Mercuric Reductase Research Articles

Computational insights into mercuric reductase from Pseudomonas fluorescens: a bioinformatic and molecular dynamics approach for mercury detoxification

Mercury (Hg) pollution poses significant threats to human health and ecosystems worldwide due to its persistence, bioaccumulation, and toxic effects. This study focuses on mercuric reductase from Pseudomonas fluorescens (UniProt ID Q51772), a key enzyme involved in detoxifying mercury through reduction to its less toxic elemental form, Hg(0). This study aims to explore the potential of this enzyme for bioremediation applications, focusing on structural insights, functional mechanisms, and biotechnological enhancements to facilitate mercury detoxification. The protein sequence of Q51772 was analyzed using bioinformatics tools to determine its structural and functional attributes. Physiochemical properties, including molecular weight, isoelectric point, and secondary structure predictions, were assessed. Virulence prediction tools confirmed the protein’s nontoxic and nonpathogenic nature. Homology modeling and docking studies provided insights into its three-dimensional structure and binding interactions with mercury substrates. Q51772 consists of 548 amino acids and belongs to the Pyridine nucleotide-disulphide oxidoreductase family, class I. It features specific domains crucial for mercury reduction, identified through Pfam and InterPro analyses. Physiochemical analysis indicated a stable protein with hydrophilic tendencies conducive to enzymatic function. Structural modeling validated by ProQ and PROCHECK confirmed the reliability of the predicted structure. Molecular docking of organic mercury compound with wild-type and mutant mercuric reductase revealed strong binding affinities, with key hydrogen bonds involving residues Gly95, Thr122, and Asp390. Mercuric reductase Q51772 from Pseudomonas fluorescens emerges as a promising candidate for bioremediation of mercury-contaminated environments. Future research should focus on optimizing its performance under diverse environmental conditions to advance sustainable solutions for mercury pollution control.

Further Evidence of Anthropogenic Impact: High Levels of Multiple-Antimicrobial-Resistant Bacteria Found in Neritic-Stage Sea Turtles.

Marine turtles are globally threatened and face daily anthropogenic threats, including pollution. Water pollution from emerging contaminants such as antimicrobials is a major and current environmental concern. This study investigated the phenotypic antimicrobial resistance and heavy metal resistance genes of 47 Vibrio isolates from different stages of sea turtles (oceanic stage vs neritic stage) from the Taiwanese coast. The results show that a high proportion (48.9%; 23/47) of the Vibrio species isolated from sea turtles in our study had a multiple antimicrobial resistance (MAR) pattern. It was found that Vibrio spp. isolates with a MAR pattern and those with a MAR index value greater than 0.2 were both more likely to be observed in neritic-stage sea turtles. Furthermore, isolates from neritic-stage sea turtles exhibited greater resistance to the majority of antimicrobials tested (with the exception of beta-lactams and macrolides) than isolates from the oceanic-stage groups. Isolates from neritic sea turtles were found to be more resistant to nitrofurans and aminoglycosides than isolates from oceanic sea turtles. Furthermore, isolates with a MAR pattern (p = 0.010) and those with a MAR index value greater than 0.2 (p = 0.027) were both found to be significantly positively associated with the mercury reductase (merA) gene. The findings of our study indicate that co-selection of heavy metals and antimicrobial resistance may occur in aquatic bacteria in the coastal foraging habitats of sea turtles in Taiwan.

Impacts of Selenium Supplementation on Soil Mercury Speciation, Soil Properties and Mercury-Resistant Microorganisms and Resistant Genes

