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Engineering a bioluminescent bioreporter from an environmentally‐sourced mercury‐resistant Enterobacter cloacae strain for the detection of bioavailable mercury
Abstract
Aim:
Escherichia coli is the conventional choice as the host strain for whole-cell bioreporter construction due to its well-understood genetics and well-established cloning protocols. However, for real-world environmental biosensing applications, it is often beneficial to use a bacterial strain derived directly from the environment under study to better ensure chemical target specificity and optimal response time. The aim of this study was to develop a whole-cell bioreporter for detection of bioavailable mercury by replacing Escherichia coli with a wild type bacterial host derived from a soil environment.
Materials and results:
In this study, an Enterobacter cloacae strain isolated from soil derived from a municipal and electronic waste dumping site was engineered to serve as a bioluminescent bioreporter for mercury toxicity by linking its merR-like gene and promoter sequence to a reorganized luxABCDE gene cassette from Photorhabdus luminescens. This bioreporter, designated as E. cloacae DWH4lux , detected mercury (HgCl2 ) at a minimum concentration of 0·2 μg l-1 with a linear response profile being maintained between a range of 0·4 to 1600 μg l-1 (R2 = 0·9604) with a peak bioluminescent response occurring within one hour after exposure. No significant synergistic or antagonistic influences were observed on the bioluminescent response by other contaminating metal elements. E. cloacae DWH4lux was also demonstrated to detect mercury effectively in artificially contaminated water sample with linear correlation (R2 =0·9623).
Conclusions:
The results indicated that E. cloacae DWH4lux could detect mercury in quantities below the U.S. Environmental Protection Agency's permitted limit values (2 μg l-1 ). Hence, it is concluded that E. cloacae DWH4lux has the potential to serve as an effective whole-cell bioreporter for the environmental monitoring of mercury contamination.
Significance and impact of study:
This study provides new insight into the recruitment of mercury tolerant bacterial hosts derived from environmental samples over the conventional lab-based E. coli host for the construction of mercury bioreporters. With improved response time and selectivity, the environmentally sourced bacteria can serve as an alternative host choice to improve biosensing technology in the near future. This article is protected by copyright. All rights reserved.
... Classical tools (e.g., selective extractions) are inefficient in extracting targeted Hg fractions, and are unsuitable for in situ monitoring (Bloom et al., 2003). Recently, several novel methods, including the stable dilution isotope technique (Janssen et al., 2016;Shetaya et al., 2017), whole-cell biosensors (Din et al., 2019;Mahbub et al., 2017;Ndu et al., 2015;Solovyev et al., 2015;Wang et al., 2015;Xu et al., 2015), and diffusive gradient thin film technique (DGT) (Ndu et al., 2018;Neal-Walthall et al., 2022;Pelcova et al., 2014), show great potentials in quantifying Hg bioavailability in complex natural environments (Table 2). Specifically, the labile Hg content quantified by the stable dilution isotope technique is much higher than that by chemical extraction (12-25 % versus <0.32 % of THg), indicating that the bioavailability of Hg might be largely underestimated in previous studies using the classical method (Shetaya et al., 2017). ...
... The main disadvantage of this method is the high cost (Shetaya et al., 2017). The whole-cell biosensor has been proven to be effective in the quantitative detection of the labile Hg(II) in the laboratory, but is seldom used for environmental samples because it is greatly affected by complex environmental conditions (Din et al., 2019;Mahbub et al., 2017;Ndu et al., 2015). DGT has recently been proven to help quantify the bioavailable Hg fractions in environments, which is converted from the amount of Hg on the adsorption membrane according to the law of free diffusion (Pelcova et al., 2014;Turull et al., 2018;Wang et al., 2016;Xu et al., 2019b). ...
