Mercury-resistant Bacterial Strains Research Articles

Gut heavy metal and antibiotic resistome of humans living in the high Arctic.

Contaminants, such as heavy metals (HMs), accumulate in the Arctic environment and the food web. The diet of the Indigenous Peoples of North Greenland includes locally sourced foods that are central to their nutritional, cultural, and societal health but these foods also contain high concentrations of heavy metals. While bacteria play an essential role in the metabolism of xenobiotics, there are limited studies on the impact of heavy metals on the human gut microbiome, and it is so far unknown if and how Arctic environmental contaminants impact the gut microbes of humans living in and off the Arctic environment. Using a multiomics approach including amplicon, metagenome, and metatranscriptome sequencing, we identified and assembled a near-complete (NC) genome of a mercury-resistant bacterial strain from the human gut microbiome, which expressed genes known to reduce mercury toxicity. At the overall ecological level studied through α- and β-diversity, there was no significant effect of heavy metals on the gut microbiota. Through the assembly of a high number of NC metagenome-assembled genomes (MAGs) of human gut microbes, we observed an almost complete overlap between heavy metal-resistant strains and antibiotic-resistant strains in which resistance genes were all located on the same genetic elements.

The chemical species of mercury accumulated by Pseudomonas idrijaensis, a bacterium from a rock of the Idrija mercury mine, Slovenia.

A mercury-resistant bacterial strain has been isolated from a rock of the Idrija mercury mine in Slovenia. The rock had 19 g carbon and 2952 mg mercury (Hg) per kg. Mass spectrometry and DNA sequencing showed that the bacterium belongs to the Pseudomonas genus. It is called Pseudomonas idrijaensis. This bacterial strain is sensitive to methylmercury (MeHg) like the reference P. aeruginosa strain PAO1, and is resistant to divalent mercury (Hg(II)) in contrast to PAO1. This difference could be attributed to the presence of the mer operon yet deprived of the merB gene encoding the organomercurial lyase, on the basis of whole genome sequencing. The P. idrijaensis mer operon displays the RTPCADE organization and is contained in the Tn5041 transposon. This transposon identified here occurs in other Gram-negative Hg-resistant strains isolated from mercury ores, aquatic systems and soils, including Pseudomonas strains from 15,000 to 40,000 years old Siberian permafrost. When P. idrijaensis was exposed to mercury chloride, two intracellular Hg species were identified by high energy-resolution XANES spectroscopy, a dithiolate Hg(SR)2 and a tetrathiolate Hg(SR)4 complex. P. idrijaensis had a much higher [Hg(SR)2]/[Hg(SR)4] molar ratio than bacteria lacking the mer operon when exposed to 4 μg Hg2+/L - resulting in an intracellular accumulation of 4.3 μg Hg/g dw. A higher amount of the Hg(SR)2 complex provides a chemical signature for the expression of the dicysteinate Mer proteins in response to mercury toxicity.

Multiple Lines of Evidences Reveal Mechanisms Underpinning Mercury Resistance and Volatilization by Stenotrophomonas sp. MA5 Isolated from the Savannah River Site (SRS), USA.

