一株高抗汞细菌的分离鉴定及其抗性基因的克隆与表达

摘要：【目的】本研究旨在从重金属汞抗性细菌中分离鉴定汞抗性基因【方法】从北京凉水河河床底泥中分离抗汞细菌,采用16S rRNA基因序列分析结合生理生化特征对菌株进行鉴定。根据GenBank中已发表的多种抗汞细菌的merA基因序列设计引物,以抗性细菌基因组DNA为模板,扩增merA基因,并在大肠杆菌（Escherichia coli）BL21(DE3)表达。同时对表达菌株的重金属汞抗性进行测定。【结果】分离得到一株能在含HgCl2为70 mg/L的平板上生长良好的高抗汞细菌,编号为KHg2。16S rRNA     

Diversity of mercury resistance plasmids obtained by exogenous isolation from the bacteria of sugar beet in three successive years

Abstract: Self-transmissible plasmids conferring mercury resistance were exogenously isolated from the bacterial populations of sugar beet roots (rhizoplane) and leaves (phyllosphere) into a Pseudomonas putida recipient. Fifty rhizoplane plasmids and 29 phyllosphere plasmids (60–383 kb) were purified. Numerical analysis of plasmid DNA restriction enzyme digest patterns identified five distinct groups. Three of these plasmid groups were isolated from sugar beet crops grown at the same site over three consecutive years, demonstrating their established presence. Each group of plasmids comprised individual isolates with structural additions or deletions. The frequency of exogenous isolation correlated with factors likely to influence plant growth, bacterial activity and the physiological state of donors prior to sampling. All plasmids investigated conferred narrow spectrum mercury resistance with a reductase detoxification mechanism. None of the plasmids conferred resistance to a range of antibiotics, other heavy metals, or to UV, and following transfer to recipient bacteria the range of carbon source utilisation was not altered. This is the first report of the persistence of Pseudomonas spp. plasmid structural types isolated over several years from a terrestrial habitat.     

Mercuric reductase in environmental Gram-positive bacteria sensitive to mercury

According to existing data, mercury resistance operons (mer operons) are in general thought to be rare in bacteria, other than those from mercury-contaminated sites. We have found that a high proportion of strains in environmental isolates of Gram-positive bacteria express mercuric reductase (MerA protein): the majority of these strains are apparently sensitive to mercury. The expression of MerA was also inducible in all cases. These results imply the presence of phenotypically cryptic mer resistance operons, with both the merA (mercuric reductase) and merR (regulatory) genes still present, but the possible absence of the transport function required to complete the resistance mechanism. This indicates that mer operons or parts thereof are more widely spread in nature than is suggested by the frequency of mercury-resistant bacteria.     

Removal of mercury from mercury-contaminated sediments using a combined method of chemical leaching and volatilization of mercury by bacteria

A method for the removal of mercury sulfide frommercury-contaminated sediments was developed, whichconsists of chemical leaching and volatilization ofmercury by bacteria. More than 85% of the mercury insediment containing 0.11–37.4 mg/kg of mercury wasefficiently extracted with 3 M HCl and 74 mMFeCl₃. Subsequent volatilization by bacteriaresulted in the removal of 62.9–75.1% of mercury frommercury-contaminated Minamata Bay sediments. Methylmercury was also eliminated from soil at a highefficiency. Thus, this combined method of chemicaland microbial treatments could be used for efficientremoval of both organic and inorganic mercurials fromnatural sediments.     

The isolation and initial characterization of mercury resistant chemolithotrophic thermophilic bacteria from mercury rich geothermal springs

Mercury rich geothermal springs are likely environments where mercury resistance is critical to microbial life and where microbe-mercury interactions may have evolved. Eleven facultative thermophilic and chemolithoautotrophic, thiosulfate oxidizing bacteria were isolated from thiosulfate enrichments of biofilms from mercury rich hot sulfidic springs in Mount Amiata, Italy. Some strains were highly resistant to mercury (≥200 μM HgCl₂) regardless of its presence or absence during primary enrichments, and three reduced ionic mercury to its elemental form. The gene encoding for the mercuric reductase enzyme (MerA), was amplified by PCR from seven strains. However, one highly resistant strain did not reduce mercury nor carried merA, suggesting an alternative resistance mechanism. All strains were members of the order Bacillales and were most closely related to previously described thermophiles belonging to the Firmicutes. Phylogenetic analyses clustered the MerA of the isolates in two supported novel nodes within the Firmicutes lineage and a comparison with the 16S rRNA gene tree suggested at least one case of horizontal gene transfer. Overall, the results show that the thermophilic thiosulfate oxidizing isolates were adapted to life in presence of mercury mostly, but not exclusively, by possessing MerA. These findings suggest that reduction of mercury by chemolithotrophic thermophilic bacteria may mobilize mercury from sulfur and iron deposits in geothermal environments.     