Soil mercury (Hg) contamination is a serious threat to local ecology and public health. Exogenous selenium (Se) supplementation can effectively reduce the toxicity of Hg. However, the mechanisms affecting the changes in soil Hg speciation, soil properties and the microbial Hg-resistant system during the Se–Hg interaction after exogenous Se supplementation are not clear. Therefore, in this study, soil culture experiments were conducted to analyze the effects of different Se additions on the transformation of Hg speciation, soil properties and Hg-resistant microorganisms and resistant genes (mer operon). The results indicated that Se supplementation facilitated the transformation of soil Hg from bioavailable (exchangeable and carbonate-bound) to stable forms (organic material-bound and residual), significantly reducing Hg bioavailability. Se supplementation notably decreased the electrical conductivity of Hg-contaminated soil, but had no significant effect on the soil pH, organic matter content, cation exchange capacity or alkaline phosphatase and catalase activities. The maximum activity levels of soil sucrase and urease were observed when 1 mg kg−1 Se was added. Se significantly inhibited soil peroxidase and ascorbate oxidase activities, thereby alleviating the oxidative stress in the soil system caused by Hg. Additionally, Se significantly activated the Hg-resistant system in soil microorganisms by either decreasing or increasing the regulatory genes merD and merR, and it significantly upregulated the cytoplasmic protein gene merP and the membrane protein genes merC, merF and merT. This further increased the abundance of the organomercury lyase gene merB and the mercuric reductase gene merA, promoting the conversion of Hg species to Hg⁰. Furthermore, the abundance of mer operon-containing microorganisms, such as Thiobacillus ferrooxidants, Pseudomonas, Streptomyces and Cryptococcus, significantly increased with Se addition, explaining the role of soil microorganisms in mitigating soil Hg stress via Se supplementation.

Labile carbon inputs boost microbial contribution to legacy mercury reduction and emissions from industry-polluted soils

Soils is a crucial reservoir influencing mercury (Hg) emissions and soil-air exchange dynamics, partially modulated by microbial reducers aiding Hg reduction. Yet, the extent to which microbial engagements contribute to soil Hg volatilization remains largely unknown. Here, we characterized Hg-reducing bacterial communities in natural and anthropogenically perturbed soil environments and quantified their contribution to soil Hg(0) volatilization. Our results revealed distinct Hg-reducing bacterial compositions alongside elevated mercuric reductase (merA) gene abundance and diversity in soils adjacent to chemical factories compared to less-impacted ecosystems. Notably, solely industry-impacted soils exhibited increased merA gene abundance along Hg gradients, indicating microbial adaption to Hg selective pressure through quantitative changes in Hg reductase and genetic diversity. Microcosm studies demonstrated that glucose inputs boosted microbial involvement and induced 2–8 fold increments in cumulative Hg(0) volatilization in industry-impacted soils. Microbially-mediated Hg reduction contributed to 41.6% of soil Hg(0) volatilization in industry-impacted soils under 25% water-holding capacity and glucose input conditions over a 21-day incubation period. Alcaligenaceae, Moraxellaceae, Nitrosomonadaceae and Shewanellaceae were identified as potential contributors to Hg(0) volatilization in the soil. Collectively, our study provides novel insights into microbially-mediated Hg reduction and soil-air exchange processes, with important implications for risk assessment and management of industrial Hg-contaminated soils.

Biochemical and structural basis of mercuric reductase, GbsMerA, from Gelidibacter salicanalis PAMC21136

Heavy metals, including mercury, are non-biodegradable and highly toxic to microorganisms even at low concentrations. Understanding the mechanisms underlying the environmental adaptability of microorganisms with Hg resistance holds promise for their use in Hg bioremediation. We characterized GbsMerA, a mercury reductase belonging to the mercury-resistant operon of Gelidibacter salicanalis PAMC21136, and found its maximum activity of 474.7 µmol/min/mg in reducing Hg+2. In the presence of Ag and Mn, the enzyme exhibited moderate activity as 236.5 µmol/min/mg and 69 µmol/min/mg, respectively. GbsMerA exhibited optimal activity at pH 7.0 and a temperature of 60 °C. Moreover, the crystal structure of GbsMerA and structural comparison with homologues indicated that GbsMerA contains residues, Tyr437´ and Asp47, which may be responsible for metal transfer at the si-face by providing a hydroxyl group (−OH) to abstract a proton from the thiol group of cysteine. The complex structure with NADPH indicated that Y174 in the re-face can change its side chain direction upon NADPH binding, indicating that Y174 may have a role as a gate for NADPH binding. Moreover, the heterologous host expressing GbsMerA (pGbsMerA) is more resistant to Hg toxicity when compared to the host lacking GbsMerA. Overall, this study provides a background for understanding the catalytic mechanism and Hg detoxification by GbsMerA and suggests the application of genetically engineered E. coli strains for environmental Hg removal.

The provenance of microorganisms adapted to extreme salinity, extreme temperature, and toxic metals within the Montney shale formation.

