Synthetic oligonucleotide probes for detection of mercury-resistance genes in environmental freshwater microbial communities in response to pollutants

Mercury-resistance genes were detected byin situ hybridization using new synthetic oligonucleotide probes specific formerA andmerB genes according to the published sequences of the corresponding enzymes. These DNA probes were used for the detection of specific mercury-resistant microorganisms isolated from the Rhine River which had been polluted 3 years previously in 1986. Mercuric reductase and organomercurial lyase genes persist in the bacterial genome even after the disappearance of the pollutant but are absent in axenic amoebae. A total of 49 bacterial isolates showed DNA homologies with the³²P-labelled DNA probes and 15 free-living amoebae were selected due to their harboured symbiotic mercury-resistant bacteria.     

Nucleotide sequence and expression of the organomercurial-resistance determinants from a Pseudomonas K-62 plasmid pMR26

pMRA17 cloned from Pseudomonas K-62 plasmid pMR26 specified the resistance to both organic and inorganic mercurials. DNA sequence of this broad-spectrum resistant mer operon was determined. The 5504-bp sequence includes six open reading frames (ORFs), five of which were identified as merR, merT, merP, merA and merB in order by analysis of deletion mutants and by comparison with the DNA and amino acid (aa) sequences of previously sequenced mer operons. The merB encoding organomercurial lyase showed a less identity than the other mer genes with those from other broad-spectrum resistance operons. The remaining ORF named merE, located between merA and merB, had no significant homology with the published mer genes and seemed to be a new gene which may involve in phenylmercury resistance. Induction experiments and maxicell analyses of the mer-polypeptides revealed that pMRA17 mer operon expressed mercurial-inducible phenotype and the merB and merE as well as the merA were under the control of MerR which could activate not only by mercuric ion but also by organomercurials.© 1997 Elsevier Science B.V. All rights reserved.     

Characterization of a mercury-reducing Bacillus cereus strain isolated from the Pulicat Lake sediments, south east coast of India

Pulicat Lake sediments are often severely polluted with the toxic heavy metal mercury. Several mercury-resistant strains of Bacillus species were isolated from the sediments and all the isolates exhibited broad spectrum resistance (resistance to both organic and inorganic mercuric compounds). Plasmid curing assay showed that all the isolated Bacillus strains carry chromosomally borne mercury resistance. Polymerase chain reaction and southern hybridization analyses using merA and merB3 gene primers/probes showed that five of the isolated Bacillus strains carry sequences similar to known merA and merB3 genes. Results of multiple sequence alignment revealed 99% similarity with merA and merB3 of TnMERI1 (class II transposons). Other mercury resistant Bacillus species lacking homology to these genes were not able to volatilize mercuric chloride, indicating the presence of other modes of resistance to mercuric compounds.     

Varied resonses in gene expression of culturable heterotrophic bacteria isolated from the environment

Summary Gene expression in heterotrophic bacteria isolated from environmental samples was studied using a combination of non-selective and selective plating techniques and gene probe methodology. The gene probes and their respective phenotypes were nahAB, for naphthalene degradation, merA, for narrowlspectrum mercury resistance, and merB, for broad-spectrum mercury resistance. Gene-probe-positive organisms could be placed into one of three categories: (1) organisms that could express their genetic information immediately upon isolation from the environment; (2) organisms that expressed their genotype only after cultivation before selection for the genotype; and (3) organisms that did not express their genotype at all in our hands. For all three probes it was found that most organisms fell into category 2. This phenomenon was also observed with bacteria isolated from lake water that probed positive with the nitrogenase (nifHDK) gene probe. The data suggest that the numbers of isolates identified by gene probes merely reflect the genetic potential of a community whereas various expression data suggest that differences in the actual activity of those genotypes exist in the natural environment. Correspondence to: B. H. Olson     

Expression of organomercurial lyase in eastern cottonwood enhances organomercury resistance

