Interaction of bacteria with mercuric compounds

We investigated the interaction of mercuric compounds with the bacteriaCorynebacterium ammoniagenes, Micmcoccus luteus, andMycobacterium smegmatus capable of producing hydroxy lamines (R-NOH) and 2-C-methyl-D-erythritol-2,4-cyclopvrophosphate (MECP), which are prone to form free radicals. The interaction of these substances with Hg²⁺ ions and their dynamics during the mercuric poisoning of bacteria was studied by EPR and NMR. Under stress conditions induced by lowering pH or generation of active oxygen species, the bacteria and, especially, their mutants with enhanced sensitivity to oxidative stress, were found to respond to exposure to 1–3 μ/ml HgCl₂ andp-chloromercuribenzoate by a several-fold increase in their viability. The data obtained were interpreted in terms of the involvement of the sulfhydryl groups of bacterial surface proteins in this phenomenon. The interaction of bacteria with mercuric compounds may affect the pathogenesis of tuberculosis and other diseases.     

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.     

Studies on the effect of mercury and organomercurial on the growth and nitrogen fixation by mercury-resistant Azotobacter strains

Five nitrogen-fixing Azotobacter strains isolated from agricultural farms in West Bengal, India, were resistant to mercuric ion and organomercurials. Resistance of Hg-resistant bacteria to mercury compounds is mediated by the activities of mercuric reductase and organomercurial lyase in the presence of NADPH and GSH as cofactors. These bacteria showed an extended lag phase in the presence of 10–50 μmol 1^-1 HgCl₂. Nitrogen-fixing ability of these isolates was slightly inhibited when the mercuryresistant bacterial cells were preincubated with 10 μmol 1^-1 HgCl₂. Acetylene reduction by these bacteria was significantly inhibited (91-97%) by 50 μmol 1^-1 HgCl₂. However, when GSH and NADPH were added to the acetylene reduction assay mixture containing 50 μmol 1^-1 HgCl₂, only 42–50% inhibition of nitrogenase activity was observed. NADPH and GSH might have a role in suppressing the inhibition of N₂-fixation in the presence of Hg compounds either by assisting Hg-detoxifying enzymes to lower Hg concentration in the assay mixture or by formation of adduct comprising Hg and GSH which is unable to inhibit nitrogen fixation.     

Luminescent bacteria toxicity assay in the study of mercury speciation

The toxicities of solutions of 10 mercury compounds to luminescent bacteria were measured using the Microtox Toxicity Bioassay. The aim of this study was to assess the influence that the counter-ions have on the aquatic toxicity of mercury salts. The toxicities of these mercury compounds were very similar, except for mercurous tannate and mercuric salicylate. This can be attributed to differences in the ionization and speciation patterns of these compounds relative to the other compounds tested. In general, the toxicity of the solutions at pH 5 was not significantly different from the toxicity of these solutions at pH 6, but a clear reduction in toxicity was observed when the pH of the solution was adjusted to pH 9. Significant differences were found between the toxicity of Hg(I) and Hg(II) salts of the same anion at pH 9. When cysteine was added to a mercuric nitrate solution (at pH 6), a reduction in the toxicity was observed. This can be explained in terms of the strong binding of mercury to cysteine, thus reducing the concentration of mercury species available to cause an observable toxic effect to the bioluminescent bacteria.     