A largely understudied microbially mediated mercury (Hg) bioremediative pathway includes the volatilization of Hg2+ to Hg0. Therefore, studies on Hg resistant bacteria (HgR), isolated from historically long-term contaminated environments, can serve as models to understand mechanisms underpinning Hg cycling. Towards this end, a mercury resistant bacterial strain, identified as Stenotrophomonas sp., strain MA5, was isolated from Mill Branch on the Savannah River Site (SRS); an Hg-impacted ecosystem. Minimum inhibitory concentration (MIC) analysis showed Hg resistance of up to 20 µg/mL by MA5 with 95% of cells retaining viability. Microcosm studies showed that the strain depleted more than 90% of spiked Hg2+ within the first 24 h of growth and the detection of volatilized mercury indicated that the strain was able to reduce Hg2+ to Hg0. To understand molecular mechanisms of Hg volatilization, a draft whole genome sequence was obtained, annotated and analyzed, which revealed the presence of a transposon-derived mer operon (merRTPADE) in MA5, known to transport and reduce Hg2+ into Hg0. Based on the whole genome sequence of strain MA5, qRT-PCR assays were designed on merRTPADE, we found a ~40-fold higher transcription of mer T, P, A, D and E when cells were exposed to 5 µg/mL Hg2+. Interestingly, strain MA5 increased cellular size as a function of increasing Hg concentrations, which is likely an evolutionary response mechanism to cope with Hg stress. Moreover, metal contaminated environments are shown to co-select for antibiotic resistance. When MA5 was screened for antibiotic resistance, broad resistance against penicillin, streptomycin, tetracycline, ampicillin, rifampicin, and erythromycin was found; this correlated with the presence of multiple gene determinants for antibiotic resistance within the whole genome sequence of MA5. Overall, this study provides an in-depth understanding of the underpinnings of Stenotrophomonas-mercury interactions that facilitate cellular survival in a contaminated soil habitat.

Bioremediation of Mercury by Vibrio fluvialis Screened from Industrial Effluents.

Thirty-one mercury-resistant bacterial strains were isolated from the effluent discharge sites of the SIPCOT industrial area. Among them, only one strain (CASKS5) was selected for further investigation due to its high minimum inhibitory concentration of mercury and low antibiotic susceptibility. In accordance with 16S ribosomal RNA gene sequences, the strain CASKS5 was identified as Vibrio fluvialis. The mercury-removal capacity of V. fluvialis was analyzed at four different concentrations (100, 150, 200, and 250 μg/ml). Efficient bioremediation was observed at a level of 250 μg/ml with the removal of 60% of mercury ions. The interesting outcome of this study was that the strain V. fluvialis had a high bioremediation efficiency but had a low antibiotic resistance. Hence, V. fluvialis could be successfully used as a strain for the ecofriendly removal of mercury.

Mercury remediation potential of a mercury resistant strain Sphingopyxis sp. SE2 isolated from contaminated soil

A mercury resistant bacterial strain SE2 was isolated from contaminated soil. The 16s rRNA gene sequencing confirms the strain as Sphingopyxis belongs to the Sphingomonadaceae family of the α-Proteobacteria group. The isolate showed high resistance to mercury with estimated concentrations of Hg that caused 50% reduction in growth (EC50) of 5.97 and 6.22mg/L and minimum inhibitory concentrations (MICs) of 32.19 and 34.95mg/L in minimal and rich media, respectively. The qualitative detection of volatilized mercury and the presence of mercuric reductase enzyme proved that the strain SE2 can potentially remediate mercury. ICP-QQQ-MS analysis of the remaining mercury in experimental broths indicated that a maximum of 44% mercury was volatilized within 6hr by live SE2 culture. Furthermore a small quantity (23%) of mercury was accumulated in live cell pellets. While no volatilization was caused by dead cells, sorption of mercury was confirmed. The mercuric reductase gene merA was amplified and sequenced. Homology was observed among the amino acid sequences of mercuric reductase enzyme of different organisms from α-Proteobacteria and ascomycota groups.

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

ABSTRACTIn 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.

Mercury resistance and volatilization by Pseudoxanthomonas sp. SE1 isolated from soil

Abstract A mercury resistant bacterial strain SE1 isolated from contaminated soil was identified as Pseudoxanthomonas based on 16s rRNA sequencing. The Hg resistance was examined in both nutrient-rich media as well as low nutrient media and expressed as EC50 and MIC values. Estimated EC50 and MIC values in nutrient-rich media and low nutrient media had the following respective recordings — 22.6 mg L−1; 23.1 mg L−1 and 1.4 mg L−1 and 1.7 mg L−1. The isolate was able to volatilize inorganic mercury demonstrated by a modified photographic film experiment and subsequently revealed its ability to remove mercury from the solution. The ICP-QQQ-MS analysis of SE1 inoculated solution showed almost 60% of 1.5 mg L−1 mercury was volatilized in 6 h and almost 40% were accumulated in cell pellets. The mercuric reductase gene merA was identified in the genome of isolate SE1 and sequenced. The deduced amino acid sequence of merA gene indicated a sequence homology with different organisms from the alpha proteobacteria group and eukaryotic fungi. merA encoded enzyme mercuric reductase activity was evident in the crude protein of the isolate. The isolate’s ability to resist Hg, it’s Hg volatilization potential and the presence of merA gene and mercuric reductase enzyme demonstrates the potential application of this strain in mercury bioremediation.

