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.     

Cell-free mercury volatilization activity from three marine caulobacter strains

Three mercury-resistant marine Caulobacter strains showed an inducible mercury volatilization activity. Cell-free mercury volatilization (mercuric reductase) from these three marine Caulobacter strains was characterized and compared with enzyme activities determined by plasmids of Escherichia coli and Staphylococcus aureus. The temperature sensitivity of the Caulobacter mercuric reductase was greater than that of mercuric reductase from other gram-negative sources. Cell-free enzyme activity required NADH or NADPH, with NADPH functioning much better at lower concentrations than NADH. The Km for the Caulobacter enzyme was 4 microM Hg2+. Ag+ was a competitive inhibitor of Caulobacter mercuric reductase (Ki = 0.2 microM Ag+), as with previously studied enzymes. Arsenite was a noncompetitive inhibitor of the Caulobacter enzyme with a Ki of 75 microM AsO2-.     

Cell-free mercury volatilization activity from three marine caulobacter strains.

Three mercury-resistant marine Caulobacter strains showed an inducible mercury volatilization activity. Cell-free mercury volatilization (mercuric reductase) from these three marine Caulobacter strains was characterized and compared with enzyme activities determined by plasmids of Escherichia coli and Staphylococcus aureus. The temperature sensitivity of the Caulobacter mercuric reductase was greater than that of mercuric reductase from other gram-negative sources. Cell-free enzyme activity required NADH or NADPH, with NADPH functioning much better at lower concentrations than NADH. The Km for the Caulobacter enzyme was 4 microM Hg2+. Ag+ was a competitive inhibitor of Caulobacter mercuric reductase (Ki = 0.2 microM Ag+), as with previously studied enzymes. Arsenite was a noncompetitive inhibitor of the Caulobacter enzyme with a Ki of 75 microM AsO2-.     

Plasmid-encoded mercuric reductase in Mycobacterium scrofulaceum.

A Chesapeake Bay water isolate of Mycobacterium scrofulaceum containing a 115-megadalton plasmid (pVT1) grew in the presence of 100 microM HgCl2 and converted soluble 203Hg2+ to volatile mercury at a rate of 50 pmol/10(8) cells per min. Cell extracts contained a soluble mercuric reductase whose activity was not dependent on exogenously supplied thiol compounds. The enzyme displayed nearly identical activity when either NADH or NADPH served as the electron donor. A spontaneously cured derivative lacking pVT1 failed to grow in the presence of 100 microM HgCl2 and possessed no detectable mercuric reductase activity.     

In vivo and in vitro studies of nitrate reductase regulation in Asperillus nidulans.

Summary Induced wildtype cells ofA. nidulans rapidly lost NADPH — linked nitrate reductase activity when subjected to carbon and or nitrogen starvation. A constitutive mutant at the regulatory gene for nitrate reductase,nirA ^c1, rapidly lost nitrate reductase activity upon carbon starvation. This loss of activity is thought to be due to a decrease in the NADPH concentration in the cells. Cell free extracts from wild-type cells grown in the presence of nitrate, rapidly lost their nitrate reductase activity when incubated at 25° C. NADPH prevented this loss of activity. Wildtype cells grown in the presence of nitrate and urea have a higher initial NADPH: NADP⁺ ratio and cell free extracts from such cells lost their nitrate reductase activity slower than extracts of cells grown with nitrate alone.The Pentose Phosphate Pathway mutant,pppB-1, had a lower NADPH concentration compared with the wildtype grown under the same conditions and cell free extracts lost their nitrate reductase activity more rapidly than the wildtype. Cell free extracts ofnirA ^c-1 and a non-inducible mutant for nitrate reductase,nirA ^--14, upon incubation lost little of their nitrate reductase activity.     

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.     

Specificity for nicotinamide adenine dinucleotide by nitrate reductase from leaves

Preliminary work revealed that nitrate reductase in crude extracts prepared from leaves of certain corn genotypes as well as soybeans could utilize NADPH as well as NADH as the electron donor. Isoelectric focusing and diethylaminoethyl cellulose chromatography confirmed previous findings that NADH and NADPH activities could not be separated, which suggests the involvement of a single enzyme. Nitrate reduction with both cofactors varies with plant species, plant age, and assay conditions. The ability of the nitrate reductase from a given genotype to utilize NADPH was associated with the amount of NADPH-phosphatase in the extract. While diethylaminoethyl cellulose chromatography of plant extracts separated nitrate reductase from the bulk (90%) of the phosphatase and caused a decrease in the NADPH activity, the residual level of phosphatase was sufficient to account for the apparent NADPH nitrate reductase activity. Addition of KH₂PO₄ and KF, inhibitors of NADPH-phosphatase activity in in vitro assays, caused a drastic reduction or abolishment of NADPH-mediated nitrate reductase activity but were without effect on NADH nitrate reductase activity. It is concluded that NADPH-nitrate reduction, in soybean and certain corn genotypes, is an artifact resulting from the conversion of NADPH to NADH by a phosphatase and that the enzyme in leaf tissue is NADH-dependent (E.C.1.6.6.1).     