Plasmid-borne mercury resistance in aquatic bacteria

Abstract Bacteria isolated from the River Mersey were analysed for their tolerance to mercury (HgCl₂). About 40% of the population was tolerant to mercury and in 13 of 52 mercury-tolerant isolates tested the mercury resistance (Hg^®) was transferred to Escherichia coli in conjugal matings. These 13 isolates represented a range of gram-negative genera and in each case mercury resistance was coded by a conjugative plasmid. These plasmids (75 kb to > 250 kb in size) all expressed mercury resistance of the narrow spectrum variety, volatilised HgCl₂ to elemental Hg° vapour and showed some degree of temperature sensitivity of transfer. None expressed resistance to nine different antibiotics. These 13 Hg^R plasmids were classified by restriction mapping into three distinct groups typified by pMER11, pMER327 and pMER610. The eight pMER610 group plasmids are identical and belong to the IncHI-2 group. Two of the four pMER327 group plasmids are closely related while the other two contain some common restriction fragments. pMER11 is quite distinct from the other groups. These results imply that within this aquatic environment plasmids play an important role in the response of bacteria to contaminating mercury and that there is widespread plasmid transfer and considerable genetic rearrangement.     

Plasmid-encoded mercury resistance in a Pseudomonas stutzeri strain that degrades o-xylene

Abstract A Pseudomonas stutzeri strain, previously isolated for its ability to utilize o -xylene, bears a plasmid, pPB, of about 80 kbp. pPB was found to encode resistance to mercuric chloride and organomercury compounds. Loss of the plasmid resulted in a simultaneous loss of the metal resistance, but not of the ability to degrade o -xylene. Transfer of the Hg^r phenotype to an Hg^s strain was achieved by mobilizing pPB with RP4. Mercury reductase activity was induced by mercuric chloride and by phenylmercuric acetate and Thimerosal. pPB may be considered a broad spectrum resistance plasmid.     

Evaluation of mercury resistance and accumulation characteristics in wheat using a modified membership function

Mercury pollution in fields has become a potential threat to human health. Planting wheat cultivars with low mercury accumulation in slight or medium mercury-polluted fields is an efficient solution to ensure food safety. Therefore, this study evaluated the mercury resistance and accumulation characteristics of 30 generalized wheat cultivars in major wheat-producing areas of China. A modified membership function that considers the weight of each trait was used.Results demonstrated that the plant height of wheat significantly increased under both low mercury and high mercury stresses. The uppermost internode length significantly increased while the spikelet number significantly decreased under low mercury stress. Yield-related traits, including total grain number, fresh grain yield, and dry grain yield, significantly decreased under high mercury stress. The mercury concentrations in wheat grains presented a significant negative correlation with the mercury resistance coefficients of plant height (−0.38*), spike length (−0.39*), and fresh grain yield (−0.38*) under high mercury stress. The heritability of all traits reached medium to high levels, ranging from 0.31 to 0.68. This finding suggested that the investigated traits are stable and suitable for the assessment system. Selection criteria for wheat mercury resistance were established using discriminant analysis, which integrated the mercury resistance coefficients of effective tiller number, fresh grain yield, and dry biomass into the discriminant function under low mercury stress and the mercury resistance coefficients of dry grain yield and dry biomass under high mercury stress. Ultimately, Liangxing-99, Nongda-3163, and Gaocheng-8901 were screened for high mercury resistance and low mercury accumulation. These wheat cultivars could be planted in fields with low or medium mercury pollution to obtain safe grains.     

Biosorption of lead, cadmium and mercury by immobilized Microcystis aeruginosa in a column

Immobilized Microcystis aeruginosa in a flow-through sorption column was evaluated for the potential to remove Pb²⁺, Cd²⁺ and Hg²⁺ from aqueous solutions. M. aeruginosa showed high affinity for the three heavy metals with removal efficiency of 90% for Cd²⁺ and Hg²⁺, and 80% for Pb²⁺ at saturation conditions. Competitive sorption experiments conducted in paired and ternary systems indicated that Pb²⁺ was sequestered preferentially over Cd²⁺ and Hg²⁺. The presence of Cd²⁺ interfered only slightly with the uptake of Hg²⁺, as Pb²⁺ and Hg²⁺ did with Cd²⁺. In contrast, Hg²⁺ sorption was affected by Pb²⁺ to a great extent. Desorption with 1 M HCl was completed within 25 min with high efficiency and effectiveness for the three metals. The results of this study indicate that M. aeruginosa is to be a potential biosorbent material except when Hg²⁺ and Pb²⁺ are in the same contaminated solution.     

