Mercury adaptation among bacteria from a deep-sea hydrothermal vent

Since deep-sea hydrothermal vent fluids are enriched with toxic metals, it was hypothesized that (i) the biota in the vicinity of a vent is adapted to life in the presence of toxic metals and (ii) metal toxicity is modulated by the steep physical-chemical gradients that occur when anoxic, hot fluids are mixed with cold oxygenated seawater. We collected bacterial biomass at different distances from a diffuse flow vent at 9 degrees N on the East Pacific Rise and tested these hypotheses by examining the effect of mercuric mercury [Hg(II)] on vent bacteria. Four of six moderate thermophiles, most of which were vent isolates belonging to the genus Alcanivorax, and six of eight mesophiles from the vent plume were resistant to >10 microM Hg(II) and reduced it to elemental mercury [Hg(0)]. However, four psychrophiles that were isolated from a nearby inactive sulfide structure were Hg(II) sensitive. A neighbor-joining tree constructed from the deduced amino acids of a PCR-amplified fragment of merA, the gene encoding the mercuric reductase (MR), showed that sequences obtained from the vent moderate thermophiles formed a unique cluster (bootstrap value, 100) in the MR phylogenetic tree, which expanded the known diversity of this locus. The temperature optimum for Hg(II) reduction by resting cells and MR activity in crude cell extracts of a vent moderate thermophile corresponded to its optimal growth temperature, 45 degrees C. However, the optimal temperature for activity of the MR encoded by transposon Tn501 was found to be 55 to 65 degrees C, suggesting that, in spite of its original isolation from a mesophile, this MR is a thermophilic enzyme that may represent a relic of early evolution in high-temperature environments. Results showing that there is enrichment of Hg(II) resistance among vent bacteria suggest that these bacteria have an ecological role in mercury detoxification in the vent environment and, together with the thermophilicity of MR, point to geothermal environments as a likely niche for the evolution of bacterial mercury resistance.     

Expression of mercuric ion reductase in Eastern cottonwood (Populus deltoides) confers mercuric ion reduction and resistance

Mercury is one of the most hazardous heavy metals and is a particular problem in aquatic ecosystems, where organic mercury is biomagnified in the food chain. Previous studies demonstrated that transgenic model plants expressing a modified mercuric ion reductase gene from bacteria could detoxify mercury by converting the more toxic and reductive ionic form [Hg(II)] to less toxic elemental mercury [Hg(0)]. To further investigate if a genetic engineering approach for mercury phytoremediation can be effective in trees with a greater potential in riparian ecosystems, we generated transgenic Eastern cottonwood (Populus deltoides) trees expressing modified merA9 and merA18 genes. Leaf sections from transgenic plantlets produced adventitious shoots in the presence of 50 microm Hg(II) supplied as HgCl2, which inhibited shoot induction from leaf explants of wild-type plantlets. Transgenic shoots cultured in a medium containing 25 microm Hg(II) showed normal growth and rooted, while wild-type shoots were killed. When the transgenic cottonwood plantlets were exposed to Hg(II), they evolved 2-4-fold the amount of Hg(0) relative to wild-type plantlets. Transgenic merA9 and merA18 plants accumulated significantly higher biomass than control plants on a Georgia Piedmont soil contaminated with 40 p.p.m. Hg(II). Our results indicate that Eastern cottonwood plants expressing the bacterial mercuric ion reductase gene have potential as candidates for in situ remediation of mercury-contaminated soils or wastewater.     