Introduction Shale oil reservoirs are hypothesized to be sterile due to the extremely high temperature, pressure and salinity within these formations (Evans et al. 2018). High concentrations of toxic metals also pose challenges that demand specific microbial adaptions (Boyd and Barkay 2012, White and Gadd 1998, Ben Fekih et al. 2018). While some microorganisms are introduced into and are selected for within shale formations during hydraulic fracturing, the possibility that certain microorganisms are pre-existing inhabitants of these formations is less clear. Here, we followed the microbial diversity of input and output fluids injected into a Montney formation shale reservoir to assess the distribution and transport of microbial populations during hydraulic fracturing. Enrichment cultures distinguished various metabolisms in the microbial populations found in different sample types, and adaptations allowing them to colonize such niches. Material and methods Fracturing fluid, drilling muds (3302 m, 3350 m and 3400 m depths), shale cuttings (rinsed from the drillings muds), shale core plugs and produced water samples (12-month period) were sampled from a Montney shale oil reservoir. Microbial community compositions were analyzed by amplicon sequencing. Metal content was analyzed by inductively coupled plasma-mass spectrophotometry. High salinity enrichments at 90°C of the drilling muds or rinsed shale samples were set up in triplicate and amended with glucose and guar gum (a mannose/galactose-based polymer used during hydraulic fracturing). Sugars were measured through spectrophotometric assays. Metagenomic analyses were performed to assess microbial gene content. Results/Discussion Provenance of microorganisms from the Montney shale formation Provenance of microorganisms from the Montney shale formation Input fluids (fracturing fluid, drilling muds) were revealed to be the likely source of most of the microbial diversity. However, some microorganisms were only detected in the subsurface samples. ASVs affiliated with Aurantimonas, Caminicella, BRH-c8a (Family Desulfallas) and Geotoga exhibited occurrence patterns consistent with being derived from subsurface shale formations. Geotoga has only ever been reported from oil reservoirs (Semenova et al. 2020). Analysis of produced water revealed ASVs from these groups increasing in abundance during hydraulic fracturing operations, suggesting selective pressure from oil reservoir conditions (e.g., toxic metal presence, input of saline water, temperature and pressure fluctuations). Incubations set up from drilling muds showed a preference for glucose while incubations of the rinsed shale cuttings showed a microbial preference for guar gum (i.e., mannose production; Fig. 0), reinforcing the presence of different populations being derived from surface and subsurface samples. Adaptations for life in Montney shale Adaptations for life in Montney shale When considering adaptations of microorganisms for environmental conditions found in oil reservoirs, it is relevant to note the presence of toxic metals such as arsenic, cadmium and mercury. Levels of all three metals were found to vary over time within the 28-day shale microbial enrichments and 12-month produced water time course analyses (Suppl. material 1). Metagenomics revealed various genes for the internalization and metabolism of all three metals within the shale microbiome (i.e., arsenate reductases, arsenite transporters, metallothioneins, mercuric reductases). In conclusion, the results of this study suggest that shale reservoirs thus might not be sterile environments, and host microorganisms are able to contend with major perturbations.

Study of mercury resistance and Fourier transform infrared spectroscopy-based metabolic profiling of a potent Bacillus tropicus strain from forest soil.

Mercury (Hg) is a highly toxic heavy metal and Hg-resistant indigenous bacterial isolates may offer a green and cost-effective bioremediation strategy to counter Hg contamination. In this study, a potent Hg-resistant bacterium was isolated from the forest soil of a bird sanctuary. Identification using matrix-assisted laser desorption ionization-time of flight mass spectrometry depicted the isolate as a strain of Bacillus tropicus, validated by morphological, biochemical, and molecular studies. The isolate demonstrated biological Hg removal efficiency and capacity of 50.67% and 19.76 mg g-1 , respectively. The plasmid borne resistance determinant, merA, encoding mercuric reductase, was detected in the bacterium endowing it with effective Hg volatilization and resistance capability. A Fourier-transform infrared spectroscopic comparative metabolic profiling revealed the involvement of various functional groups like -COOH, -CH2 , -OH, PO4 - and so on, resulting in differential spectral patterns of the bacterium both in control and Hg-exposed situations. A temporal variance in metabolic signature was also observed during the early and mid-log phase of growth in the presence of Hg. The bacterium described in this study is the first indigenous Hg-resistant strain isolated from the Uttar Dinajpur region, which could be further explored and exploited as a potent bioresource for Hg remediation.

In silico explorations of bacterial mercuric reductase as an ecofriendly bioremediator for noxious mercuric intoxications.