Summary Release of inorganic mercury pollutants into shallow aquatic environments has resulted in the bacterial production of a more toxic organic mercury species, methylmercury. The bacterial organomercurial lyase (MerB) catalyses the protonolysis of the carbon-mercury bond and releases Hg(II), a less toxic, non-biomagnified form of mercury. Our objective was to engineer eastern cottonwood (Populus deltoides), a fast-growing tree adapted to growth in riparian environments, with the merB gene to explore its potential for phytoremediation of mercury. We produced multiple eastern cottonwood clones expressing a modified bacterial merB gene, confirmed that the gene was expressed in the transclones and tested the regenerated plants for their ability to tolerate exposure to an organic mercury source, phenylmercuric acetate (PMA), in vitro and in hydroponic culture, compared to wild-type control trees. Transgenic merB plants expressed high levels of MerB protein and showed some evidence of higher resistance to the organic mercury than wild-type plants, producing longer roots under exposure to PMA in vitro, although hydroponic culture results were inconclusive. Our results indicate that in order for merB to be useful in eastern cottonwood trees designed to degrade methylmercury at mercury-contaminated aquatic sites, it will probably need to be combined with other genes such as merA.     

Conjugal Gene Transfer to Aquatic Bacteria Detected by the Generation of a New Phenotype

An experimental approach based on the assembly of genes of a catabolic pathway was used to detect transconjugants in aquatic communities. Resistance to phenylmercury acetate was established in transconjugants when wide-host-range conjugal plasmids containing merB, the gene encoding organomercurial lyase, were transferred to strains from aquatic communities that had been acclimated to inorganic mercury and thus enriched for populations containing merA, the gene encoding mercuric reductase (T. Barkay, Appl. Environ. Microbiol. 53:2725-2732, 1987). Conjugation was confirmed by using the plasmids' encoded antibiotic resistance patterns and by hybridization with a eukaryotic gene. Three merB-conjugal plasmids, belonging to incompatibility groups W (pGTE16), P1 (pGTE26), and N (pGTE25), were prepared. Transfers by filter matings of pGTE16 and pGTE26 from Pseudomonas aeruginosa PA01 to indigenous strains were at efficiencies of 4.5 × 10^-2 and 4.8 × 10^-3 transconjugant per potential recipient, respectively. These efficiencies were from 1 to 2 orders of magnitude below those observed for intraspecies matings with genetically marked recipients. The third plasmid, pGTE25, was not stably maintained in P. aeruginosa donors, and its transfer from Escherichia coli donors was below the level of detection. Characterized transconjugant strains were shown to be Pseudomonas spp. Potential applications of the described experimental approach in the creation of bacterial populations with new catabolic capabilities in hazardous waste sites and in the detection of transfer of recombinant DNA from engineered microorganisms to indigenous bacteria are discussed.     

First Description of Various Bacteria Resistant to Heavy Metals and Antibiotics Isolated from Polluted Sites in Tunisia

Environmental bacteria belonging to various families were isolated from polluted water collected from ten different sites in Tunisia. Sites were chosen near industrial and urban areas known for their high degree of pollution. The aim of this study was to investigate cross-resistance between heavy metals (HM), i.e., silver, mercury and copper (Ag, Hg, and Cu), and antibiotics. In an initial screening, 80 isolates were selected on ampicillin, and 39 isolates, retained for further analysis, could grow on a Tris-buffered mineral medium with gluconate as carbon source. Isolates were identified based on their 16S rRNA gene sequence. Results showed the prevalence of antibiotic resistance genes, especially all isolates harbored the bla_TEM gene. Some of them (15.38%) harbored bla_SHV. Moreover, several were even ESBLs and MBLs-producers, which can threaten the human health. On the other hand, 92.30%, 56.41%, and 51.28% of the isolates harbored the heavy metals resistance genes silE, cusA, and merA, respectively. These genes confer resistance to silver, copper, and mercury. A cross-resistance between antibiotics and heavy metals was detected in 97.43% of our isolates.     

