Enhanced Mercury Biosorption by Bacterial Cells with Surface-Displayed MerR

The metalloregulatory protein MerR, which exhibits high affinity and selectivity toward mercury, was exploited for the construction of microbial biosorbents specific for mercury removal. Whole-cell sorbents were constructed with MerR genetically engineered onto the surface of Escherichia coli cells by using an ice nucleation protein anchor. The presence of surface-exposed MerR on the engineered strains enabled sixfold-higher Hg²⁺ biosorption than that found in the wild-type JM109 cells. Hg²⁺ binding via MerR was very specific, with no observable decline even in the presence of 100-fold excess Cd²⁺ and Zn²⁺. The Hg²⁺ binding property of the whole-cell sorbents was also insensitive to different ionic strengths, pHs, and the presence of metal chelators. Since metalloregulatory proteins are currently available for a wide variety of toxic heavy metals, our results suggest that microbial biosorbents overexpressing metalloregulatory proteins may be used similarly for the cleanup of other important heavy metals.     

A sensitive assay of mercury using fluorescence correlation spectroscopy of gold nanoparticles

We described a new and sensitive method for the determination of mercury ions (Hg²⁺) on the basis of fluorescence correlation spectroscopy (FCS) and recognition of oligonucleotides. In this assay, 30‐nm gold nanoparticles (GNPs) were modified with oligonucleotides containing thymine bases (T) as fluorescent probes, and the principle of this assay was based on the specific binding of Hg²⁺ by two DNA thymine bases. When two GNPs labelled with different oligonucleotides were mixed with a sample containing Hg²⁺, the T‐Hg²⁺‐T binding reaction should cause GNPs to form dimers (or oligomers), which would lead to a significant increase in the characteristic diffusion time of GNPs in the detection volume. The FCS method is a single molecule detection method and can sensitively detect the change in the characteristic diffusion time of GNPs before and after binding reactions. The quantitative analysis was performed according to the relation between the change in the characteristic diffusion time of GNPs and the concentration of Hg²⁺. Under optimal conditions, the linear range of this method was from 0.3 nM to 100 nM, and the detection limit was 0.14 nM for Hg²⁺. This new method was successfully applied for direct determination of Hg²⁺ levels in water and cosmetics samples. Copyright © 2014 John Wiley & Sons, Ltd.     

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

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

Biosorption of inorganic and methyl mercury by a biosorbent from Aspergillus niger

A biosorbent prepared by alkaline extraction of Aspergillus niger biomass was evaluated for its potential to remove mercury species – inorganic (Hg²⁺) and methyl mercury (CH₃Hg⁺) – from aqueous solutions. Batch experiments were carried out to determine the pH and time profile of sorption for both species in the pH range 2–7. The Hg²⁺ exhibited more rapid sorption and higher capacity than the CH₃Hg⁺. Further, removal of both mercury species from spiked ground water samples was efficient and not influenced by other ions. Sorption studies with esterified biosorbent indicated loss of binding of both mercury species (>80%), which was regained when the ester groups were removed by alkaline hydrolysis, suggesting the involvement of carboxyl groups in binding. Further, no interconversion of sorbed species occurred on the biomass. The biosorbent was reusable up to six cycles without serious loss of binding capacity. Our results suggest that the biosorbent from Aspergillus niger can be used for removal of mercury and methyl mercury ions from polluted aqueous effluents.     

Characterization of mercury(II)-induced inhibition of photochemistry in the reaction center of photosynthetic bacteria

Mercuric contamination of aqueous cultures results in impairment of viability of photosynthetic bacteria primarily by inhibition of the photochemistry of the reaction center (RC) protein. Isolated reaction centers (RCs) from Rhodobacter sphaeroides were exposed to Hg²⁺ ions up to saturation concentration (~?10³ [Hg²⁺]/[RC]) and the gradual time- and concentration-dependent loss of the photochemical activity was monitored. The vast majority of Hg²⁺ ions (about 500 [Hg²⁺]/[RC]) had low affinity for the RC [binding constant K_b?~?5 mM^?1] and only a few (~?1 [Hg²⁺]/[RC]) exhibited strong binding (K_b?~?50 μM^?1). Neither type of binding site had specific and harmful effects on the photochemistry of the RC. The primary charge separation was preserved even at saturation mercury(II) concentration, but essential further steps of stabilization and utilization were blocked already in the 5 < [Hg²⁺]/[RC]?<?50 range whose locations were revealed. (1) The proton gate at the cytoplasmic site had the highest affinity for Hg²⁺ binding (K_b?~?0.2 μM^?1) and blocked the proton uptake. (2) Reduced affinity (K_b?~?0.05 μM^?1) was measured for the mercury(II)-binding site close to the secondary quinone that resulted in inhibition of the interquinone electron transfer. (3) A similar affinity was observed close to the bacteriochlorophyll dimer causing slight energetic changes as evidenced by a?~?30 nm blue shift of the red absorption band, a 47 meV increase in the redox midpoint potential, and a?~?20 meV drop in free energy gap of the primary charge pair. The primary quinone was not perturbed upon mercury(II) treatment. Although the Hg²⁺ ions attack the RC in large number, the exertion of the harmful effect on photochemistry is not through mass action but rather a couple of well-defined targets. Bound to these sites, the Hg²⁺ ions can destroy H-bond structures, inhibit protein dynamics, block conformational gating mechanisms, and modify electrostatic profiles essential for electron and proton transfer.     

