerB complexes that we attempted to prepare, the most stable was a complex consisting of the organomercurial lyase (MerB), a mercuric ion, and a molecule of the MerB inhibitor dithiothreitol (DTT). Nuclear magnetic resonance (NMR) spectroscopy and extended X-ray absorption fine structure spectroscopy of the MerB/Hg/DTT complex have shown that the ligands to the mercuric ion in the complex consist of both sulfurs from the DTT molecule and one cysteine ligand, C96, from the protein. The stability of the MerB/Hg/DTT complex, even in the presence of a large excess of competing cysteine, has been demonstrated by NMR and dialysis. We used an enzyme buffering test to determine that the MerB/Hg/DTT complex acts as a substrate for the mercuric reductase MerA. The observed MerA activity is higher than the expected activity assuming free diffusion of the mercuric ion from MerB to MerA. This suggests that the mercuric ion can be transferred between the two enzymes by a direct transfer mechanism.     

Detection of mercuric ions in water by ELISA with a mercury-specific antibody

An immunoassay that detects mercuric ions in water at concentrations of 0.5 ppb and above is described. The assay utilizes a monoclonal antibody that binds specifically to mercuric ions immobilized in wells of microtiter plates. Within the range of 0.5-10 ppb mercury, the absorbance in the enzyme-linked immunosorbent assay (ELISA) is linear to the log of the mercuric ion concentration. The quantitation of mercury by ELISA correlates closely with results from cold-vapor atomic absorption. Other divalent metal cations do not interfere with the assay, although there is interference in the presence of 1 mM chloride ions. The optimum pH for mercury detection is 7.0, although 2 ppb mercury can be detected over a wide pH range. The assay is as sensitive as cold-vapor atomic absorption for mercury detection and can be performed with only 100 microliters of sample.     

Biotoxicity of mercury as influenced by mercury(II) speciation.

Integration of physicochemical procedures for studying mercury(II) speciation with microbiological procedures for studying the effects of mercury on bacterial growth allows evaluation of ionic factors (e.g., pH and ligand species and concentration) which affect biotoxicity. A Pseudomonas fluorescens strain capable of methylating inorganic Hg(II) was isolated from sediment samples collected at Buffalo Pound Lake in Saskatchewan, Canada. The effect of pH and ligand species on the toxic response (i.e., 50% inhibitory concentration [IC50]) of the P. fluorescens isolated to mercury were determined and related to the aqueous speciation of Hg(II). It was determined that the toxicities of different mercury salts were influenced by the nature of the co-ion. At a given pH level, mercuric acetate and mercuric nitrate yielded essentially the same IC50s; mercuric chloride, on the other hand, always produced lower IC50s. For each Hg salt, toxicity was greatest at pH 6.0 and decreased significantly (P = 0.05) at pH 7.0. Increasing the pH to 8.0 had no effect on the toxicity of mercuric acetate or mercuric nitrate but significantly (P = 0.05) reduced the toxicity of mercuric chloride. The aqueous speciation of Hg(II) in the synthetic growth medium M-IIY (a minimal salts medium amended to contain 0.1% yeast extract and 0.1% glycerol) was calculated by using the computer program GEOCHEM-PC with a modified data base. Results of the speciation calculations indicated that complexes of Hg(II) with histidine [Hg(H-HIS)HIS+ and Hg(H-HIS)2(2+)], chloride (HgCl+, HgCl2(0), HgClOH0, and HgCl3-), phosphate (HgHPO4(0), ammonia (HgNH3(2+), glycine [Hg(GLY)+], alanine [Hg(ALA)+], and hydroxyl ion (HgOH+) were the Hg species primarily responsible for toxicity in the M-IIY medium. The toxicity of mercuric nitrate at pH 8.0 was unaffected by the addition of citrate, enhanced by the addition of chloride, and reduced by the addition of cysteine. In the chloride-amended system, HgCl+, HgCl2(0), and HgClOH0 were the species primarily responsible for observed increases in toxicity. In the cysteine-amended system, formation of Hg(CYS)2(2-) was responsible for detoxification effects that were observed. The formation of Hg-citrate complexes was insignificant and had no effect on Hg toxicity.     