Wetlands are considered the hotspots for mercury (Hg) biogeochemistry, garnering global attention. Therefore, it is important to review the research progress in this field and predict future frontiers. To achieve that, we conducted a literature analysis by collecting 15,813 publications about Hg in wetlands from the Web of Science Core Collection. The focus of wetland Hg research has changed dramatically over time: 1) In the initial stage (i.e., 1959–1990), research mainly focused on investigating the sources and contents of Hg in wetland environments and fish. 2) For the next 20 years (i.e., 1991–2010), Hg transformation (e.g., Hg reduction and methylation) and environmental factors that affect Hg bioaccumulation have attracted extensive attention. 3) In the recent years of 2011–2022, hot topics in Hg study include microbial Hg methylators, Hg bioavailability, methylmercury (MeHg) demethylation, Hg stable isotope, and Hg cycling in paddy fields. Finally, we put forward future research priorities, i.e., 1) clarifying the primary factors controlling MeHg production, 2) uncovering the MeHg demethylation process, 3) elucidating MeHg bioaccumulation process to better predict its risk, and 4) recognizing the role of wetlands in Hg circulation. This research shows a comprehensive knowledge map for wetland Hg research and suggests avenues for future studies.
... As mentioned earlier, WCB-based bioassays hold great promise for low-cost, time-effective, and versatile field (on-site) monitoring and have the ability to measure bioavailability of metals (Huang et al., 2015;Sánchez et al., 2021). Various microbial biosensors for heavy metal detection have been constructed by combining metal responding regulatory genes (e.g., merR, arsR) with multifarious reporter genes: for mercury (Din et al., 2019), arsenite (Elcin & Öktem, 2020a), cadmium (Elcin & Öktem, 2020b), lead (Zhang et al., 2022), chromate (Zhang et al., 2019), and nickel (Patyi et al., 2021). For As biosensors, the regulatory gene arsR from the ars operon is often applied to compose the sensing element of biosensors for the detection of arsenic (Bereza-Malcolm et al., 2018;Wang et al., 2021). ...
Arsenic contamination is a critical global problem, and its widespread environmental detection is becoming a prominent issue. Herein, electrospun fibers of cellulose acetate (CA) and polycaprolactone (PCL) were successfully fabricated and used as the support material for immobilization of arsenic-sensing bacterial bioreporter for the first time. To date, no attempt has been made to immobilize fluorescent whole-cell bioreporter cells on electrospun fibers for arsenic detection. CA and PCL electrospun fibers were fabricated via traditional electrospinning technique and characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), and contact angle meter. Following immobilization of the bacterial bioreporter cells, the immobilized bacteria were also characterized by viability assay using AlamarBlue. The effects of growth phase and cell concentration on the fluorescence response of fiber-immobilized arsenic bioreporters to arsenic were also investigated. After immobilization of arsenic bioreporters on 10 wt% PCL fiber, 91% of bacterial cells remained viable, while this value was 55.4% for cells immobilized on 12.5 wt% CA fiber. Bioreporter cells in the exponential growth phase were shown to be more sensitive to arsenic compared to aged cells. While both the electropsun PCL- and CA-immobilized bioreporters successfully detected 50 and 100 µg/L of arsenite (As (III)) concentrations, the PCL-immobilized bioreporter showed better fluorescence performance which should be investigated in future studies. This study helps to fill some gaps in the literature and demonstrates the potential for using electrospun fiber-immobilized arsenic whole-cell bioreporter for arsenic detection in water.
... However, the biosensor sensitivity is usually determined by genetic circuit engineering and types of reporter genes (Gupta et al., 2019;Hui et al., 2020). Metalloregulator MerR is a Hg(II)-responsive transcriptional factor, which has been employed to develop bacterial whole-cell biosensors using luciferase (Din et al., 2019), β-galactosidase (Hansen and Sorensen, 2000), fluorescence protein , and visual pigments Hui et al., 2021a) as the signal outputs. All of these above bacterial biosensors responded to Hg(II) with high selectivity and variable sensitivity. ...