Staphylococcus aureus - induced tumor necrosis factor - related apoptosis - inducing ligand expression mediates apoptosis and caspase-8 activation in infected osteoblasts

Background

Bacterial mercury resistance is based on enzymatic reduction of ionic mercury to elemental mercury and has recently been demonstrated to be applicable for industrial wastewater clean-up. The long-term monitoring of such biocatalyser systems requires a cultivation independent functional community profiling method targeting the key enzyme of the process, themerAgene coding for the mercuric reductase. We report on the development of a profiling method formerAand its application to monitor changes in the functional diversity of the biofilm community of a technical scale biocatalyzer over 8 months of on-site operation.

Results

Based on an alignment of 30merAsequences from Gram negative bacteria, conserved primers were designed for amplification ofmerAfragments with an optimized PCR protocol. The resulting amplicons of approximately 280 bp were separated by thermogradient gelelectrophoresis (TGGE), resulting in strain specific fingerprints for mercury resistant Gram negative isolates with differentmerAsequences. ThemerAprofiling of the biofilm community from a technical biocatalyzer showed persistence of some and loss of other inoculum strains as well as the appearance of new bands, resulting in an overall increase of the functional diversity of the biofilm community. One predominant new band of themerAcommunity profile was also detected in a biocatalyzer effluent isolate, which was identified asPseudomonas aeruginosa. The isolated strain showed lower mercury reduction rates in liquid culture than the inoculum strains but was apparently highly competitive in the biofilm environment of the biocatalyzer where moderate mercury levels were prevailing.

Conclusions

ThemerAprofiling technique allowed to monitor the ongoing selection for better adapted strains during the operation of a biocatalyzer and to direct their subsequent isolation. In such a way, a predominant mercury reducingPs. aeruginosastrain was identified by its unique mercuric reductase gene.     

Identification by MALDI-TOF Mass Spectrometry of Mercury-resistant Bacteria Associated with the Rhizosphere of an Apple Orchard

In this work, mercury-resistant bacterial strains were isolated from the rhizosphere of an apple orchard, growing in a soil with high levels of mercury (Nuevo San Joaquin, Queretaro State, Mexico). Analysis of the soil in this region by the Cold Vapor Atomic Absortion Spectroscopy method showed that it contained 637 ± 51 mg mercury per kg. Mercury accumulation by fresh apples from this orchard amounted to 15.44 ± 4.33 mg/kg. The bacterial isolates were identified by application of proteomic technique of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). They were found to be strains of Bacillus muralis and Bacillus simplex. All strains showed the ability to catalyze the volatilization of Hg as measured via the nonradioactive X-ray method. In all strains merR and merA genes were detected by polymerase chain reaction. Nucleotide sequence analysis showed that merR from B. simplex was 435 bp in length and that its sequence was similar to merR sequences reported for other bacteria such as Cupriavidus, Ralstonia, Pseudomonas and Serratia. To our knowledge, this is the first report of mercury-resistant Bacillus strains isolated from the rhizosphere of an apple orchard and the first merR gene sequence from such Bacilli.     

Molecular Cloning and Genetic Analysis of Functional merB Gene from Indian Isolates of Escherichia coli