Lignin biomarkers as tracers of mercury sources in lakes water column

This study presents the role of specific terrigenous organic compounds as important vectors of mercury (Hg) transported from watersheds to lakes of the Canadian boreal forest. In order to differentiate the autochthonous from the allochthonous organic matter (OM), lignin derived biomarker signatures [Lambda, S/V, C/V, P/(V + S), 3,5-Bd/V and (Ad/Al)v] were used. Since lignin is exclusively produced by terrigenous plants, this approach can give a non equivocal picture of the watershed inputs to the lakes. Moreover, it allows a characterization of the source of OM and its state of degradation. The water column of six lakes from the Canadian Shield was sampled monthly between June and September 2005. Lake total dissolved Hg concentrations and Lambda were positively correlated, meaning that Hg and ligneous inputs are linked (dissolved OM r ² = 0.62, p < 0.0001; particulate OM r ² = 0.76, p < 0.0001). Ratios of P/(V + S) and 3,5-Bd/V from both dissolved OM and particulate OM of the water column suggest an inverse relationship between the progressive state of pedogenesis and maturation of the OM in soil before entering the lake, and the Hg concentrations in the water column. No relation was found between Hg levels in the lakes and the watershed flora composition—angiosperm versus gymnosperm or woody versus non-woody compounds. This study has significant implications for watershed management of ecosystems since limiting fresh terrestrial OM inputs should reduce Hg inputs to the aquatic systems. This is particularly the case for large-scale land-use impacts, such as deforestation, agriculture and urbanization, associated to large quantities of soil OM being transferred to aquatic systems.     

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.     

Diversity of purple nonsulfur bacteria in shrimp ponds with varying mercury levels

This research aimed to study the diversity of purple nonsulfur bacteria (PNSB) and to investigate the effect of Hg concentrations in shrimp ponds on PNSB diversity. Amplification of the pufM gene was detected in 13 and 10 samples of water and sediment collected from 16 shrimp ponds in Southern Thailand. In addition to PNSB, other anoxygenic phototrophic bacteria (APB) were also observed; purple sulfur bacteria (PSB) and aerobic anoxygenic phototrophic bacteria (AAPB) although most of them could not be identified. Among identified groups; AAPB, PSB and PNSB in the samples of water and sediment were 25.71, 11.43 and 8.57%; and 27.78, 11.11 and 22.22%, respectively. In both sample types, Roseobacter denitrificans (AAPB) was the most dominant species followed by Halorhodospira halophila (PSB). In addition two genera, observed most frequently in the sediment samples were a group of PNSB (Rhodovulum kholense, Rhodospirillum centenum and Rhodobium marinum). The UPGMA dendrograms showed 7 and 6 clustered groups in the water and sediment samples, respectively. There was no relationship between the clustered groups and the total Hg (Hg_T) concentrations in the water and sediment samples used (<0.002–0.03 μg/L and 35.40–391.60 μg/kg dry weight) for studying the biodiversity. It can be concluded that there was no effect of the various Hg levels on the diversity of detected APB species; particularly the PNSB in the shrimp ponds.     

Diversity of mercury resistance determinants among Bacillus strains isolated from sediment of Minamata Bay

Thirty mercury-resistant (Hg R) Bacillus strains were isolated from mercury-polluted sediment of Minamata Bay, Japan. Mercury resistance phenotypes were classified into broad-spectrum (resistant to inorganic Hg(2+) and organomercurials) and narrow-spectrum (resistant to inorganic Hg(2+) and sensitive to organomercurials) groups. Polymerase chain reaction (PCR) product sizes and the restriction nuclease site maps of mer operon regions from all broad-spectrum Hg R Bacillus were identical to that of Bacillus megaterium MB1. On the other hand, the PCR products of the targeted merP (extracellular mercury-binding protein gene) and merA (intracellular mercury reductase protein gene) regions from the narrow-spectrum Hg R Bacillus were generally smaller than those of the B. megaterium MB1 mer determinant. Diversity of gene structure configurations was also observed by restriction fragment length polymorphism (RFLP) profiles of the merA PCR products from the narrow-spectrum Hg R Bacillus. The genetic diversity of narrow-spectrum mer operons was greater than that of broad-spectrum ones.     