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.     

Effect of metal binding on the intrinsic fluorescence of bovine carbonic anhydrase

Bovine carbonic anhydrase shows an intrinsic fluorescence which results from tryptophans located in different microenvironments. It is possible to attribute the whole fluorescence to at least two types of tryptophan.This fluorescence is differently affected by the binding of different metals. In fact while Zn²⁺ causes an increase of the fluorescence yield, the binding of Co²⁺, Cu²⁺ and Hg²⁺ is followed by a quenching of the fluorescence. The quenching is about 40% for the cobalt, 80% for the copper and 60% for the mercury derivative. The binding of Cu²⁺ and Hg²⁺ induces also a change in the shape of the fluorescence emission spectrum. This fact suggests a different influence of the metals on the various types of tryptophan.The fluorescence quenching induced by iodide which can bind to the metal and act as a fluorescence perturbing agent is also indicative of the presence of different tryptophans.     

An amplified surface plasmon resonance "turn-on" sensor for mercury ion using gold nanoparticles

Inorganic mercury ion (Hg²⁺) has been shown to coordinate to DNA duplexes that feature thymine–thymine (T–T) base pair mismatches. This observation suggests that an Hg²⁺-induced conformational change in a single-stranded DNA molecule can be used to detect aqueous Hg²⁺. Here, we have developed an analytical method using surface plasmon resonance (SPR) to develop a highly selective and sensitive detection technique for Hg²⁺ that takes advantage of T–Hg²⁺–T coordination chemistry. The general concept used in this approach is that the “turn-on” reaction of a hairpin probe via coordination of Hg²⁺ by the T–T base pair results in a substantial increase in the SPR response, followed by specific hybridization with a gold nanoparticle probe to amplify the sensor performance. Meanwhile, the limit of detection is 1 nM, which is lower than other recently developed techniques. A linear correlation is observed between the measured SPR reflectivity and the logarithm of the Hg²⁺ concentration over the concentration range of 5–5000 nM. Additionally, the SPR system provides high selectivity for Hg²⁺ in the presence of other divalent metal ions up to micromolar concentration levels. The proposed approach is also successfully utilized for the determination of Hg²⁺ in water samples.     

Influence of Mercury on the Green Alga Haematococcus lacustris. Inhibition Effects and Recovery of Impact

The influence of mercury ions on germination of the resting cells (aplanospores) and on cell division, cell structure, phototaxis, and photosynthesis during the flagellate stage of Hoemotococcus lacustris was investigated. Aplanospores possess a higher tolerance against mercury ions than flagellates. The reason could be seen in the thicker wall of the resting cells which possibly provide a detoxifying effect by immobilisation of Hg²⁺. This is confirmed by a normal phototactic orientation of flagellates formed from Hg ²⁺-influenced aplanospores. In contrast, a direct addition of Hg²⁺ (0.1 to 1 /*M) to the flagellate stage induced an immediate loss of the flagellates to react phototactically, but it was interesting that the inhibition was overcome with time. Obviously, these Hg²⁺ concentrations influence only the sensory transduction chain, whereas the energetic background is not injured because the velocity of movement and the percentage of motile cells were scarcely affected. This is supported by the high level of the photochemical efficiency of photosystem II, which remains unchanged at 1 uM Hg²⁺. Recovery of photosynthesis from inhibition by 10/iM Hg²⁺ suggests a connection between Hg²⁺ influence and metabolism of the D, protein in the reaction centre of photosystem II. The Hg²⁺ effect was reversed with time in light, but not in darkness, and streptomycin, an inhibitor of chloroplast protein synthesis, prevented recovery. In flagellates, showing no reactivation, exposure to 10uA/l Hg²⁺ caused cell swelling, a loss of the flagella, and a disorganisation in structure of the chloroplast and of nucleus.     

Engineering expression of bacterial polyphosphate kinase in tobacco for mercury remediation

To develop the potential of plants to sequester and accumulate mercurials from the contaminated sites, we engineered a tobacco (Nicotiana tabacum) plant to express a bacterial ppk gene, encoding polyphosphate kinase (PPK), under control of a plant promoter. The designated plant expression plasmid pPKT116 that contains the entire coding region of ppk was used for Agrobacterium-mediated gene transfer into tobacco plants. A large number of independent transgenic tobacco plants were obtained, in some of which the ppk gene was stably integrated in the plant genome and substantially translated to the expected PPK protein in the transgenic tobacco. The presence of Hg²⁺ did not cause considerable morphological abnormalities in the transgenic tobacco, which grew, flowered, and set seed similarly to the wild-type tobacco on the medium containing normally toxic levels of Hg²⁺. The ppk-transgenic tobacco showed more resistance to Hg²⁺ and accumulated more mercury than its wild-type progenitors. These results suggest that ppk-specified polyphosphate has abilities to reduce mercury toxicity, probably via chelation mechanism, and also to accumulate mercury in the transgenic tobacco. Based on the results obtained in the present study, the expression of ppk gene in transgenic tobacco plants might provide a means for phytoremediation of mercury pollution.     