Microbial Life at 90 C: the Sulfur Bacteria of Boulder Spring

The physiology of the bacteria living in Boulder Spring (Yellowstone National Park) at 90 to 93 C was studied with radioactive isotope techniques under conditions approximating natural ones. Cover slips were immersed in the spring; after a fairly even, dense coating of bacteria had developed, these cover slips were incubated with radioactive isotopes under various conditions and then counted in a gas flow or liquid scintillation counter. Uptake of labeled compounds was virtually completely inhibited by formaldehyde, hydrochloric acid, and mercuric bichloride, and inhibition was also found with streptomycin and sodium azide. The water of Boulder Spring contains about 3 mug of sulfide per ml. Uptake of labeled compounds occurs only if sulfide or another reduced sulfur compound is present during incubation. The pH optimum for uptake of radioactive compounds by Boulder Spring bacteria is 9.2, a value near that of the natural spring water (8.9). Many experiments with a variety of compounds were performed to determine the temperature optimum for uptake of labeled compounds. The results with all the compounds were generally similar, with broad temperature optima between 80 and 90 C, and with significant uptake in boiling (93 C) but not in superheated water (97 C). The results show that the bacteria of Boulder Spring are able to function at the temperature of their environment, although they function better at temperatures somewhat lower. The fine structure of these bacteria has been studied by allowing bacteria in the spring to colonize glass slides or Mylar strips which were immediately fixed, and the bacteria were then embedded and sectioned. The cell envelope structure of these bacteria is quite different from that of other mesophilic or thermophilic bacteria. There is a very distinct plasma membrane, but no morphologically distinct peptidoglycan layer was seen outside of the plasma membrane. Instead, a rather thick diffuse layer was seen, within which a subunit structure was often distinctly visible, and connections frequently occurred between this outer layer and the plasma membrane. The thick outer layer usually consisted of two parts, the outer part of which was sometimes missing. Within the cells, structures resembling ribosomes were seen, and regions lacking electron density which probably contained deoxyribonucleic acid were also visible.     

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%.     

The ecology of mercury-resistant bacteria in Chesapeake Bay

Total ambient mercury concentrations and numbers of mercury resistant, aerobic heterotrophic bacteria at six locations in Chesapeake Bay were monitored over a 17 month period. Mercury resistance expressed as the proportion of the total, viable, aerobic, heterotrophic bacterial population reached a reproducible maximum in spring and was positively correlated with dissolved oxygen concentration and sediment mercury concentration and negatively correlated with water turbidity. A relationship between mercury resistance and metabolic capability for reduction of mercuric ion to the metallic state was established by surveying a number of HgCl₂-resistant cultures. The reaction was also observed in microrganisms isolated by differential centrifugation of water and sediment samples. Mercuric ion exhibited an average half-life of 12.5 days in the presence of approximately 10⁵ organisms/ml. Cultures resistant to 6 ppm of mercuric chloride and 3 ppm of phenylmercuric acetate (PMA) were classified into eight generic categories.Pseudomonas spp. were the most numerous of those bacteria capable of metabolizing both compounds; however, PMA was more toxic and was more selective forPseudomonas. The mercury-resistant generic distribution was distinct from that of the total bacterial generic distribution and differed significantly between water and sediment, positionally and seasonally. The proportion of nonglucose-utilizing mercury-resistantPsuedomonas spp. was found to be positively correlated with total bacterial mercury resistance. It is concluded from this study that numbers of mercury-resistant bacteria as established by plate count can serve as a valid index ofin situ Hg²⁺ metabolism.     

Hematotoxicity and Genotoxicity of Mercuric Chloride Following Subchronic Exposure Through Drinking Water in Male Rats

Erythrocytes are a convenient model to understand the subsequent oxidative deterioration of biological macromolecules in metal toxicities. The present study examined the variation of hematoxic and genotoxic parameters following subchronic exposure of mercuric chloride via drinking water and their possible association with oxidative stress. Male rats were exposed to 50 ppm (HG1) and 100 ppm (HG2) of mercuric chloride daily for 90 days. A significant dose-dependent decrease was observed in red blood cell count, hemoglobin, hematocrit, and mean cell hemoglobin concentration in treated groups (HG1 and HG2) compared with controls. A significant dose-dependent increase was observed in lipid peroxidation; therefore, a significant variation was found in the antioxidant enzyme activities, such as superoxide dismutase, catalase, and glutathione peroxidase. Interestingly, mercuric chloride treatment showed a significant dose-dependent increase in frequency of total chromosomal aberration and in percentage of aberrant bone marrow metaphase of treated groups (p < 0.01). The oxidative stress induced by mercury treatment may be the major cause for chromosomal aberration as free radicals lead to DNA damage. These data will be useful in screening the antioxidant activities of natural products, which may be specific to the bone marrow tissue.     