Growth and Phylogenetic Properties of Novel Bacteria Belonging to the Epsilon Subdivision of the Proteobacteria Enriched from Alvinella pompejana and Deep-Sea Hydrothermal Vents

Recent molecular characterizations of microbial communities from deep-sea hydrothermal sites indicate the predominance of bacteria belonging to the epsilon subdivision of Proteobacteria (epsilon Proteobacteria). Here, we report the first enrichments and characterizations of four epsilon Proteobacteria that are directly associated with Alvinella pompejana, a deep sea hydrothermal vent polychete, or with hydrothermal vent chimney samples. These novel bacteria were moderately thermophilic sulfur-reducing heterotrophs growing on formate as the energy and carbon source. In addition, two of them (Am-H and Ex-18.2) could grow on sulfur lithoautrotrophically using hydrogen as the electron donor. Optimal growth temperatures of the bacteria ranged from 41 to 45°C. Phylogenetic analysis of the small-subunit ribosomal gene of the two heterotrophic bacteria demonstrated 95% similarity to Sulfurospirillum arcachonense, an epsilon Proteobacteria isolated from an oxidized marine surface sediment. The autotrophic bacteria grouped within a deeply branching clade of the epsilon Proteobacteria, to date composed only of uncultured bacteria detected in a sample from a hydrothermal vent along the mid-Atlantic ridge. A molecular survey of various hydrothermal vent environments demonstrated the presence of two of these bacteria (Am-N and Am-H) in more than one geographic location and habitat. These results suggest that certain epsilon Proteobacteria likely fill important niches in the environmental habitats of deep-sea hydrothermal vents, where they contribute to overall carbon and sulfur cycling at moderate thermophilic temperatures.     

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.     

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%.     

Biotransformation of Hg(II) by Cyanobacteria

The biotransformation of Hg(II) by cyanobacteria was investigated under aerobic and pH-controlled culture conditions. Mercury was supplied as HgCl₂ in amounts emulating those found under heavily impacted environmental conditions where bioremediation would be appropriate. The analytical procedures used to measure mercury within the culture solution, including that in the cyanobacterial cells, used reduction under both acid and alkaline conditions in the presence of SnCl₂. Acid reduction detected free Hg(II) ions and its complexes, whereas alkaline reduction revealed that meta-cinnabar (β-HgS) constituted the major biotransformed and cellularly associated mercury pool. This was true for all investigated species of cyanobacteria: Limnothrix planctonica (Lemm.), Synechococcus leopoldiensis (Racib.) Komarek, and Phormidium limnetica (Lemm.). From the outset of mercury exposure, there was rapid synthesis of β-HgS and Hg(0); however, the production rate for the latter decreased quickly. Inhibitory studies using dimethylfumarate and iodoacetamide to modify intra- and extracellular thiols, respectively, revealed that the former thiol pool was required for the conversion of Hg(II) into β-HgS. In addition, increasing the temperature enhanced the amount of β-HgS produced, with a concomitant decrease in Hg(0) volatilization. These findings suggest that in the environment, cyanobacteria at the air-water interface could act to convert substantial amounts of Hg(II) into β-HgS. Furthermore, the efficiency of conversion into β-HgS by cyanobacteria may lead to the development of applications in the bioremediation of mercury.     

Isolation and Characterization of Bacteria Capable of Tolerating the Extreme Conditions of Clean Room Environments

In assessing the bacterial populations present in spacecraft assembly, spacecraft test, and launch preparation facilities, extremophilic bacteria (requiring severe conditions for growth) and extremotolerant bacteria (tolerant to extreme conditions) were isolated. Several cultivation approaches were employed to select for and identify bacteria that not only survive the nutrient-limiting conditions of clean room environments but can also withstand even more inhospitable environmental stresses. Due to their proximity to spacefaring objects, these bacteria pose a considerable risk for forward contamination of extraterrestrial sites. Samples collected from four geographically distinct National Aeronautics and Space Administration clean rooms were challenged with UV-C irradiation, 5% hydrogen peroxide, heat shock, pH extremes (pH 3.0 and 11.0), temperature extremes (4°C to 65°C), and hypersalinity (25% NaCl) prior to and/or during cultivation as a means of selecting for extremotolerant bacteria. Culture-independent approaches were employed to measure viable microbial (ATP-based) and total bacterial (quantitative PCR-based) burdens. Intracellular ATP concentrations suggested a viable microbial presence ranging from below detection limits to 10⁶ cells/m². However, only 0.1 to 55% of these viable cells were able to grow on defined culture medium. Isolated members of the Bacillaceae family were more physiologically diverse than those reported in previous studies, including thermophiles (Geobacillus), obligate anaerobes (Paenibacillus), and halotolerant, alkalophilic species (Oceanobacillus and Exiguobacterium). Non-spore-forming microbes (α- and β-proteobacteria and actinobacteria) exhibiting tolerance to the selected stresses were also encountered. The multiassay cultivation approach employed herein enhances the current understanding of the physiological diversity of bacteria housed in these clean rooms and leads us to ponder the origin and means of translocation of thermophiles, anaerobes, and halotolerant alkalophiles into these environments.     