Genetically programmed biosensors have been widely used to monitor bioavailable heavy metal pollutions in terms of their toxicity to living organisms. Most bacterial biosensors were initially designed to detect specific heavy metals such as mercury and cadmium. However, most available biosensors failed to distinguish cadmium from various heavy metals, especially mercury. Integrating diverse sensing elements into a single genetic construct or a single host strain has been demonstrated to quantify several heavy metals simultaneously. In this study, a dual-sensing construct was assembled by employing mercury-responsive regulator (MerR) and cadmium-responsive regulator (CadR) as the separate sensory elements and enhanced fluorescent protein (eGFP) and mCherry red fluorescent protein (mCherry) as the separate reporters. Compared with two corresponding single-sensing bacterial sensors, the dual-sensing bacterial sensor emitted differential double-color fluorescence upon exposure to 0-40 µM toxic Hg(II) and red fluorescence upon exposure to toxic Cd(II) below 200 µM. Bioavailable Hg(II) could be quantitatively determined using double-color fluorescence within a narrow concentration range (0-5 µM). But bioavailable Cd(II) could be quantitatively measured using red fluorescence over a wide concentration range (0-200 µM). The dual-sensing biosensor was applied to detect bioavailable Hg(II) and Cd(II) simultaneously. Significant higher red fluorescence reflected the predominant pollution of Cd(II), and significant higher green fluorescence suggested the predominant pollution of Hg(II). Our findings show that the synergistic application of various sensory modules contributes to an efficient biological device that responds to concurrent heavy metal pollutants in the environment.
... Over recent decades, the E. cloacae have taken on clinical significance and have emerged as nosocomial pathogens from intensive care patients [2] because these bacteria have an intrinsic resistance to broad-spectrum cephalosporins [3]. However, strains of this group of bacteria were widely found in nature and were isolated from different sources: human clinical specimens [4], water [5], soil [6] (include polluted soil [7]), plants [8] and other environmental sources [9]. In plants, Enterobacter spp. ...
The O-antigen (O-polysaccharide) is an essential component of lipopolysaccharide on the surface of Gram-negative bacteria and plays an important role in interaction with host organisms. In this study, we investigated the chemical structure and characterized the gene cluster of Enterobacter cloacae K7 O-antigen. As judged by sugar analyses along with NMR spectroscopy data, E. cloacae K7 antigen has a tetrasaccharide O-unit with the following structure:
→8)-β-Psep5Ac7Ac-(2→2)-β-l-Rhap-(1→4)-α-l-Rhap-(1→3)-α-d-Galp-(1→
The O-antigen gene cluster of E. cloacae K7 between conserved genes galF and gnd was sequenced. Most genes necessary for the O-antigen synthesis were found in the cluster and their functions were tentatively assigned by comparison with sequences in the available databases.
... A variety of microbial bioluminescent biosensors have been designed to detect metal contaminants in the environment (Lajoie et al., 2002;Kelly et al., 2004;Ren;Frymier, 2005;Harpaz et al., 2018;Din et al., 2019;Du et al., 2019). These biosensor systems are becoming appropriate alternatives to the traditional analytical methods including respirometry, measurement of bacterial growth inhibition, chemical analysis, and microscopic analysis. ...
Recombinant bioluminescent bacteria are frequently directed towards use as environmental biosensors because it has high sensitivity, selectivity, costless, easy to use and function as rapid measurement to detect heavy metals. The bioluminescence of DF4/PUTK2 assay is based on using the bio-reporter Acinetobacter DF4/PUTK2 carrying luciferase genes luxCDABE which emit light constitutively. This can be measured in time intervals by luminometer to determine the behavior of bio-reporter against lead. The light emitted in the lead treated samples was equal or increased than the control. Therefore, the bio-reporter DF4/PUTK2 was subjected to intensive studies to elucidate its behavior with lead and if it was possible to be employed as a lead light-on assay in water in reverse order. Time exposure (5 to 990 min), lead concentration (0 to 30000 ppm), and lead salts (acetate-chloride and nitrate) were included in this study. The bio-reporter DF4/PUTK2 was more sensitive to lead concentrations range (19.25 to 15000 ppm). However, at high concentrations of lead, the light was being decreased due to cell death and/or metabolic burden simultaneously. It was possible to detect the presence of lead in water samples through light-induction in specific concentrations which add another advantage to the bio-reporter DF4/PUTK2.