The ecology of mercury-resistant bacteria in Chesapeake Bay

Total ambient mercury concentrations and numbers of mercury resistant, aerobic heterotrophic bacteria at six locations in Chesapeake Bay were monitored over a 17 month period. Mercury resistance expressed as the proportion of the total, viable, aerobic, heterotrophic bacterial population reached a reproducible maximum in spring and was positively correlated with dissolved oxygen concentration and sediment mercury concentration and negatively correlated with water turbidity. A relationship between mercury resistance and metabolic capability for reduction of mercuric ion to the metallic state was established by surveying a number of HgCl₂-resistant cultures. The reaction was also observed in microrganisms isolated by differential centrifugation of water and sediment samples. Mercuric ion exhibited an average half-life of 12.5 days in the presence of approximately 10⁵ organisms/ml. Cultures resistant to 6 ppm of mercuric chloride and 3 ppm of phenylmercuric acetate (PMA) were classified into eight generic categories.Pseudomonas spp. were the most numerous of those bacteria capable of metabolizing both compounds; however, PMA was more toxic and was more selective forPseudomonas. The mercury-resistant generic distribution was distinct from that of the total bacterial generic distribution and differed significantly between water and sediment, positionally and seasonally. The proportion of nonglucose-utilizing mercury-resistantPsuedomonas spp. was found to be positively correlated with total bacterial mercury resistance. It is concluded from this study that numbers of mercury-resistant bacteria as established by plate count can serve as a valid index ofin situ Hg²⁺ metabolism.     

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.     

Oral bacteria resistant to mercury and to antibiotics are present in children with no previous exposure to amalgam restorative materials

Dental plaque from 76 children without amalgam restorations was screened for bacteria resistant to mercuric chloride. Seventy-one per cent of the children harboured mercury-resistant oral bacteria and the median percentage of the total oral microflora resistant to mercuric chloride was 0.007% (range 0-5.3%). Eighty-seven mercury-resistant bacteria were isolated and 86% of these were streptococci with Streptococcus mitis predominating. Sixty per cent of the mercury-resistant isolates were also resistant to at least one of the four antibiotics tested (penicillin, ampicillin, erythromycin and tetracycline) with resistance to tetracycline (40% of isolates) most frequently encountered.     

Adaptive responses and cellular behaviour of biphenyl-degrading bacteria toward polychlorinated biphenyls

Polychlorinated biphenyls (PCBs) are one of the most widely distributed classes of chlorinated chemicals in the environment. For cleanup of large areas of PCB-contaminated environments, bioremediation seems to be a promising approach. However, the multitude of PCB congeners, their low bioavailability and high toxicity are important factors that affect the cleanup progression. Elucidating how the PCB-degrading microorganisms involved in the process adapt to and deal with the stressing conditions caused by this class of compounds may help to improve the bioremediation process. Also specific physiological characteristics of biphenyl-utilizing bacteria involved in the degradation of PCBs may enhance their availability to these compounds and therefore contribute to a better microbial mineralization. This review will focus in the stress responses caused in aerobic biphenyl-utilizing bacteria by PCBs and its metabolic intermediates and will also analyze bacterial properties such as motility and chemotaxis, adherence to solid surfaces, biosurfactant production and biofilm development, all properties found to enhance bacteria-pollutant interaction.     

Comparison of methods to measure acute metal and organometal toxicity to natural aquatic microbial communities

Microbial communities in water from Baltimore Harbor and from the mainstem of Chesapeake Bay were examined for sensitivity to mercuric chloride, monomethyl mercury, stannic chloride, and tributyltin chloride. Acute toxicity was determined by measuring the effects of [3H]thymidine incorporation, [14C]glutamate incorporation and respiration, and viability as compared with those of controls. Minimum inhibitory concentrations were low for all metals (monomethyl mercury, less than 0.05 microgram liter-1; mercuric chloride, less than 1 microgram liter-1; tributyltin chloride, less than 5 micrograms liter-1) except stannic chloride (5 mg liter-1). In some cases, mercuric chloride and monomethyl mercury were equally toxic at comparable concentrations. The Chesapeake Bay community appeared to be slightly more sensitive to metal stress than the Baltimore Harbor community, but this was not true for all treatments or assays. For culturable bacteria the opposite result was found. Thymidine incorporation and glutamate metabolism were much more sensitive indicators of metal toxicity than was viability. To our knowledge, this is the first use of the thymidine incorporation method for ecotoxicology studies. We found it the easiest and fastest of the three methods; it is at least equal in sensitivity to metabolic measurements, and it likely measures the effects on greater portion of the natural community.     