In vitro binding of inorganic mercury to the plasma membrane of rat platelet affects Na+-K+-ATPase activity and platelet aggregation

Hg²⁺ binding to ouabain-sensitive Na⁺-K⁺-ATPase of rat platelet membrane was specific with a K_a of 1.3×10⁹ moles and B_max of 3.8 nmoles/mg protein. The binding of mercury to Na⁺-K⁺-ATPase also inhibits the enzyme significantly (P<0.001), which is greater than its ouabain sensitivity. Further in the cytosol of washed platelets conjugation of reduced glutathione (GSH) to Hg²⁺ is correlated dose dependently (25, 50 and 100 pmoles) to enhanced GSH-S-transferase (GST) activity. It may be concluded from the present in vitro experiments that mercury binds specifically to thiol groups present in the platelet membrane Na⁺-K⁺-ATPase, inhibits the enzyme and induces changes in platelet function, namely, platelet aggregation by interfering with the sodium pump.     

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.     

Naphthalimide-based fluorescent probe for Hg2+ detection and imaging in living cells and zebrafish

A simple naphthalimide-based fluorescent probe was designed and synthesized for the determination of mercury ion (Hg²⁺). The probe showed a noticeable fluorescence quenching response for Hg²⁺. When added with Hg²⁺, the fluorescence intensity of the probe at 560 nm was remarkably decreased with the color changed from yellow to colorless under ultraviolet (UV) light. The probe had a notable selectivity and sensitivity for Hg²⁺ and displayed an excellent sensing performance when detecting Hg²⁺ at low concentration (19.5 nM). The binding phenomenon between the probe and Hg²⁺ was identified by Job's method and high-resolution mass spectrometry (HRMS). Moreover, the probe was not only utilized to identify Hg²⁺ in real samples with satisfactory results (92.00%–110.00%) but also was successfully used for bioimaging in cells and zebrafish. The recognition mechanism has been verified by transmission electron microscopy (TEM) for the first time. All the results showed that the probe could be used as a potent useful tool for detection of Hg²⁺.     

Mercury Increases Light Susceptibility in the Green Alga Haematococcus lacustris

The influence of mercury ions (Hg²⁺, 10 μM) on photosynthesis was investigated in flagellates and aplanospores of Haematococcus lacustris. Hg²⁺ stress resulted in a fast decrease of chlorophyll fluorescence yield. This was initially caused by an increase in reversible non-photochemical quenching of chlorophyll fluorescence. During further exposure to Hg²⁺, an increasing contribution of pH independent non-photochemical quenching and a parallel rise in the content of the xanthophyll cycle pigment zeaxanthin was detected. An increase of the initial chlorophyll fluorescence as a final sign of Hg²⁺ induced adverse effects on photosynthesis supports our hypothesis that mercury ions predispose to non-reversible, “chronic” photoinhibition.     

Effects of Cadmium and Mercury on the Upper Part of Skeletal Muscle Glycolysis in Mice

The effects of pre-incubation with mercury (Hg²⁺) and cadmium (Cd²⁺) on the activities of individual glycolytic enzymes, on the flux and on internal metabolite concentrations of the upper part of glycolysis were investigated in mouse muscle extracts. In the range of metal concentrations analysed we found that only hexokinase and phosphofructokinase, the enzymes that shared the control of the flux, were inhibited by Hg²⁺ and Cd²⁺. The concentrations of the internal metabolites glucose-6-phosphate and fructose-6-phosphate did not change significantly when Hg²⁺ and Cd²⁺ were added. A mathematical model was constructed to explore the mechanisms of inhibition of Hg²⁺ and Cd²⁺ on hexokinase and phosphofructokinase. Equations derived from detailed mechanistic models for each inhibition were fitted to the experimental data. In a concentration-dependent manner these equations describe the observed inhibition of enzyme activity. Under the conditions analysed, the integral model showed that the simultaneous inhibition of hexokinase and phosphofructokinase explains the observation that the concentrations of glucose-6-phosphate and fructose-6-phosphate did not change as the heavy metals decreased the glycolytic flux.     

A new proposal regarding the transport mechanism of mercury in biological membranes

The interactions of mercury (Hg²⁺) with biological membranes have been investigated. The experimental results indicate that Hg²⁺ induces a rapid alkalinization in energized Lysosomes from rat liver. The interpretation of the process is that the mercury enters the Lysosomes as a Hg(OH)₂ electroneutral compound, thus inducing alkalinization in the matrix.