... Thereby, it is essential to elucidate the genetic mechanism of the response of host to specific metal to construct whole-cell bioreporters (Xu et al., 2013). In addition, the selectivity of typical whole-cell bioreporters is poor because of the non-specific cellular response to metal ions (Martin-Betancor et al., 2015;Din et al., 2019). For these reasons, the application of whole-cell bioreporters is restricted and in slow development. ...
The whole-cell bioreporters based on the cop-operon sensing elements have been proven specifically useful in the assessment of bioavailable copper ions in water environments. In this study, a series of experiments was conducted to further improve the sensitivity and robustness of bioreporters. First, an Escherichia coli △copA△cueO△cusA mutant with three copper transport genes knocked out was constructed. Then, the copAp::gfpmut2 sensing element was inserted into the chromosome of E. coli △copA△cueO△cusA by gene knock-in method to obtain the bioreporter strain E. coli WMC-007. In optimized assay conditions, the linear detection range of Cu2+ was 0.025–5 mg/L (0.39–78.68 μM) after incubating E. coli WMC-007 in Luria–Bertani medium for 5 h. The limit of detection of Cu2+ was 0.0157 mg/L (0.25 μM). Moreover, fluorescence spectrometry and flow cytometry experiments showed more environmental robustness and lower background fluorescence signal than those of the sensor element based on plasmids. In addition, we found that the expression of GFPmut2 in E. coli WMC-007 was induced by free copper ions, rather than complex-bound copper, in a dose-dependent manner. Particularly, the addition of 40 mM 3-(N-Morpholino)propanesulfonic acid buffer to E. coli WMC-007 culture enabled accurate quantification of bioavailable copper content in aqueous solution samples within a pH range from 0.87 to 12.84. The copper recovery rate was about 95.88–113.40%. These results demonstrate potential applications of E. coli WMC-007 as a bioreporter to monitor copper contamination in acidic mine drainage, industrial wastewater, and drinking water. Since whole-cell bioreporters are relatively inexpensive and easy to operate, the combination of this method with other physicochemical techniques will in turn provide more specific information on the degree of toxicity in water environments.
Mercury (Hg) and its organic compounds, especially monomethylmercury (MeHg), cause major damage to the ecosystem and human health. In surface water or sediments, microorganisms play a crucial role in the methylation and demethylation of Hg. Given that Hg transformation processes are intracellular reactions, accurate assessment of the bioavailability of Hg(II)/MeHg in the environment, particularly for microorganisms, is of major importance. Compared with traditional analytical methods, bacterial whole-cell biosensors (BWCBs) provide a more accurate, convenient, and cost-effective strategy to assess the environmental risks of Hg(II)/MeHg. This Review summarizes recent progress in the application of BWCBs in the detection of bioavailable Hg(II)/MeHg, providing insight on current challenges and strategies. The principle and components of BWCBs for Hg(II)/MeHg bioavailability analysis are introduced. Furthermore, the impact of water chemical factors on the bioavailability of Hg is discussed as are future perspectives of BWCBs in bioavailable Hg analysis and optimization of BWCBs.
The toxicity of mercury (Hg) mainly depends on its form. Whole-cell biosensors respond selectively to toxic Hg(ii), efficiently transformed by environmental microbes into methylmercury, a highly toxic form that builds up in aquatic animals. Metabolically engineered Escherichia coli (E. coli) have successfully produced rainbow colorants. By de novo reconstruction of the carotenoid synthetic pathway, the Hg(ii)-responsive production of lycopene and β-carotene enabled programmed E. coli to potentially become an optical biosensor for the qualitative and quantitative detection of ecotoxic Hg(ii). The red color of the lycopene-based biosensor cell pellet was visible upon exposure to 49 nM Hg(ii) and above. The orange β-carotene-based biosensor responded to a simple colorimetric assay as low as 12 nM Hg(ii). A linear response was observed at Hg(ii) concentrations ranging from 12 to 195 nM. Importantly, high specificity and good anti-interference capability suggested that metabolic engineering of the carotenoid biosynthesis was an alternative to developing a visual platform for the rapid analysis of the concentration and toxicity of Hg(ii) in environmentally polluted water.