Comparison of mercury sulfides with mercury chloride and methylmercury on hepatic P450, phase-2 and transporter gene expression in mice

Zuotai (mainly β-HgS) and Zhusha (also called as cinnabar, mainly α-HgS) are used in traditional medicines in combination with herbs or even drugs in the treatment of various disorders, while mercury chloride (HgCl₂) and methylmercury (MeHg) do not have known medical values but are highly toxic. This study aimed to compare the effects of mercury sulfides with HgCl₂ and MeHg on hepatic drug processing gene expression. Mice were orally administrated with Zuotai (β-HgS, 30 mg/kg), α-HgS (HgS, 30 mg/kg), HgCl₂ (33.6 mg/kg), or MeHg (3.1 mg/kg) for 7 days, and the expression of genes related to phase-1 drug metabolism (P450), phase-2 conjugation, and phase-3 (transporters) genes were examined. The mercurials at the dose and duration used in the study did not have significant effects on the expression of cytochrome P450 1–4 family genes and the corresponding nuclear receptors, except for a slight increase in PPARα and Cyp4a10 by HgCl₂. The expressions of UDP-glucuronosyltransferase and sulfotransferase were increased by HgCl₂ and MeHg, but not by Zuotai and HgS. HgCl₂ decreased the expression of organic anion transporter (Oatp1a1), but increased Oatp1a4. Both HgCl₂ and MeHg increased the expression of multidrug resistance-associated protein genes (Mrp1, Mrp2, Mrp3, and Mrp4). Zuotai and HgS had little effects on these transporter genes. In conclusion, Zuotai and HgS are different from HgCl₂ and MeHg in hepatic drug processing gene expression; suggesting that chemical forms of mercury not only affect their disposition and toxicity, but also affect their effects on the expression of hepatic drug processing genes.     

Rhizosphere bacteria enhance the accumulation of selenium and mercury in wetland plants

The role of rhizosphere bacteria in facilitating Se and Hg accumulation in two wetland plants, saltmarsh bulrush (Scirpus robustus Pursh) and rabbitfoot grass (Polypogon monspeliensis (L.) Desf.), was studied. Ampicillin-amended plants (i.e., with inhibited rhizosphere bacteria) supplied with Na₂SeO₄ or HgCl₂ had significantly lower concentrations of Se and Hg, respectively, in roots than plants without ampicillin. These results were confirmed by inoculating axenic saltmarsh bulrush plants with bacteria isolated from the rhizosphere of plants collected from the field; these plants accumulated significantly more Se and Hg compared to axenic controls. Therefore, rhizosphere bacteria can increase the efficiency of Se and Hg phytoremediation by promoting the accumulation of Se and Hg in tissues of wetland plants. Received: 9 April 1999 / Accepted: 11 May 1999     

Review of characteristics of mercury speciation and mobility from areas of mercury mining in semi-arid environments

The speciation of mercury—including most phase minerals, secondary phases, gaseous and aqueous species—is very important for evaluating the environmental impact and mobilization of this contaminant. Mining activities produce mercury mine waste, which includes several types of material (mainly mine waste and calcines) with varying mercury content and speciation depending on the ore deposit and processing technology. The main phase minerals are cinnabar, metacinnabar, metallic Hg⁰, corderoite, livingstonite, calomel and schuetteite. The aqueous mobilization of mercury is controlled by complex formation, pH-Eh conditions, the primary phase mineral of mercury, and organic-matter and iron oxyhydroxide content. The possibility of colloidal transport of mercury from mine waste is influenced by the atmospheric emission of metallic Hg⁰ and the leaching of waste by episodic high-intensity precipitations. In these climatic conditions, mercury can be mobilized to pore water, surface water or groundwater by the dissolution of metallic Hg⁰ and cinnabar in acidic conditions, and by the colloidal transport. The presence of Hg-soluble phases (chlorides and oxychlorides) may enhance the mobilization of mercury. In the semi-arid conditions of the Valle del Azogue (SE Spain) the atmospheric emissions of the Hg⁰ present in calcines and mine waste may be significant and the dissolution of Hg⁰ and metal-sulfate salts during episodic runoff events may explain the mobilization of Hg, Fe, Pb, Zn and other heavy metals.