Genotoxicity of mercury compounds. A review

This article reviews literature data concerning the genotoxicity of 29 mercury-containing agents, including laboratory compounds as well as ingredients of preparations used as fungicides, dyes, disinfectants and drugs. A variety of genetic end-points were investigated in bacteria, yeasts, moulds, plants, insects, cultured cells from fishes, rodents or humans, aquatic organisms, amphibians, mammalia and exposed humans. The overall evaluation is quite complex. Mercury compounds failed to induce point mutations in bacteria but often exerted clastogenic effects in eukaryotes, especially by binding SH groups and acting as spindle inhibitors, thereby causing c-mitosis and consequently aneuploidy and/or polyploidy. Inorganic mercury compounds were also found to induce the generation of reactive oxygen species and glutathione depletion in cultured mammalian cells. Although different mercury compounds tended to produce qualitatively comparable genetic effects, which suggests the involvement of a common toxic entity, methylmercury derivatives and other ionizable organomercury compounds were more active in short-term tests than either non-ionizable mercury compounds (e.g., dimethylmercury) or inorganic mercury salts (e.g., mercuric chloride). The results of cytogenetic monitoring in peripheral blood lymphocytes of individuals exposed to elemental mercury or mercury compounds from accidental, occupational or alimentary sources were either negative or borderline or uncertain as to the actual role played by mercury in some positive findings. Both genotoxic and non-genotoxic mechanisms may contribute to the renal carcinogenicity of mercury, which so far has been convincingly demonstrated only in male rodents treated with methylmercury chloride.     

Mode of in vitro interaction of mercuric mercury with selenite to form high-molecular weight substance in rabbit blood

Mode of interaction of mercuric mercury and selenite in rabbit blood was investigated in vitro. After the incubation of rabbit blood with 10^?5 M each of ²⁰³HgCl₂ and Na₂⁷⁵SeO₃, the amounts of both ²⁰³Hg and ⁷⁵Se incorporated into erythrocytes were markedly larger than the case where the blood was treated separately with one of these compounds. Most of ²⁰³Hg and ⁷⁵Se distributed into plasma and erythrocytes were found in high-molecular weight substance(s) (HMWS) fractionated by gel filtration at a molar ratio of 1:1. The ²⁰³Hg and ⁷⁵Se in HMWS found in plasma and erythrocytes were hardly diffusable through the erythrocytes membrane. The formation of the HMWS containing mercury and selenium was observed in stroma-free hemolysate incubated with mercuric chloride and selenite, but not in plasma. Addition of reduced glutathione (GSH) to the plasma, however, gave the HMWS as reaction products containing equimolar amounts of mercury and selenium. Further the binding properties of selenium to proteins were studied in the plasma incubated with selenodiglutathione (GSSeSG) or with selenite in the presence of GSH. The results indicated that GSH, a cellular component, is essential for the formation of an active selenium compound from selenite and that the interaction of mercuric mercury and selenite in plasma in the presence of GSH may occur through the other mechanism than the formation of GSSeSG.     

Comparison of methods to measure acute metal and organometal toxicity to natural aquatic microbial communities.

Microbial communities in water from Baltimore Harbor and from the mainstem of Chesapeake Bay were examined for sensitivity to mercuric chloride, monomethyl mercury, stannic chloride, and tributyltin chloride. Acute toxicity was determined by measuring the effects of [3H]thymidine incorporation, [14C]glutamate incorporation and respiration, and viability as compared with those of controls. Minimum inhibitory concentrations were low for all metals (monomethyl mercury, less than 0.05 microgram liter-1; mercuric chloride, less than 1 microgram liter-1; tributyltin chloride, less than 5 micrograms liter-1) except stannic chloride (5 mg liter-1). In some cases, mercuric chloride and monomethyl mercury were equally toxic at comparable concentrations. The Chesapeake Bay community appeared to be slightly more sensitive to metal stress than the Baltimore Harbor community, but this was not true for all treatments or assays. For culturable bacteria the opposite result was found. Thymidine incorporation and glutamate metabolism were much more sensitive indicators of metal toxicity than was viability. To our knowledge, this is the first use of the thymidine incorporation method for ecotoxicology studies. We found it the easiest and fastest of the three methods; it is at least equal in sensitivity to metabolic measurements, and it likely measures the effects on greater portion of the natural community.     