Comparative Study of Uptake and Cellular Distribution of Hg203-Labeled Phenyl-Mercuric Acetate and Mercuric Acetate by Pea Roots

Uptake and cellular distribution of mercury²⁰³ from dilute mercuric acetate or phenylmercuric acetate solutions by excised pea roots (Pisum sativum) have been investigated. The time course of uptake showed that the amount of mercury uptake was increased with the time of incubation, and was similar for inorganic mercury or phenylmercuric acetate. The trend of mercury²⁰³ incorporation into cellular components from mercuric acetate and phenylmercuric acetate differed greatly as the time of incubation increased. The concentrations of mercuric acetate and phenylmercuric acetate solutions or the temperature of incubation also affected the mercury²⁰³ uptake as well as its cellular distribution. Longer time of exposure or higher concentration resulted in a greater mercury incorporation into mitochondrial fraction from phenylmercuric acetate than from inorganic mercury. This difference in intracellular distribution may be responsible for the degree of toxicity between inorganic mercury and phenylmercuric acetate in biological systems.     

Pyridine nucleotide specificity of barley nitrate reductase

NADPH nitrate reductase activity in higher plants has been attributed to the presence of NAD(P)H bispecific nitrate reductases and to the presence of phosphatases capable of hydrolyzing NADPH to NADH. To determine which of these conditions exist in barley (Hordeum vulgare L. cv. Steptoe), we characterized the NADH and NADPH nitrate reductase activities in crude and affinity-chromatography-purified enzyme preparations. The pH optima were 7.5 for NADH and 6 to 6.5 for the NADPH nitrate reductase activities. The ratio of NADPH to NADH nitrate reductase activities was much greater in crude extracts than it was in a purified enzyme preparation. However, this difference was eliminated when the NADPH assays were conducted in the presence of lactate dehydrogenase and pyruvate to eliminate NADH competitively. The addition of lactate dehydrogenase and pyruvate to NADPH nitrate reductase assay media eliminated 80 to 95% of the NADPH nitrate reductase activity in crude extracts. These results suggest that a substantial portion of the NADPH nitrate reductase activity in barley crude extracts results from enzyme(s) capable of converting NADPH to NADH. This conversion may be due to a phosphatase, since phosphate and fluoride inhibited NADPH nitrate reductase activity to a greater extent than the NADH activity. The NADPH activity of the purified nitrate reductase appears to be an inherent property of the barley enzyme, because it was not affected by lactate dehydrogenase and pyruvate. Furthermore, inorganic phosphate did not accumulate in the assay media, indicating that NADPH was not converted to NADH. The wild type barley nitrate reductase is a NADH-specific enzyme with a slight capacity to use NADPH.     

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.     

Ferrous Iron-Dependent Volatilization of Mercury by the Plasma Membrane of Thiobacillus ferrooxidans

Of 100 strains of iron-oxidizing bacteria isolated, Thiobacillus ferrooxidans SUG 2-2 was the most resistant to mercury toxicity and could grow in an Fe²⁺ medium (pH 2.5) supplemented with 6 μM Hg²⁺. In contrast, T. ferrooxidans AP19-3, a mercury-sensitive T. ferrooxidans strain, could not grow with 0.7 μM Hg²⁺. When incubated for 3 h in a salt solution (pH 2.5) with 0.7 μM Hg²⁺, resting cells of resistant and sensitive strains volatilized approximately 20 and 1.7%, respectively, of the total mercury added. The amount of mercury volatilized by resistant cells, but not by sensitive cells, increased to 62% when Fe²⁺ was added. The optimum pH and temperature for mercury volatilization activity were 2.3 and 30°C, respectively. Sodium cyanide, sodium molybdate, sodium tungstate, and silver nitrate strongly inhibited the Fe²⁺-dependent mercury volatilization activity of T. ferrooxidans. When incubated in a salt solution (pH 3.8) with 0.7 μM Hg²⁺ and 1 mM Fe²⁺, plasma membranes prepared from resistant cells volatilized 48% of the total mercury added after 5 days of incubation. However, the membrane did not have mercury reductase activity with NADPH as an electron donor. Fe²⁺-dependent mercury volatilization activity was not observed with plasma membranes pretreated with 2 mM sodium cyanide. Rusticyanin from resistant cells activated iron oxidation activity of the plasma membrane and activated the Fe²⁺-dependent mercury volatilization activity of the plasma membrane.     