A Bioremediation Approach to Mercury Removal in a Shake Flask Culture Using Pseudomonas putida (ATCC 49128)

Mercury is one of the most poisonous elements found on earth bonded to sulfhydryl groups of enzymes and proteins, thereby inactivating vital cell functions. Indeed this has drawn the attention of many environmental researchers who have been attempting through various mean to expunging mercury from these contaminated medium. Biological approach provided a satisfactory outcome in the clean-up of mercury contaminated soil and water due to its high potential for greater performance, environment friendliness and cost effectiveness. Mercuryresistant bacterial strain (P. putida ATCC 49128), was experimented on its potential to grow and reduce mercury to a permissible level under optimum conditions of nutrient, pH and other related physical factors in an incubator shake flask. It was observed that P. putida displayed a usual growth pattern when tried at low level mercury concentration of 1.0 μM, 6.0 μM and 19.0 μM, by exponentially growing during the first 4 hours of inoculation, but drastically decreased by the end of 24 hours’ time. This was indicated by the mercury removal rate of 99.0%, 99.83% and 98.58% in the three mercury concentrations used. Also, under the same optimum condition of growth, mercury concentration of 1000 μM was reduced by 92.0% after the first initial 1 hour to 98.0% at the end of 28 hour study. Comparably, similar trend was also observed when P. putida was used as bioaugmented organism to treat mercury contaminated samples from two petroleum industry based wastewater. A reduction rate of 84% was observed at the initial first 4 hours to about 90.5% after 96 hour experiment for plant P1 wastewater. While results from wastewater plant P2 indicated reduction rate of 97.2%, followed by 94.09% and lastly 56.8% respectively. The result affirmed the ability of this strain to optimally utilize the optimal conditional factors to grow and reduce mercury concentration overwhelmingly within a shortest time of less than 30 hours.

Bioremediation potential of a highly mercury resistant bacterial strain Sphingobium SA2 isolated from contaminated soil

A mercury resistant bacterial strain, SA2, was isolated from soil contaminated with mercury. The 16S rRNA gene sequence of this isolate showed 99% sequence similarity to the genera Sphingobium and Sphingomonas of α-proteobacteria group. However, the isolate formed a distinct phyletic line with the genus Sphingobium suggesting the strain belongs to Sphingobium sp. Toxicity studies indicated resistance to high levels of mercury with estimated EC50 values 4.5 mg L−1 and 44.15 mg L−1 and MIC values 5.1 mg L−1 and 48.48 mg L−1 in minimal and rich media, respectively. The strain SA2 was able to volatilize mercury by producing mercuric reductase enzyme which makes it potential candidate for remediating mercury. ICP-QQQ-MS analysis of Hg supplemented culture solutions confirmed that almost 79% mercury in the culture suspension was volatilized in 6 h. A very small amount of mercury was observed to accumulate in cell pellets which was also evident according to ESEM-EDX analysis. The mercuric reductase gene merA was amplified and sequenced. The deduced amino acid sequence demonstrated sequence homology with α-proteobacteria and Ascomycota group.