Genes encoding mercuric reductases from selected gram-negative aquatic bacteria have a low degree of homology with merA of transposon Tn501

An investigation of the Hg2+ resistance mechanism of four freshwater and four coastal marine bacteria that did not hybridize with a mer operonic probe was conducted (T. Barkay, C. Liebert, and M. Gillman, Appl. Environ. Microbiol. 55:1196-1202, 1989). Hybridization with a merA probe, the gene encoding the mercuric reductase polypeptide, at a stringency of hybridization permitting hybrid formation between evolutionarily distant merA genes (as exists between gram-positive and -negative bacteria), detected merA sequences in the genomes of all tested strains. Inducible Hg2+ volatilization was demonstrated for all eight organisms, and NADPH-dependent mercuric reductase activities were detected in crude cell extracts of six of the strains. Because these strains represented random selections of bacteria from three aquatic environments, it is concluded that merA encodes a common molecular mechanism for Hg2+ resistance and volatilization in aerobic heterotrophic aquatic communities.     

Genes encoding mercuric reductases from selected gram-negative aquatic bacteria have a low degree of homology with merA of transposon Tn501.

An investigation of the Hg2+ resistance mechanism of four freshwater and four coastal marine bacteria that did not hybridize with a mer operonic probe was conducted (T. Barkay, C. Liebert, and M. Gillman, Appl. Environ. Microbiol. 55:1196-1202, 1989). Hybridization with a merA probe, the gene encoding the mercuric reductase polypeptide, at a stringency of hybridization permitting hybrid formation between evolutionarily distant merA genes (as exists between gram-positive and -negative bacteria), detected merA sequences in the genomes of all tested strains. Inducible Hg2+ volatilization was demonstrated for all eight organisms, and NADPH-dependent mercuric reductase activities were detected in crude cell extracts of six of the strains. Because these strains represented random selections of bacteria from three aquatic environments, it is concluded that merA encodes a common molecular mechanism for Hg2+ resistance and volatilization in aerobic heterotrophic aquatic communities.     

Mercuric reductase activity in the adaptation to cationic mercury, phenyl mercuric acetate and multiple antibiotics of a gram-negative population isolated from an aerobic fixed-bed reactor.

Eighty-eight strains, isolated from an aerobic fixed-bed reactor and identified to the genus level, were examined for resistance to 21 antibiotics, cationic mercury and phenylmercuric acetate. All except three were able to grow on Mueller-Hinton agar plates containing 8 micrograms/ml mercuric chloride, but only 42 exhibited a mercuric reductase and an organomercurial lyase activity. Furthermore, 82 of them were multiply-antibiotic resistant, whereas no positive correlation between this property and cationic mercury volatilization capacity was found. It was concluded that this bacterial community-adapted response to these selective agents, which has been most often shown to be mediated by R plasmids, was the result of two independent phenomena. Moreover, the high percentage of multiple antibiotic and mercury resistance found in this population suggested that simultaneous selections occurred on filters of bacteria which exhibited mucoid colonies and tolerance to these two categories of stress agents.     

Inactivation and sedimentation of mercury from organomercurials by Klebsiella pneumoniae

Abstract A susceptibility of 63 clinical isolates of Klebsiella pneumoniae to inorganic and organic mercuric compounds was determined. 18 of them were found to be resistant to fluorescein mercuric acetate (FMA) and merbromin (MB). Moreover, all the resistant strains inactivate the antibacterial effect of FMA. The changes in the amount of organic mercury at the time of inactivation of the drug and the structures of the end products were examined in detail with the plasmid-bearing strain JK9 and its transconjugants of Escherichia coli .
The results showed that FMA was inactivated by an intracellular enzyme produced inducively and was degraded to fluorescein (sodium salt, uranine), which led to the sedimentation of metallic mercury. The discovery of the genes conferring inducible organic mercury-inactivating enzymes determined by plasmids was the next step and their application in the recovery of metallic mercury from organomercurials is now imminent.