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.     

Light-dependent Reduction of Oxidized Glutathione by Ruptured Chloroplasts

Crude extracts of pea shoots (Pisum sativum) catalyzed oxidized glutathione (GSSG)-dependent oxidation of NADPH which was attributed to NADPH-specific glutathione reductase. The pH optimum was 8 and the K_m values for GSSG and NADPH were 23 μm and 4.9 μm, respectively. Reduced glutathione (GSH) inhibited the reaction. Crude extracts also catalyzed NADPH-dependent reduction of GSSG; the ratio of the rate of NADPH oxidized to GSH formed was 0.49. NADH and various substituted mono- and disulfides would not substitute for NADPH and GSSG respectively. Per mg of chlorophyll, enzyme activity of isolated chloroplasts was 69% of the activity of crude extracts.     

Role of NADPH and the NADPH-dependent methemoglobin reductase in the hydroxylase activity of human erythrocytes

Human erythrocytes were shown previously to catalyze the oxyhemoglobin-requiring hydroxylation of aniline, and the reaction was stimulated apparently preferentially by NADPH in the presence of methylene blue (K. S. Blisard and J. J. Mieyal,J. Biol. Chem.254, 5104, 1979). The current study provides a further characterization of the involvement of the NADPH-dependent electron transport system in this reaction. In accordance with the role of NADPH, the hydroxylase activity of erythrocytes or hemolysates from individuals with glucose-6-phosphate dehydrogenase deficiency (i.e., with diminished capacity to form NADPH) displayed decreased responses to glucose or glucose 6-phosphate, respectively, in the presence of methylene blue in comparison to samples from normal adults; maximal activity could be restored by direct addition of NADPH to the deficient hemolysates. Kinetic studies of the methylene blue-stimulated aniline hydroxylase activity of normal hemolysates revealed a biphasic dependence on NADPH concentrations: a plateau was observed at relatively low concentrations (K_m^NADPH ~ 20 μm), whereas saturation was not achieved at the higher concentrations of NADPH. The latter low efficiency phase (i.e., at the higher concentrations of NADPH) could be ascribed to a direct transfer of electrons from NADPH to methylene blue to hemoglobin. The high efficiency phase suggested involvement of the NADPH-dependent methemoglobin reductase; accordingly 2′-AMP, an analog of NADP⁺, effectively inhibited this reaction, but the pattern was noncompetitive. This behavior is suggestive of a mechanism by which both NADPH and methylene blue are substrates for the reductase and interact with it in a sequential fashion. The kinetic patterns observed for variation in NADPH concentration at several fixed concentrations of methylene blue, and vice versa, are consistent with this interpretation.     

Biosynthesis of alkane-2,3-diols: enzymatic reduction of 3-hydroxyoctadecane-2-one to octadecane-2,3-diol by a NADPH (B-side) specific microsomal reductase from the uropygial glands of ring-necked pheasants (Phasianus colchicus).

Cell-free preparations from the uropygial gland of ring-necked pheasant catalyzed the reduction of a synthetic R,S-mixture of 3-hydroxyl[3-¹⁴C]octadecane-2-one (acyloin) to a mixture of threo- and erythro-[3-¹⁴C]octadecane-2,3-diol, the final step in the postulated pathway for the biosynthesis of alkane-2,3-diols. The product of enzymatic reduction was identified by Chromatographic techniques and chemical degradation studies. The acyloin reductase showed a pH optimum near 4.0 and specificity for NADPH. With stereospecifically labeled [³H]NADPH, it was shown that acyloin reductase preferentially transferred hydride from the B-side of the nicotinamide ring to the acyloin. A typical Michaelis-Menten substrate saturation was observed for the acyloin and an apparent K_m of 70 μm was calculated from linear double reciprocal plots. Acyloin reductase was inhibited by thioldirected reagents such as p-chloromercuribenzoate and N-ethylmaleimide. Subcellular fractionation of the gland homogenates using sucrose density gradient centrifugation showed that acyloin reductase activity coincided with NADPH:cytochrome c reductase activity, strongly suggesting that acyloin reductase is localized in the microsomal membranes.     

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.     