Mercury is a major pollutant in the environment due to its high concentration in the soil. In this study, a mercuric reductase was extracted from Pseudomonas aeruginosa. The sequence of the enzyme was retrieved from the literature and structural homologs were identified. The protein bonded with Mercuric compounds and their interaction was briefly studied. Autodock Vina was used to perform a molecular docking with the target protein. Results showed that the sequence consists of most of the random coil 44.74% followed by α-helix and B-turns. Moreover, the protein was predicted to have a FAD/NAD(P)-binding domain. The virulence factor prediction using different approaches of Virulentpred and VICMpred suggested that P00392 is non-toxic. Next, the mutational analyses were performed to predict the active site residues in the resulting models and to determine mutants. The results show that the enzyme is involved in the bioremediation of mercury by using in-silico techniques. Finally, molecular docking studies were conducted on the best-selected model to find the active site residues and to generate a pattern of interaction to understand the mode of action of the substrate and its catalytic activity which refers to the binding with mercury.

Quantitative Proteomic Analysis of Cyanide and Mercury Detoxification by Pseudomonas pseudoalcaligenes CECT 5344.

The cyanide-degrading bacterium Pseudomonas pseudoalcaligenes CECT 5344 uses cyanide and different metal-cyanide complexes as the sole nitrogen source. Under cyanotrophic conditions, this strain was able to grow with up to 100 μM mercury, which was accumulated intracellularly. A quantitative proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/MS) has been applied to unravel the molecular basis of the detoxification of both cyanide and mercury by the strain CECT 5344, highlighting the relevance of the cyanide-insensitive alternative oxidase CioAB and the nitrilase NitC in the tolerance and assimilation of cyanide, independently of the presence or absence of mercury. Proteins overrepresented in the presence of cyanide and mercury included mercury transporters, mercuric reductase MerA, transcriptional regulator MerD, arsenate reductase and arsenical resistance proteins, thioredoxin reductase, glutathione S-transferase, proteins related to aliphatic sulfonates metabolism and sulfate transport, hemin import transporter, and phosphate starvation induced protein PhoH, among others. A transcriptional study revealed that from the six putative merR genes present in the genome of the strain CECT 5344 that could be involved in the regulation of mercury resistance/detoxification, only the merR2 gene was significantly induced by mercury under cyanotrophic conditions. A bioinformatic analysis allowed the identification of putative MerR2 binding sites in the promoter regions of the regulatory genes merR5, merR6, arsR, and phoR, and also upstream from the structural genes encoding glutathione S-transferase (fosA and yghU), dithiol oxidoreductase (dsbA), metal resistance chaperone (cpxP), and amino acid/peptide extruder involved in quorum sensing (virD), among others. IMPORTANCE Cyanide, mercury, and arsenic are considered very toxic chemicals that are present in nature as cocontaminants in the liquid residues generated by different industrial activities like mining. Considering the huge amounts of toxic cyanide- and mercury-containing wastes generated at a large scale and the high biotechnological potential of P. pseudoalcaligenes CECT 5344 in the detoxification of cyanide present in these industrial wastes, in this work, proteomic, transcriptional, and bioinformatic approaches were used to characterize the molecular response of this bacterium to cyanide and mercury, highlighting the mechanisms involved in the simultaneous detoxification of both compounds. The results generated could be applied for developing bioremediation strategies to detoxify wastes cocontaminated with cyanide, mercury, and arsenic, such as those generated at a large scale in the mining industry.

Mercury remediation potential of mercury-resistant strain Rheinheimera metallidurans sp. nov. isolated from a municipal waste dumping site