Studies were carried out to characterize organomercurial lyase genes from wild type mercury-resistant Escherichia coli isolates, previously collected from five geographically distinct regions of the Indian subcontinent. PCR amplification followed by DNA sequencing of amplified fragments showed three merB identical to the previously characterized mer B from E. coli pR831b that were thus considered as the same gene. The remaining two genes derived from E. coli isolates of an almost mercury-free site (Dal lake, Kashmir) and designated as pIAAD₃ merB and pIAAD₁₄ merB showed slight variation (2%) at base. However, this variation in pIAAD₃ due to the absence of base “T” at 479 position results in complete frame shift and the predicted MerB-like polypeptide derived from it showed 21.53% divergent at its C terminal end from the previously characterized pR831b MerB. The expression profile of pIAAD₃ merB in pQE30 and pUC18 vectors each demonstrated 22.2 kDa proteins. The induced DH5α E. coli cells possessing pIAAD₃ merB cloned in pUC18 vector split phenyl mercuric acetate (PMA) into benzene and inorganic mercury efficiently, thus giving a clue that the expressed gene product is biologically active. The current study suggests that such genetic changes may take place in the continued absence of mercury pressure, and with such modifications, they finally break down to act as vestigial remnants. Further work is going on in our lab to exploit pIAAD₃ merB for the bioremediation of mercury-polluted sites.     

Bacterial <Emphasis Type="Italic">mer</Emphasis> operon-mediated detoxification of mercurial compounds: a short review

Mercury pollution has emerged as a major problem in industrialized zones and presents a serious threat to environment and health of local communities. Effectiveness and wide distribution of mer operon by horizontal and vertical gene transfer in its various forms among large community of microbe reflect importance and compatibility of this mechanism in nature. This review specifically describes mer operon and its generic molecular mechanism with reference to the central role played by merA gene and its related gene products. The combinatorial action of merA and merB together maintains broad spectrum mercury detoxification system for substantial detoxification of mercurial compounds. Feasibility of mer operon to coexist with antibiotic resistance gene (amp ^r , kan ^r , tet ^r ) clusters enables extensive adaptation of bacterial species to adverse environment. Flexibility of the mer genes to exist as intricate part of chromosome, plasmids, transposons, and integrons enables high distribution of these genes in wider microbial gene pool. Unique ability of this system to manipulate oligodynamic property of mercurial compounds for volatilization of mercuric ions (Hg²⁺) makes it possible for a wide range of microbes to tolerate mercury-mediated toxicity.     

Community Analysis of a Mercury Hot Spring Supports Occurrence of Domain-Specific Forms of Mercuric Reductase

Mercury is a redox-active heavy metal that reacts with active thiols and depletes cellular antioxidants. Active resistance to the mercuric ion is a widely distributed trait among bacteria and results from the action of mercuric reductase (MerA). Protein phylogenetic analysis of MerA in bacteria indicated the occurrence of a second distinctive form of MerA among the archaea, which lacked an N-terminal metal recruitment domain and a C-terminal active tyrosine. To assess the distribution of the forms of MerA in an interacting community comprising members of both prokaryotic domains, studies were conducted at a naturally occurring mercury-rich geothermal environment. Geochemical analyses of Coso Hot Springs indicated that mercury ore (cinnabar) was present at concentrations of parts per thousand. Under high-temperature and acid conditions, cinnabar may be oxidized to the toxic form Hg²⁺, necessitating mercury resistance in resident prokaryotes. Culture-independent analysis combined with culture-based methods indicated the presence of thermophilic crenarchaeal and gram-positive bacterial taxa. Fluorescence in situ hybridization analysis provided quantitative data for community composition. DNA sequence analysis of archaeal and bacterial merA sequences derived from cultured pool isolates and from community DNA supported the hypothesis that both forms of MerA were present. Competition experiments were performed to assess the role of archaeal merA in biological fitness. An essential role for this protein was evident during growth in a mercury-contaminated environment. Despite environmental selection for mercury resistance and the proximity of community members, MerA retains the two distinct prokaryotic forms and avoids genetic homogenization.     