Water contamination has become increasingly a critical global environmental issue that threatens human and ecosystems’ health. Monitoring and risk assessment of toxic pollutants in water bodies is essential to identifying water pollution treatment needs. Compared with the traditional monitoring approaches, environmental biosensing via whole-cell bioreporters (WCBs) has exhibited excellent capabilities for detecting bioavailability of multiple pollutants by providing a fast, simple, versatile and economical way for environmental risk assessment. The performance of WCBs is determined by its elements of construction, such as host strain, regulatory and reporter genes, as well as experimental conditions. Previously, numerous studies have focused on the design and construction of WCB rather than improving the detection process and commercialization of this technology. For investigators working in the environmental field, WCB can be used to detect pollutants is more important than how they are constructed. This work provides a review of the development of WCBs and a brief introduction to genetic construction strategies and aims to summarize key studies on the application of WCB technology in detection of water contaminants, including organic pollutants and heavy metals. In addition, the current status of commercialization of WCBs is highlighted.
Engineered microorganisms have proven to be a highly effective and robust tool to specifically detect heavy metals in the environment. In this study, a highly specific pigment-based whole-cell biosensor has been investigated for the detection of bioavailable Hg(II) based on an artificial heavy metal resistance operon. The basic working principle of biosensors is based on the violacein biosynthesis under the control of mercury resistance (mer) promoter and mercury resistance regulator (MerR). Engineered biosensor cells have been demonstrated to selectively respond to Hg(II), and the specific response was not influenced by interfering metal ions. The response of violacein could be recognized by the naked eye, and the time required for the maximum response of violacein (5 h) was less than that of enhanced green fluorescence protein (eGFP) (8 h) in the single-signal output constructs. The response of violacein was almost unaffected by the eGFP in a double-promoter controlled dual-signals output construct. However, the response strength of eGFP was significantly decreased in this genetic construct. Exponentially growing violacein-based biosensor detected concentrations as low as 0.39 μM Hg(II) in a colorimetric method, and the linear relationship was observed in the concentration range of 0.78–12.5 μM. Non-growing biosensor cells responded to concentrations as low as 0.006 μM Hg(II) in a colorimetric method and in a Hg(II) containing plate sensitive assay, and the linear relationship was demonstrated in a very narrow concentration range. The developed biosensor was finally validated for the detection of spiked bioavailable Hg(II) in environmental water samples.
The environmental release of mercury is continuously increasing with high degree of mobility, transformation and amplified toxicity. Improving remediation strategies is becoming increasingly important to achieve more stringent environmental safety standards. This study develops a laboratory-scale reactor for bioremediation of aqueous mercury using a biofilm-producing bacterial strain, KBH10, isolated from mercury-polluted soil. The strain was found resistant to 80 mg/L of HgCl2 and identified as Bacillus nealsonii via 16S rRNA gene sequence analysis. The strain KBH10 was characterized for optimum growth parameters and its mercury biotransformation potential was validated through mercuric reductase assay. A packed-bed column bioreactor was designed for biofilm-mediated mercury removal from artificially contaminated water and residual mercury was estimated. Strain KBH10 could grow at a range of temperature (20–50 °C) and pH (6.0–9.0) with optimum temperature established at 30 °C and pH 7.0. The optimum mercuric reductase activity (77.8 ± 1.7 U/mg) was reported at 30 °C and was stable at a temperature range of 20–50 °C. The residual mercury analysis of artificially contaminated water indicated 60.6 ± 1.5% reduction in mercury content within 5 h of exposure. This regenerative process of biofilm-mediated mercury removal in a packed-bed column bioreactor can provide new insight into its potential use in mercury bioremediation. HIGHLIGHTS
Bioaccumulation of aqueous mercury in the food web is a matter of grave concern.;
Enzymatic reduction of mercury by resistant bacteria reduces its potential toxicity.;
Bacillus nealsonii possess the inbuilt mechanisms of necessary adaptation.;
Mercury content was reduced up to 60.6 ± 1.5% within 5 h of exposure in packed-bed bioreactor.;
Biofilm-mediated mercury remediation is efficient, regenerative and cost-effective.;
The molecular evolutionary genetics analysis (Mega) software implements many analytical methods and tools for phylogenomics and phylomedicine. Here, we report a transformation of Mega to enable cross-platform use on Microsoft Windows and Linux operating systems. Mega X does not require virtualization or emulation software and provides a uniform user experience across platforms. Mega X has additionally been upgraded to use multiple computing cores for many molecular evolutionary analyses. Mega X is available in two interfaces (graphical and command line) and can be downloaded from www.megasoftware.net free of charge.