A novel mercury-resistant bacterium, designated strain DCL_24T, was isolated from the legacy waste at the Daddu Majra dumping site in Chandigarh, India. It showed resistance up to 300 µM of inorganic mercury (mercuric chloride). The isolate was found to be a Gram-negative, facultative anaerobic, motile, and rod-shaped bacterium that can grow at 4 – 30 °C (optimum 25 °C), pH 6.0 – 12.0 (optimum 7.0), and 0 – 4.0 % (w/v) NaCl (optimum 0.5 – 2.0 %). The 16 S rRNA gene-based phylogenetic analysis showed that DCL_ 24 T shared a 97.53 % similarity with itsºlosest type strain Rheinheimera muenzenbergensis E-49T. Insilico DNA-DNA hybridization and average nucleotide identity values were found to be 18.60 % and 73.77 %, respectively, between the genomes of DCL_24T and R. muenzenbergensis E-49T. The strain DCL_24T has 44.33 DNA G+C content (mol %). Based on the phenotypic, chemotaxonomic, and genotypic data, the strain DCL_24T represents a novel species within the genus Rheinheimera, for which the name Rheinheimera metallidurans sp. nov is proposed. The type strain is DCL_24T (MTCC13203T = NBRC115780T = JCM 35551 T). The isolate was found to volatilize and remove mercury efficiently, as demonstrated by X-ray film and dithizone-based colorimetric methods. Around 92 % of mercury removal was observed within 48 h. The mercury-resistant determinant mer operon consisting of merA, encoding the mercuric reductase enzyme, and transport and regulatory genes (merT, merP, merD, and merR) were found in the isolate. Relative expression analysis of merA at increasing concentrations of HgCl2 was confirmed by quantitative real-time PCR. These data indicate the merA-mediated reduction of toxic Hg2+ into a non-toxic volatile Hg0. The phytotoxicity assay performed using Arabidopsis thaliana seeds further demonstrated the mercury toxicity reduction potential of DCL_24T. The study shows that this novel isolate, DCL_24T, is an interesting candidate for mercury bioremediation. However, further studies are required to assess the bioremediation efficacy of the strain under the harsh environmental conditions prevailing in polluted sites.

Bacterial Metal-Scavengers Newly Isolated from Indonesian Gold Mine-Impacted Area: Bacillus altitudinis MIM12 as Novel Tools for Bio-Transformation of Mercury.

Selikat river, located in the north part of Bengkulu Province, Indonesia, has critical environmental and ecological issues of contamination by mercury due to artisanal small-scale gold mining (ASGM) activities. The present study focused on the identification and bioremediation efficiency of the mercury-resistant bacteria (MRB) isolated from ASGM-impacted areas in Lebong Tambang village, Bengkulu Province, and analyzed their merA gene function in transforming Hg2+ to Hg0. Thirty-four MRB isolates were isolated, and four out of the 34 isolates exhibited not only the highest degree of resistance to Hg (up to 200ppm) but also to cadmium (Cd), chromium (Cr), copper (Cu), and lead (Pb). Further analysis shows that all four selected isolates harbor a merA operon-encoded mercuric ion (Hg2+) reductase enzyme, with the Hg bioremediation efficiency varying from 71.60 to 91.30%. Additionally, the bioremediation efficiency for Cd, Cr, Cu, and Pb ranged from 54.36 to 98.37%. Among the 34, two isolates identified as Bacillus altitudinis possess effective and superior multi-metal degrading capacity up to 91.30% for Hg, 98.07% for Cu, and 54.36% for Cr. A pilot-scale study exhibited significant in situ bioremediation of Hg from gold mine tailings of 82.10 and 95.16% at 4- and 8-day intervals, respectively. Interestingly, translated nucleotide blast against bacteria and Bacilli merA sequence databases suggested that B. altitudinis harbor merA gene is the first case among Bacilli with the possibility exhibits a novel mechanism of bioremediation, considering our new finding. This study is the first to report the structural and functional Hg-resistant bacterial diversity of unexplored ASGM-impacted areas, emphasizing their biotechnological potential as novel tools for the biological transformation and adsorption of mercury and other toxic metals.

Cloneable inorganic nanoparticles.

When a defined protein/peptide (or combinations thereof) control and define the synthesis of an inorganic nanoparticle, the result is a cloneable NanoParticle (cNP). This is because the protein sequence/structure/function is encoded in DNA, and therefore the physicochemical properties of the nanoparticle are also encoded in DNA. Thus the cloneable nanoparticle paradigm can be considered as an extension of the central dogma of molecular biology (e.g. DNA → mRNA → protein → cNP); modifications to the DNA encoding a cNP can modify the resulting properties of the cNP. Inorganic ion oxidoreductases (e.g., mercuric reductase, tellurite reductase, etc.) can select and reduce specific inorganic oxyanions and coordination complexes, creating zerovalent precipitates. Other proteins/peptides (often genetically concatenated to the parent oxidoreductase) serve as ligands, directing the size, shape, crystal structure and other properties of the nanoparticle. The DNA encoding a cNP can be recombinantly transferred into any organism. Ideally, this enables recombinant production of cNPs with the same defined physiochemical properties. Such cNPs are of interest for applications ranging from molecular imaging, bio-remediation, catalysis, and biomining. In this Feature Article we detail and define the cNP concept, and retrace the story of our creation of a cloneable Se NanoParticle (cSeNP). We also describe our more preliminary work that we expect to result in cloneable semiconductor quantum dots, cloneable Te nanoparticles, and other cNP formulations. We highlight the application of cNPs in cellular electron microscopy and compare this approach to other cloneable imaging contrast approaches.