The nucleotide sequence of the mercuric resistance operons of plasmid R100 and transposon Tn501: further evidence for mer genes which enhance the activity of the mercuric ion detoxification system

Summary The DNA sequences of the mercuric resistance determinants of plasmid R100 and transposon Tn501 distal to the gene (merA) coding for mercuric reductase have been determined. These 1.4 kilobase (kb) regions show 79% identity in their nucleotide sequence and in both sequences two common potential coding sequences have been identified. In R100, the end of the homologous sequence is disrupted by an 11.2 kb segment of DNA which encodes the sulfonamide and streptomycin resistance determinants of Tn21. This insert contains terminal inverted repeat sequences and is flanked by a 5 base pair (bp) direct repeat. The first of the common potential coding sequences is likely to be that of the merD gene. Induction experiments and mercury volatilization studies demonstrate an enhancing but non-essential role for these merA-distal coding sequences in mercury resistance and volatilization. The potential coding sequences have predicted codon usages similar to those found in other Tn501 and R100 mer genes.     

Environmental Conditions Constrain the Distribution and Diversity of Archaeal <Emphasis Type="Italic">merA</Emphasis> in Yellowstone National Park,Wyoming, U.S.A.

The distribution and phylogeny of extant protein-encoding genes recovered from geochemically diverse environments can provide insight into the physical and chemical parameters that led to the origin and which constrained the evolution of a functional process. Mercuric reductase (MerA) plays an integral role in mercury (Hg) biogeochemistry by catalyzing the transformation of Hg(II) to Hg(0). Putative merA sequences were amplified from DNA extracts of microbial communities associated with mats and sulfur precipitates from physicochemically diverse Hg-containing springs in Yellowstone National Park, Wyoming, using four PCR primer sets that were designed to capture the known diversity of merA. The recovery of novel and deeply rooted MerA lineages from these habitats supports previous evidence that indicates merA originated in a thermophilic environment. Generalized linear models indicate that the distribution of putative archaeal merA lineages was constrained by a combination of pH, dissolved organic carbon, dissolved total mercury and sulfide. The models failed to identify statistically well supported trends for the distribution of putative bacterial merA lineages as a function of these or other measured environmental variables, suggesting that these lineages were either influenced by environmental parameters not considered in the present study, or the bacterial primer sets were designed to target too broad of a class of genes which may have responded differently to environmental stimuli. The widespread occurrence of merA in the geothermal environments implies a prominent role for Hg detoxification in these environments. Moreover, the differences in the distribution of the merA genes amplified with the four merA primer sets suggests that the organisms putatively engaged in this activity have evolved to occupy different ecological niches within the geothermal gradient.     

The merG Gene Product Is Involved in Phenylmercury Resistance in Pseudomonas Strain K-62

The physiological function of a new gene, hereby designated merG, located between merA and merB on the broad-spectrum mer operon of Pseudomonas strain K-62 plasmid pMR26 was investigated. The 654-bp merG gene encodes a protein with a canonical leader sequence at its N terminus. The processing of the signal peptide of this protein was dose-dependently inhibited by sodium azide, a potent inhibitor of protein export. These results suggest that the mature MerG protein (ca. 20 kDa) may be located in the periplasm. Deletion of the merG gene from the broad-spectrum mer operon of pMR26 had no effect on the inorganic mercury resistance phenotype, but rendered the bacterium more sensitive to phenylmercury than its isogenic wild-type strain. Escherichia coli cells bearing pMU29, which carries a deletion of the merG gene, took up significantly more phenylmercury than the bacteria with the intact plasmid pMRA17. When the merG gene in a compatible plasmid was transformed into the E. coli strain carrying pMU29, the high uptake of and high sensitivity to phenylmercury were almost completely restored to their original levels. These results demonstrate that the merG gene is involved in phenylmercury resistance, presumably by reducing in-cell permeability to phenylmercury.     