Isolation and Characterization of Mercury Resistant Bacillus sp. from Soils with an Extensive History as Substrates for Mercury Extraction in Mexico

Metal phytoextraction assisted by bacteria plays an important role in bioremediation systems. In this work, mercury-resistant bacterial strains were isolated from soils with high levels of mercury (San Joaquin, Queretaro State, Mexico) and identified as Bacillus sp. based on the 16S rDNA gene sequence analysis. The bacterial strains were found to exhibit different multiple mercury-resistance and carbon source utilization characteristics. The mercury reduction ability was tested through a volatilization assay. The bacterial isolates were also evaluated for their ability to promote growth and mercury uptake in tomato plants. In a roll towel assay, the maximum vigor index of tomato plants was obtained with the inoculation of Bacillus sp. A2, A12, B11, B15 and C1, while in a pot assay, the maximum vigor index was obtained with the inoculation of Bacillus sp. A6, A7 and B20, compared with un-inoculated controls in the presence of HgCl2. Maximum Hg accumulation in the roots and shoots of tomato plants was obtained only with Bacillus sp. A7 in the roll towel assay, whereas in the pot assay, maximum accumulation was obtained with Bacillus sp. A12 compared with un-inoculated controls. Our results show that mercury accumulation in tissue is enhanced by these plant growth promoting bacterial strains, which recommends their possible use as microbe-assisted phytoremediation systems in mercury-polluted soils.

Isolation and Characterization of Mercury Resistant Bacteria from Haldia river sediments

The Mercury is known as a toxic heavy metal. The present work is aimed to isolate and characterize mercury resistant bacterial strains from the Haldia dock area river sediment. Bacterial cells are grown in 10 ppm of HgCl2 containing nutrient media. These isolates were resistant up to 150 ppm of mercury salt. The bacterial isolates were identified to belong to the genera: Serratia and Streptococcus or Enterococcus.. Many of the chosen isolates were tested for growth in a variety of antibiotics. Results of this study demonstrate the occurrence of diverse groups of marine bacteria capable of high tolerance to mercury .Mercury tolerant strains from dock area can be used to detect mercury toxicity in this type of polluted area and can be used for bioremediation of mercury toxicity.

Mercury-resistant bacterial strains Pseudomonas and Psychrobacter spp. isolated from sediments of Orbetello Lagoon (Italy) and their possible use in bioremediation processes

This study was aimed to isolate Hg-resistant bacteria from contaminated sediments of the Orbetello Lagoon in Italy and to assess their possible use as biofilms in bioremediation processes. Enrichment cultures prepared from contaminated sediments in the presence of 0.05mM of mercury and under aerobic conditions allowed the isolation of five heterotrophic bacterial strains. 16S rDNA gene sequencing assigned the isolated strains to the genera Pseudomonas and Psychrobacter. For the first time mercury-resistant bacterial strains belonging to the genus Psychrobacter were evidenced. Minimum inhibitory concentrations in the presence of HgCl2 and of CH3HgCl showed high levels of resistance. EC50 values for the isolated bacterial strains in the presence of HgCl2 and of CH3HgCl confirmed the adaptation to the metal. Hg-resistant strains ORHg1, ORHg4 and ORHg5 showed the capacity to volatilize inorganic and organic mercury to elemental mercury, and formed biofilms on pumice particles, and were shown to play a role in the removal of mercury from sediment leachates. This study reports isolation and characterization of new Hg-resistant bacterial strains and provides novel insight into their possible use in bioremediation processes of mercury polluted sediments.

A conservative region of the mercuric reductase gene (merA) as a molecular marker of bacterial mercury resistance

The most common bacterial mercury resistance mechanism is based on the reduction of Hg(II) to Hg(0), which is dependent of the mercuric reductase enzyme (MerA) activity. The use of a 431 bp fragment of a conservative region of the mercuric reductase (merA) gene was applied as a molecular marker of this mechanism, allowing the identification of mercury resistant bacterial strains.