Unraveling the potential of bacteria isolated from the equatorial region of Indian Ocean in mercury detoxification

The marine environment is most vital and flexible with continual variations in salinity, temperature, and pressure. As a result, bacteria living in such an environment maintain the adaption mechanisms that are inherent in unstable environmental conditions. The harboring of metal-resistant genes in marine bacteria contributes to their effectiveness in metal remediation relative to their terrestrial counterparts. A total of four mercury-resistant bacteria (MRB) i.e. NIOT-EQR_J7 (Alcanivorax xenomutans); NIOT-EQR_J248 and NIOT-EQR_J251 (Halomonas sp.); and NIOT-EQR_J258 (Marinobacter hydrocarbonoclasticus) were isolated from the equatorial region of the Indian Ocean (ERIO) and identified by analyzing the 16S rDNA sequence. The MRBs can reduce up to 70% of Hg(II). The mercuric reductase (merA) gene was amplified and the mercury (Hg) volatilization was confirmed by the X-ray film method. The outcomes obtained from ICP-MS validated that the Halomonas sp. NIOT-EQR_J251 was more proficient in removing the Hg from culture media than other isolates. Fourier transform infrared (FT-IR) spectroscopy results revealed alteration in several functional groups attributing to the Hg tolerance and reduction. The Gas Chromatography-Mass Spectrometry (GC-MS) analysis confirmed that strain Halomonas sp. (NIOT-EQR_J248 and NIOT-EQR_J251) released Isooctyl thioglycolate (IOTG) compound under mercury stress. The molecular docking results suggested that IOTG can efficiently bind with the glutathione S-transferase (GST) enzyme. A pathway has been hypothesized based on the GC-MS metabolic profile and molecular docking results, suggesting that the compound IOTG may mediate mercuric reduction via merA-GST related detoxification pathway.

Is the merA gene sufficient as a molecular marker of mercury bacterial resistance?

Gene encoding mercuric ion reductase, merA is a crucial component of the mer operon for reduction of nonorganic mercury ions into less toxic form. The merA gene or its fragments are commonly used as a molecular marker of bacterial resistance to mercury. In this study, it was tested whether the merA gene can be considered as a molecular marker of mercury bacterial resistance. For this purpose, the presence of the mer operon in bacteria isolated from the microbiota of Tussilago farfara L. growing in post-industrial mercury-contaminated and non-contaminated areas was verified by merA gene identification. Mercury resistance was determined by analyzing the bacterial growth parameters in standard Luria-Bertani (LB) medium with mercury concentration of 0.01% (w/v) and in medium without mercury addition. The results obtained showed that the merA gene was present in all T. farfara L. bacterial isolates growing in both mercury-contaminated and noncontaminated soils, however, only the isolates from mercury-contaminated areas were able to grow under mercury conditions. Although merA is commonly regarded as a molecular marker of bacterial mercury resistance, results of our research indicate the need for a verification of that statement/thesis and further investigation of bacterial mercury resistance to indicate other its key markers, structures, or mechanisms.

In vitro and in silico Studies Reveal Bacillus cereus AA-18 as a Potential Candidate for Bioremediation of Mercury-Contaminated Wastewater.

Mercury (Hg) pollution is a worldwide problem and increasing day by day due to natural and anthropogenic sources. In this study, mercury-resistant (HgR) bacterial isolates were isolated from industrial wastewater of Ittehad Chemicals Ltd., Kala Shah Kaku, Lahore, Pakistan. Out of 65 bacterial isolates, five isolates were screened out based on showing resistance at 30–40 μg/ml against HgCl2. Selected Hg-resistant bacterial isolates were characterized as Bacillus subtilis AA-16 (OK562835), Bacillus cereus AA-18 (OK562834), Bacillus sp. AA-20 (OK562833), Bacillus paramycoides AA-30 (OK562836), and Bacillus thuringiensis AA-35 (OK562837). B. cereus AA-18 showed promising results in the resistance of HgCl2 (40 μg/ml) due to the presence of merA gene. Scanning electron microscopy (SEM) analysis of immobilized B. cereus AA-18 showed the accumulation Hg on the cell surface. The inoculation of immobilized B. cereus AA-18 remediated 86% Hg of industrial wastewater up to 72 h at large scale (p < 0.05). In silico analysis showed structural determination of MerA protein encoded by merA gene of B. cereus AA-18 (OK562598) using ProtParam, Pfam, ConSurf Server, InterPro, STRING, Jpred4, PSIPRED, I-TASSER, COACH server, TrRosetta, ERRAT, VERIFY3D, Ramachandran plot, and AutoDock Vina (PyRx 8.0). These bioinformatics tools predicted the structural-based functional homology of MerA protein (mercuric reductase) associated with mer operon harboring bacteria involved in Hg-bioremediation system.