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.     

Reduction of Hg(II) to Hg(0) by Biogenic Magnetite from two Magnetotactic Bacteria

Understanding the biogeochemical cycle of the highly toxic element mercury (Hg) is necessary to predict its fate and transport. In this study, we determined that biogenic magnetite isolated from Magnetospirillum gryphiswaldense MSR-1 and Magnetospirillum magnetotacticum MS-1 was capable of reducing inorganic mercury [Hg(II)] to elemental mercury [Hg(0)]. These two magnetotactic bacteria (MTB) lacked mercuric resistance operons in the genomes. However, they revealed high resistance to Hg(II) under atmospheric conditions and an even higher resistance under microaerobic conditions (1% O₂ and 99% N₂). Neither strain reduced Hg(II) to Hg(0) under atmospheric conditions. However, a slow rate (0.05–0.21 µM·d^?1) of Hg(II) loss occurred from late log phase to stationary phase in two MTBs' culture media under microaerobic conditions. Increased Hg(II) entered both cells under microaerobic conditions relative to atmospheric conditions. The majority of Hg(II) was still blocked by the cell membrane. Hg(II) reduction was more effective when biogenic magnetite was extracted out, with or without the magnetosome membrane envelope. When magnetosome membrane was present, 8.55–13.53% of 250 nM Hg(II) was reduced to Hg(0) by 250 mg/L biogenic magnetite suspension within 2 hours. This ratio increased to 55.07–64.70% while magnetosome membrane was removed. We concluded that two MTBs contributed to the reduction of Hg(II) to Hg(0) at a slow rate in vivo. Such reduction was more favorable to occur when biogenic magnetite is released from dead cells. It proposed a new biotic pathway for the formation of Hg(0) in aquatic systems.     

Sulfate-Reducing Bacteria in Floating Macrophyte Rhizospheres from an Amazonian Floodplain Lake in Bolivia and Their Association with Hg Methylation

Five subgroups of sulfate-reducing bacteria (SRB) were detected by PCR in three macrophyte rhizospheres (Polygonum densiflorum, Hymenachne donacifolia, and Ludwigia helminthorriza) and three subgroups in Eichhornia crassipes from La Granja, a floodplain lake from the upper Madeira basin. The SRB community varied according to the macrophyte species but with different degrees of association with their roots. The rhizosphere of the C₄ plant Polygonum densiflorum had higher frequencies of SRB subgroups as well as higher mercury methylation potentials (27.5 to 36.1%) and carbon (16.06 ± 5.40%), nitrogen (2.03 ± 0.64%), Hg (94.50 ± 6.86 ng Hg g⁻¹), and methylmercury (8.25 ± 1.45 ng Hg g⁻¹) contents than the rhizosphere of the C₃ plant Eichhornia crassipes. Mercury methylation in Polygonum densiflorum and Eichhornia crassipes was reduced when SRB metabolism was inhibited by sodium molybdate.     