Biodegradation of thiomersal containing effluents by a mercury resistant Pseudomonas putida strain

Thiomersal, a toxic organomercurial with a strong bactericidal effect, is the most widely used preservative in vaccine production. As a result, vaccine production wastewaters are frequently polluted with thiomersal concentrations above the European limit for mercury effluent discharges for which there is, presently, no remediation technology available. This work proposes a biotechnological process for the remediation of vaccine production wastewaters based on the biological degradation of thiomersal to metallic mercury, under aerobic conditions, by a mercury resistant bacterial strain. The kinetics of thiomersal degradation by a pure culture of Pseudomonas putida spi3 was firstly investigated in batch reactors using a thiomersal amended mineral medium. Subsequently, a continuous stirred tank reactor fed with the same medium was operated at a dilution rate of 0.05 h −1, and the bioreactor performance and robustness was evaluated when exposed to thiomersal shock loads. In a second stage, the bioreactor was fed directly with a real vaccine wastewater contaminated with thiomersal and the culture ability to grow in the wastewater and remediate it was evaluated for dilution rates ranging from 0.022 to 0.1 h −1.

Mercury-resistant bacteria from permafrost sediments and prospects for their use in comparative studies of mercury resistance determinants

Mercury-resistant bacteria were isolated from permafrost sediments of Kolyma lowland and Canada existing over five thousand to two million years. Their content was shown to vary within the range 0.001-2.9% and to depend on the amount of mercury in sampling sites (coefficient of correlation 0.75). A collection of mercury-resistant bacterial strains was created. In this collection, various representatives of both Gram-positive bacteria (Bacillus, Exiguobacterium, Micrococcus, Arthrobacter) and Gram-negative bacteria (Pseudomonas, Acinetobacter, Plesiomonas, Myxobacteriales) were identified. Most resistant bacteria were found to contain determinants homologous to mer-operons of contemporary bacteria. The isolated strains of paleobacteria are proposed to be used for a comparative structural study of contemporary and ancient plasmids and transposons carrying mercury resistance determinants.

Removal of mercury from chloralkali electrolysis wastewater by a mercury-resistant Pseudomonas putida strain.

A mercury-resistant bacterial strain which is able to reduce ionic mercury to metallic mercury was used to remediate in laboratory columns mercury-containing wastewater produced during electrolytic production of chlorine. Factory effluents from several chloralkali plants in Europe were analyzed, and these effluents contained total mercury concentrations between 1.6 and 7.6 mg/liter and high chloride concentrations (up to 25 g/liter) and had pH values which were either acidic (pH 2.4) or alkaline (pH 13.0). A mercury-resistant bacterial strain, Pseudomonas putida Spi3, was isolated from polluted river sediments. Biofilms of P. putida Spi3 were grown on porous carrier material in laboratory column bioreactors. The bioreactors were continuously fed with sterile synthetic model wastewater or nonsterile, neutralized, aerated chloralkali wastewater. We found that sodium chloride concentrations up to 24 g/liter did not inhibit microbial mercury retention and that mercury concentrations up to 7 mg/liter could be treated with the bacterial biofilm with no loss of activity. When wastewater samples from three different chloralkali plants in Europe were used, levels of mercury retention efficiency between 90 and 98% were obtained. Thus, microbial mercury removal is a potential biological treatment for chloralkali electrolysis wastewater.

Molecular Analysis of merA Gene Possessed by Anaerobic Mercury-Resistant Bacteria Isolated from Sediment of Minamata Bay.

To understand the molecular basis of the mercury resistance of anaerobic bacteria, twenty-six mercury-resistant bacterial strains were anaerobically isolated from the mercury-polluted sediment of Minamata Bay, Japan. On the basis of taxonomic characteristics, all of the isolates were classified into the genus Clostridium. PCR primers designed from the core sequence of the merA genes that are involved in the inorganic mercury reduction by the aerobic mercury-resistant bacteria were used for the molecular analysis. Southern hybridization analysis confirmed that the PCR products amplified from the chromosomal DNA of the twenty-six Clostridium isolates were highly homologous to the merA gene of an aerobic mercury-resistant Gram-positive bacterium, Bacillus sp. RC607. Furthermore, the DNA sequence of the PCR product amplified from one of the isolates is 99.7% identical to the corresponding region of the merA gene of Bacillus sp. RC607. These results suggest that the same mercury resistance system developed by aerobic mercury-resistant bacteria is acquired by the anaerobic mercury-resistant bacteria.