The trafficking of HgII by alleviating its toxicity via Citrobacter sp. IITISM25 in batch and pilot-scale investigation

The study aims to see how effective the Citrobacter species strain is in removing HgII under stressful conditions. For this, a response surface methodology was chosen to optimized pH, temperature, and biomass for effective biotransformation of HgII. The optimized value for pH, temperature, and biomass were 6.5, 30 °C, and 2 mg/l with 89% HgII removal potential. TEM-EDX showed accumulated mercury onto the bacterial surface. Pot study was conducted to check the potentiality of this strain in alleviating the toxicity in Solanum lycopersicum L. under different concentrations of mercury. The enhancement in antioxidative enzymes, as well as mercury accumulation, was observed in test plants inoculated with IITISM25. Obtained result showed a greater accumulation of mercury in the root system than that of the shoot system due to poor translocation. Moreover, mercury reductase enzyme synthesis was also boosted by the addition of β-mercaptoethanol and L-cysteine. The optimized condition for maximum enzyme synthesis was at pH 7.5 and temperature 30 °C with Km = 48.07 μmol and Vmax = 9.75 μmol/min. Thus, we can say that Citrobacter species strain IITISM25 can be effectively applied in remediation of HgII stress condition as well as promotion of Solanum lycopersicum L growth under stress conditions as a promising host.

Organomercurial Lyase (MerB)-Mediated Demethylation Decreases Bacterial Methylmercury Resistance in the Absence of Mercuric Reductase (MerA).

The mer operon encodes enzymes that transform and detoxify methylmercury (MeHg) and/or inorganic mercury [Hg(II)]. Organomercurial lyase (MerB) and mercuric reductase (MerA) can act sequentially to demethylate MeHg to Hg(II) and reduce Hg(II) to volatile elemental mercury (Hg0) that can escape from the cell, conferring resistance to MeHg and Hg(II). Most identified mer operons encode either MerA and MerB in tandem or MerA alone; however, microbial genomes were recently identified that encode only MerB. However, the effects of potentially producing intracellular Hg(II) via demethylation of MeHg by MerB, independent of a mechanism to further detoxify or sequester the metal, are not well understood. Here, we investigated MeHg biotransformation in Escherichia coli strains engineered to express MerA and MerB, together or separately, and characterized cell viability and Hg detoxification kinetics when these strains were grown in the presence of MeHg. Strains expressing only MerB are capable of demethylating MeHg to Hg(II). Compared to strains that express both MerA and MerB, strains expressing only MerB exhibit a lower MIC with MeHg exposure, which parallels a redistribution of Hg from the cell-associated fraction to the culture medium, consistent with cell lysis occurring. The data support a model whereby intracellular production of Hg(II), in the absence of reduction or other forms of demobilization, results in a greater cytotoxicity than the parent MeHg compound. Collectively, these results suggest that in the context of MeHg detoxification, MerB must be accompanied by an additional mechanism(s) to reduce, sequester, or redistribute generated Hg(II). IMPORTANCE Mercury is a globally distributed pollutant that poses a risk to wildlife and human health. The toxicity of mercury is influenced largely by microbially mediated biotransformation between its organic (methylmercury) and inorganic [Hg(II) and Hg0] forms. Here, we show in a relevant cellular context that the organomercurial lyase (MerB) enzyme is capable of MeHg demethylation without subsequent mercuric reductase (MerA)-mediated reduction of Hg(II). Demethylation of MeHg without subsequent Hg(II) reduction results in a greater cytotoxicity and increased cell lysis. Microbes carrying MerB alone have recently been identified but have yet to be characterized. Our results demonstrate that mer operons encoding MerB but not MerA put the cell at a disadvantage in the context of MeHg exposure, unless subsequent mechanisms of reduction or Hg(II) sequestration exist. These findings may help uncover the existence of alternative mechanisms of Hg(II) detoxification in addition to revealing the drivers of mer operon evolution.