Biotoxicity of mercury as influenced by mercury(II) speciation

Integration of physicochemical procedures for studying mercury(II) speciation with microbiological procedures for studying the effects of mercury on bacterial growth allows evaluation of ionic factors (e.g., pH and ligand species and concentration) which affect biotoxicity. A Pseudomonas fluorescens strain capable of methylating inorganic Hg(II) was isolated from sediment samples collected at Buffalo Pound Lake in Saskatchewan, Canada. The effect of pH and ligand species on the toxic response (i.e., 50% inhibitory concentration [IC50]) of the P. fluorescens isolated to mercury were determined and related to the aqueous speciation of Hg(II). It was determined that the toxicities of different mercury salts were influenced by the nature of the co-ion. At a given pH level, mercuric acetate and mercuric nitrate yielded essentially the same IC50s; mercuric chloride, on the other hand, always produced lower IC50s. For each Hg salt, toxicity was greatest at pH 6.0 and decreased significantly (P = 0.05) at pH 7.0. Increasing the pH to 8.0 had no effect on the toxicity of mercuric acetate or mercuric nitrate but significantly (P = 0.05) reduced the toxicity of mercuric chloride. The aqueous speciation of Hg(II) in the synthetic growth medium M-IIY (a minimal salts medium amended to contain 0.1% yeast extract and 0.1% glycerol) was calculated by using the computer program GEOCHEM-PC with a modified data base. Results of the speciation calculations indicated that complexes of Hg(II) with histidine [Hg(H-HIS)HIS+ and Hg(H-HIS)2(2+)], chloride (HgCl+, HgCl2(0), HgClOH0, and HgCl3-), phosphate (HgHPO4(0), ammonia (HgNH3(2+), glycine [Hg(GLY)+], alanine [Hg(ALA)+], and hydroxyl ion (HgOH+) were the Hg species primarily responsible for toxicity in the M-IIY medium. The toxicity of mercuric nitrate at pH 8.0 was unaffected by the addition of citrate, enhanced by the addition of chloride, and reduced by the addition of cysteine. In the chloride-amended system, HgCl+, HgCl2(0), and HgClOH0 were the species primarily responsible for observed increases in toxicity. In the cysteine-amended system, formation of Hg(CYS)2(2-) was responsible for detoxification effects that were observed. The formation of Hg-citrate complexes was insignificant and had no effect on Hg toxicity.     

Role of Na+ in transport of Hg2+ and induction of the Tn21 mer operon.

The effects of sodium ions on the uptake of Hg2+ and induction of the Tn21 mer operon were studied by using Escherichia coli HMS174 harboring the reporter plasmids pRB28 and pOS14. Plasmid pRB28 carries merRT', and pOS14 carries merRTPC of the mer operon, both cloned upstream of a promoterless luciferase gene cassette in pUCD615. The bioluminescent response to 1 microM Hg2+ was significantly inhibited in E. coli HMS174(pRB28) in minimal medium supplemented with sodium ions at 10 to 140 mM. After initial acceleration, light emission declined at 50 nM Hg2+ in the presence of Na+. The mer-lux assay with resting cells carrying pRB28 and 203Hg2+ uptake experiments showed increased induction and enhanced mercury uptake, respectively, in media supplemented with sodium ions. The presence of Na+ facilitated maintenance of bioluminescence in resting HMS174(pRB28) cells induced with 50 nM Hg2+. External K+ stimulated bioluminescent response in HMS174(pRB28) and HMS174(pOS14) grown in sodium phosphate minimal medium devoid of potassium ions. Sodium ions appear to facilitate mercury transport. We propose that sodium-coupled transport of mercuric ions can be one of the mechanisms for mercury uptake by E. coli and that the Na+ gradient may energize the transport of Hg2+.