Volatilization of mercury from natural water by a broad-spectrum Hg-resistantBacillus pasteurii strain DR2

A broad-spectrum mercury-resistant bacterial strain was isolated from contaminated water and was identified as Bacillus pasteurii strain DR2. It could volatilize Hg-compounds including organomercurials from its growth media. It utilized several aromatic compounds as a sole source of carbon. The bacterial strain eliminated HgCl2 from sterile river water and the presence of benzene, toluene, naphthalene and nitrobenzene at 1 mM concentration in the system increased the rate of mercury volatilization, the volatilization rate being highest with benzene. When 1.7×107 cells of this bacterial strain were added per ml of non-sterile water the bacterial strain volatilized more than 90 percent of mercury from mercuric chloride and organo-mercurials like PMA, thiomersol and methoxy ethyl mercuric chloride (MEMC). In the absence of this bacterial strain the volatilization of PMA and MEMC due to the presence of other Hg-resistant organisms in nonsterile polluted water ranged between 20–25 percent and of HgCl2 was about 40 percent. However, in the presence of B. pasteurii DR2 volatilization of these Hg-compounds from non-sterile water increased by 20–40 percent. In the presence of 1 mM benzene the rate of mercury volatilization was even higher. In all the cases the rate of volatilization was higher in the first seven days than in the next seven days.

Response of a freshwater bacterial community to mercury contamination (HgCl2 and CH3HgCl) in a controlled system

An experimental study was made on the effects of inorganic mercury (HgCl2) and methylmercury (CH3HgCl) on freshwater aerobic heterotrophic bacterial population. The experimental system was based on two-compartment biotopes: natural sediment, from the Garonne River, and dechlorinated tap water. The response of bacterial communities to mercury additions (to the water column) was monitored by determining total Hg in water and enumerating the total number of bacteria and the evolution of the mercury resistant community. Isolation was carried out by plate count method. Enumeration of mercury resistant strains was made with a general medium (Iron-Tryp-tone agar) amended with 10 μg Hg/ml of HgCl2. The response to 2 μg Hg/L (HgCl2) was fast approximately 50% of the maximum percentage of mercury resistant bacteria being reached after one hour (21.7% after 17 h exposure). Spikes of CH3HgCl (2 μg Hg/L) in the water column caused an initial inhibition of growth of Hg-resistant and sensitive bacteria followed by a complete recovery of the background microbial population after 84 h. Seven mercury resistant bacterial strains were isolated from the experimental systems and each of them was checked for HgCl2 and CH3HgCl transformation. All were able to volatilize HgCl2 by producing elemental mercury, but none was able to degrade methylmercury. Additions of different concentrations of HgCl2 (0.02 μg Hg/L to 2 μg Hg/L) to the water column caused a proportional increase in the percentage of mercury-resistant bacteria. Low concentrations (<0.6 μg Hg/L) of CH3HgCl also induced the Hg-resistant community, whereas 2 μg Hg/L of CH3HgCl inhibited the growth of both Hg-sensitive and Hg-resistant heterotrophic bacteria.

Mercury resistance in nitrogen-fixing soil bacteria.

A large number of mercury-resistant nitrogen-fixing bacteria were isolated by screening soil samples from agricultural farms of West Bengal, India. All of the samples were found to contain significant quantities of mercury. Twenty five of the pure isolates were identified to the generic level following Bergey's Manual of Determinative Bacteriology, 8th edition. These organisms were resistant to HgCl2, and a few of them were resistant to phenylmercuric acetate (PMA) as well. Their mercury resistance was also associated with antibiotic resistance properties. Growth of two mercury-resistant N2-fixing organisms was monitored in liquid media supplemented with HgCl2. The mercury volatilizing capacity of four mercury-resistant bacterial strains was also determined.