Heavy-Metal and Antibiotic Resistance in the Bacterial Flora of Sediments of New York Bight

The New York Bight extends seaward some 80 to 100 miles (ca. 129 to 161 km) from the Long Island and New Jersey shorelines to the edge of the continental shelf. Over 14 × 10⁶ m³ of sewage sludge, dredge spoils, acid wastes, and cellar dirt are discharged into this area each year. Large populations of Bacillus sp. resistant to 20 μg of mercury per ml were observed in Bight sediments contaminated by these wastes. Resistant Bacillus populations were much greater in sediments containing high concentrations of Hg and other heavy metals than in sediments from areas further offshore where dumping has never been practiced and where heavy-metal concentrations were found to be low. Ampicillin resistance due mainly to β-lactamase production was significantly (P < 0.001) more frequent in Bacillus strains from sediments near the sewage sludge dump site than in similar Bacillus populations from control sediments. Bacillus strains with combined ampicillin and Hg resistances were almost six times as frequent at the sludge dump site as in control sediments. This observation suggests that genes for Hg resistance and β-lactamase production are simultaneously selected for in Bacillus and that heavy-metal contamination of an ecosystem can result in a selection pressure for antibiotic resistance in bacteria in that system. Also, Hg resistance was frequently linked with other heavy-metal resistances and, in a substantial proportion of Bacillus strains, involved reduction to volatile metallic Hg (Hg°).     

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.     

Mercury transport and resistance

Resistance to mercuric ions in bacteria is conferred by mercuric reductase, which reduces Hg(II) to Hg(0) in the cytoplasmic compartment. Specific mercuric ion transport systems exist to take up Hg(II) salts and deliver them to the active site of the reductase. This short review discusses the role of transport proteins in resistance and the mechanism of transfer of Hg(II) between the mercury-resistance proteins.     

Bacterial Oxidation of Mercury Metal Vapor, Hg(0)

We used metalloregulated luciferase reporter fusions and spectroscopic quantification of soluble Hg(II) to determine that the hydroperoxidase-catalase, KatG, of Escherichia coli can oxidize monatomic elemental mercury vapor, Hg(0), to the water-soluble, ionic form, Hg(II). A strain with a mutation in katG and a strain overproducing KatG were used to demonstrate that the amount of Hg(II) formed is proportional to the catalase activity. Hg(0) oxidation was much decreased in stationary-phase cells of a strain lacking KatG, suggesting that the monofunctional hydroperoxidase KatE is less effective at this reaction. Unexpectedly, Hg(0) oxidation also occurred in a strain lacking both KatE and KatG, suggesting that activities other than hydroperoxidases may carry out this reaction. Two typical soil bacteria, Bacillus and Streptomyces, also oxidize Hg(0) to Hg(II). These observations establish for the first time that bacteria can contribute, as do mammals and plants, to the oxidative phase of the global Hg cycle.     

Purification and properties of mercuric reductase from Azotobacter chroococcum

Mercury resistance determinants in bacteria are often plasmid-borne or transposon-mediated. Mercuric reductase, one of the proteins encoded by the mercury resistance operon, catalyses a unique reaction in which mercuric ions, Hg (II), are reduced to mercury metal Hg(O) using NADPH as a source of reducing power. Mercuric reductase was purified from Azotobacter chroococcum SS₂ using Red A dye matrix affinity chromatography. Freshly purified preparations of the enzyme showed a single band on polyacrylamide gel electrophoresis under non-denaturing conditions. After SDS-polyacrylamide gel electrophoresis of the freshly prepared enzyme, two protein bands, a major and a minor one, were observed with molecular weight 69 000 and 54 000, respectively. The molecular weight of the native enzyme as determined by gel filtration in Sephacryl S-300 was 142 000. The Km of Hg²⁺-reductase for HgCl₂ was 11·11 μmol l⁻¹. Titration with 5,5'-dithiobis (2-nitrobenzoate) demonstrated that two enzyme–SH groups become kinetically accessible on reduction with NADPH.