On the oxygen-sensitivity of various tetrazolium salts

Summary 1. Eight different tetrazolium salts have been chemically reduced with NADPH and PMS¹ under oxygenated and oxygen-free conditions. 2. PMS has been shown to be able to remove all of the hydrogen from NADPH very rapidly, and to transfer all of this hydrogen onto tetrazolium salts, under suitable atmospheric conditions. 3. MTT, INT, TNBT, and NBT¹ produced the same amount of formazan under both conditions; NT BT, TV, TT¹ produced formazan under oxygen-free conditions, but produced no formazan under oxygenated conditions. 4. These results are explained on the basis of competition for the NADP Hhydrogen between oxygen and the four tetrazolium salts NT, BT, TV and TT.I should like to thank The Arthritis and Rheumatism Council for financial support.     

Ferredoxin–NADP+ Reductase from Tsuga canadensis. A Procedure for Isolation and Properties

Activity of ferredoxin-NADP⁺ reductase in leaf extracts of eastern hemlock [Tsuga canadensis (L.) Carr.] was relatively low, but could be markedly increased by use of protective agents. The best method employed polyvinylpolypyrrolidone (PVP) in the extraction medium plus removal of phenolic compounds by filtering the extracts through an insoluble PVP (Polyclar AT) column. Further purification of the enzyme was achieved by means of DEAE cellulose chromatography and DEAE Sephadex chromatography. A 94-fold purification of the enzyme with a total recovery of 43% was obtained. The eastern hemlock ferredoxin-NADP⁺ reductase was characterized by its diaphorase activity, i.e. the transfer of electrons from NADPH to an electron acceptor. 2,6-dichlorophenol indophenol. The pH optimum for the oxidation of NADPH is between 8.5 and 9.0. The enzyme is highly specific for its electron donor. NADPH, but shows low specificity for electron acceptors. The apparent Michaelis constant values of the enzyme for NADPH. NADH, and 2,6-dichlorophenol indophenol are 2.4 × 10^?5, 5.4 × 10^?3, and 4.7 × 10^?5M respectively. The molecular weight of the enzyme, as estimated by gel filtration, is about 45,000. The enzyme is inhibited by both organic and inorganic mercurials and certain cations. Comparison of properties of eastern hemlock ferredoxin-NADP⁺ reductase and spinach ferredoxin-NADP⁺ reductase shows that both enzymes are similar.     

Demonstration of a NADPH-linked Δ1-pyrroline-5-carboxylate-proline shuttle in a cell-free rat liver system

These studies indicate that the interconversions of Δ¹-pyrroline-5-carboxylate and proline can function as a shuttle that generates extra-mitochondrial NADP⁺ and transfers hydride ions into mitochondria in a cell-free rat liver system. A phosphate-free buffer with high concentrations of triethanolamine and 2-mercaptoethanol prevented the cold inactivation of pyrroline-5-carboxylate reductase (ED 1.5.1.2) in liver extracts. This enzyme had an apparent K_m^NADPH that was 2% of the apparent K_m^NADH. V_max^NADPH was approx. 50% of V_max^NADH. Unlabeled proline was converted to [5-³H]proline in incubations containing liver soluble fraction, mitochondria and a [4S-³H]NADPH generating system. This demonstrated one turn of the proposed shuttle in a homologous liver system. [5-³H]Proline production increased linearly over 60 min and decreased by 87% or more when specific components were eliminated. Rotenone was required for maximal activity, suggesting that inhibition of Δ¹-pyrroline-5-carboxylate efflux would be required for significant shuttle activity in vivo. Both the relative concentrations of NADPH and NADH in liver cytosol and the kinetic characteristics of liver pyrroline-5-carboxylate reductase predict that the described shuttle should be overwhelmingly linked to NADPH rather than NADH. A NADPH-linked Δ¹-pyrroline-5-carboxylate-proline shuttle may occur in hepatocytes and function at specific times to regulate pathways limited by cytosolic [NADP⁺].     

Tn5044-conferred mercury resistance depends on temperature: the complexity of the character of thermosensitivity

Escherichia coli K12 containing the transposon Tn5044 mer operon (merR, T, P, C, and A genes) is resistant to mercuric chloride at 30°C but sensitive to this compound at 37–41.5°C. We have studied the mechanism underlying the temperature-sensitive nature of this mercury resistance phenotype, and found that the expression of the Tn5044 merA gene coding for mercuric reductase (MerA) is severely inhibited at non-permissive temperatures. Additionally, MerA showed a considerably reduced functional activity in vivo at non-permissive temperatures. However, the temperature-sensitive character of the functioning of this enzyme in cell extracts, where it interacted with one of the low-molecular weight SH compounds rather than with the transport protein MerT (as is the case in vivo), was not apparent. These data suggest that the temperature-sensitive mercury resistance phenotype should stay under control at two stages: when the merA gene is expressed and when its product interacts with MerT to accept the mercuric ion.