Identification of mercury‐resistant Streptomyces isolated from Cyperus rotundus L. rhizosphere and molecular cloning of mercury (II) reductase gene

Streptomyces is one of mercury‐resistant bacteria which can convert Hg2+ into nontoxic Hg0 . This study aimed to identify mercury‐resistant Streptomyces present in the Cyperus rotundus rhizosphere from artisanal small‐scale gold mining (ASGM) area and clone merA gene to the cloning and expression vectors. Molecular identification was conducted using 16s rRNA gene with the maximum likelihood algorithms. Results revealed that the AS1 and AS2 strains were a group of Streptomyces ardesiacus and the BR28 strain was closed to Brevibacillus agri. The AS2 merA gene was cloned to pMD20 cloning vectors, pGEX‐5x‐1 and pET‐28c expression vectors. The transformation was successfully performed in BL21 and DH5α competent cells. The full length of the merA gene was confirmed to be 1,425 bp. This study is the first research on identifying mercury‐resistant Streptomyces and cloning the full‐length merA gene in Indonesia.

Biodetoxification mercury by using a marine bacterium Marinomonas sp. RS3 and its merA gene expression under mercury stress

Mercury (Hg) pollution in water has been a problem for the ecosystem and human health, thus eco-friendly remediation methods are gaining traction around the world. In this study, a bacterial strain designated as RS3 isolated from the Red Sea (Saudi Arabia) has shown tolerance to more than 250 mg/L of Hg2+ on minimum inhibitory studies. The isolate RS3 was identified as Marinomonas sp., (Accession No: OK271312) using 16s rRNA sequencing. Tracing the growth curve for the RS3 showed that maximum growth attained at 72 h and only 10% reduction than the control medium for 50 mg/L HgCl2 supplemented seawater medium, which continued to reduce as 21% to 60 with the increment of HgCl2 from 100 to 350 mg/L. The Hg2+ removal potential of RS3 is observed to be 78% at 50 mg/L HgCl2/72 h, which is significantly altered with the addition of carbon source such as glucose (84.5%) > fructose (79.8%) > control (78%) > citrate (73.4%) > acetate (60.2%) > maltose (54.7%). Box-Behnken design (BBD) well proposed a model with R2 value of 0.8922, which predict a utmost Hg2+ removal of 89.5% by RS2 at favorable conditions (pH-7; NaC 1% and glucose 5%) at 72 h. Mercuric reductase enzyme encoded merA gene expression was found to be high in RS3 isolates cultivated in 100 mg/L of HgCl2 in comparison with other variables. Thus the seawater isolate Marinomonas sp. RS3 expressed a significant tolerance and removal potential towards the Hg2+, which would make it is a noteworthy applicant for effective mercury remediation practices.

Demethylation-The Other Side of the Mercury Methylation Coin: A Critical Review.

The public and environmental health consequences of mercury (Hg) methylation have drawn much attention and considerable research to Hg methylation processes and their dynamics in diverse environments and under a multitude of conditions. However, the net methylmercury (MeHg) concentration that accumulates in the environment is equally determined by the rate of MeHg degradation, a complex process mediated by a variety of biotic and abiotic mechanisms, about which our knowledge is limited. Here we review the current knowledge on MeHg degradation and its potential pathways and mechanisms. We describe detoxification by resistant microorganisms that employ the Hg resistance (mer) system to reductively break the carbon-mercury (C-Hg) bond producing methane (CH4) and inorganic mercuric Hg(II), which is then reduced by the mercuric reductase to elemental Hg(0). Very recent research has begun to elucidate a mechanism for the long-recognized mer-independent oxidative demethylation, likely involving some strains of anaerobic bacteria as well as aerobic methane-oxidizing bacteria, i.e., methanotrophs. In addition, photochemical and chemical demethylation processes are described, including the roles of dissolved organic matter (DOM) and free radicals as well as dark abiotic demethylation in the natural environment about which little is currently known. We focus on mechanisms and processes of demethylation and highlight the uncertainties and known effects of environmental factors leading to MeHg degradation. Finally, we suggest future research directions to further elucidate the chemical and biochemical mechanisms of biotic and abiotic demethylation and their significance in controlling net MeHg production in natural ecosystems.