DNA Probe-Mediated Detection of Resistant Bacteria from Soils Highly Polluted by Heavy Metals

Alcaligenes eutrophus CH34 DNA fragments encoding resistance to Cd²⁺, Co²⁺, Zn²⁺ (czc), or Hg²⁺ (merA) were cloned and used as probes in colony hybridization procedures with bacteria isolated from polluted environments such as a zinc factory area (desertified because of the toxic effects of zinc contamination) and from sediments from factories of nonferrous metallurgy in Belgium and mine areas in Zaire. From the different soil samples, strains could be isolated and hybridized with the czc probe (resistance to Cd²⁺, Co²⁺, and Zn²⁺ from plasmid pMOL30). Percentages of CFU isolated on nonselective plates which hybridized with the czc and the mercury resistance probes were, respectively, 25 and 0% for the zinc desert, 15 to 20 and 10 to 20% for the two Belgian factories, and 40 and 40% for the Likasi mine area. Most of these strains also carried two large plasmids of about the same size as those of A. eutrophus CH34 and shared many phenotypic traits with this strain. These findings indicated a certain correlation between the heavy-metal content in contaminated soils and the presence of heavy-metal-resistant megaplasmid-bearing A. eutrophus strains.     

Characterization of the metabolically modified heavy metal-resistant Cupriavidus metallidurans strain MSR33 generated for mercury bioremediation

Background

Mercury-polluted environments are often contaminated with other heavy metals. Therefore, bacteria with resistance to several heavy metals may be useful for bioremediation. Cupriavidus metallidurans CH34 is a model heavy metal-resistant bacterium, but possesses a low resistance to mercury compounds.

Methodology/Principal Findings

To improve inorganic and organic mercury resistance of strain CH34, the IncP-1β plasmid pTP6 that provides novel merB, merG genes and additional other mer genes was introduced into the bacterium by biparental mating. The transconjugant Cupriavidus metallidurans strain MSR33 was genetically and biochemically characterized. Strain MSR33 maintained stably the plasmid pTP6 over 70 generations under non-selective conditions. The organomercurial lyase protein MerB and the mercuric reductase MerA of strain MSR33 were synthesized in presence of Hg²⁺. The minimum inhibitory concentrations (mM) for strain MSR33 were: Hg²⁺, 0.12 and CH₃Hg⁺, 0.08. The addition of Hg²⁺ (0.04 mM) at exponential phase had not an effect on the growth rate of strain MSR33. In contrast, after Hg²⁺ addition at exponential phase the parental strain CH34 showed an immediate cessation of cell growth. During exposure to Hg²⁺ no effects in the morphology of MSR33 cells were observed, whereas CH34 cells exposed to Hg²⁺ showed a fuzzy outer membrane. Bioremediation with strain MSR33 of two mercury-contaminated aqueous solutions was evaluated. Hg²⁺ (0.10 and 0.15 mM) was completely volatilized by strain MSR33 from the polluted waters in presence of thioglycolate (5 mM) after 2 h.

Conclusions/Significance

A broad-spectrum mercury-resistant strain MSR33 was generated by incorporation of plasmid pTP6 that was directly isolated from the environment into C. metallidurans CH34. Strain MSR33 is capable to remove mercury from polluted waters. This is the first study to use an IncP-1β plasmid directly isolated from the environment, to generate a novel and stable bacterial strain useful for mercury bioremediation.     

Cadmium- and mercury-resistant Bacillus strains from a salt marsh and from Boston Harbor

Bacteria resistant to cadmium or mercury or both were isolated from the Great Sippewissett Marsh (Cape Cod, Mass.) and from Boston Harbor. Many of these metal-resistant isolates were gram-positive aerobic sporeformers, although not necessarily isolated as spores. Although several of the isolated strains bore plasmids, cadmium and mercury resistances appeared to be, for the most part, chromosomally encoded. DNA sequence homology of the gram-positive cadmium- and mercury-resistant isolates was not demonstrable with metal resistance genes from plasmids of either gram-positive (pI258) or gram-negative (pDB7) origin. Cadmium resistance of all the marsh isolates tested resulted from reduced Cd2+ transport. On the other hand, three cadmium-resistant harbor isolates displayed considerable influx but no efflux of Cd2+. Hg-resistant strains detoxified mercury by transforming Hg2+ to volatile Hg0 via mercuric reductase.