Despite the large number of bioreporters developed to date, the ability to detect heavy metal(loid)s with bioreporters has thus far been limited owing to the lack of appropriate genetic systems. We here present a novel approach to modulate the selectivity and sensitivity of microbial whole-cell bioreporters (WCBs) for sensing metal(loid)s via the znt-operon from Escherichia coli, which were applied to quantify the bioavailability of these contaminants in environmental samples. The WCB harboring the fusion gene zntAp::egfp was used as a microbial metal(loid) sensor, which was turned on by the interaction between ZntR and metal(loid) ions. This design makes it possible to modulate the selectivity and sensitivity to metal(loid)s simply by changing the metal-binding property of ZntR and by disrupting the metal efflux system of E. coli, respectively. In fact, the E. coli cell-based bioreporter harboring zntAp::egfp showed multi-target responses to Cd(II), Hg(II), and Zn(II). However, the WCBs showed responses toward only Cd(II) and Hg(II) when the amino acid sequence of the metal-binding loop of ZntR was changed to CNHEPGTVCPIC and CPGDDSADC, respectively. Moreover, the sensitivity toward both Cd(II) and Hg(II) was enhanced when copA, which is known to export copper and silver, was deleted. Thus, our findings provide a strong foundation for expanding the target of WCBs from the currently limited number of genetic systems available.
The previously described chromium resistant bacterium, Enterobacter cloacae B2-DHA, was isolated from leather manufacturing tannery landfill in Bangladesh. Here we report the entire genome sequence of this bacterium containing chromium and other heavy metal resistance genes. The genome size and the number of genes, determined by massive parallel sequencing and comparative analysis with other known Enterobacter genomes, are predicted to be 4.22 Mb and 3958, respectively. Nearly 160 of these genes were found to be involved in binding, transport, and catabolism of ions as well as efflux of inorganic and organic compounds. Specifically, the presence of two chromium resistance genes, chrR and chrA was verified by polymerase chain reaction. The outcome of this research highlights the significance of this bacterium in bioremediation of chromium and other toxic metals from the contaminated sources.
Five GES-producing
Enterobacteriaeae
displaying an Extended-spectrum ß-lactamase (ESBL) phenotype, harbored two GES variants: GES-7 ESBL, and GES-6 carbapenemase. In all isolates, both GES alleles were located on the same integron that was inserted onto an 80-kb IncM1 self-conjugative plasmid. Whole genome sequencing suggested
in vivo
horizontal gene transfer of the plasmid, along with clonal diffusion of
Enterobacter cloacae
. This is the first description in Europe of clustered
Enterobacteriaceae
isolates carrying two GES β-lactamases, among which, one has extended activity towards carbapenems.
Heavy metals, which have widespread environmental distribution and originate from natural and anthropogenic sources, are common environmental pollutants. In recent decades, their contamination has increased dramatically because of continuous discharge in sewage and untreated industrial effluents. Because they are non-degradable, they persist in the environment; accordingly, they have received a great deal of attention owing to their potential health and environmental risks. Although the toxic effects of metals depend on the forms and routes of exposure, interruptions of intracellular homeostasis include damage to lipids, proteins, enzymes and DNA via the production of free radicals. Following exposure to heavy metals, their metabolism and subsequent excretion from the body depends on the presence of antioxidants (glutathione, _-tocopherol, ascorbate, etc.) associated with the quenching of free radicals by suspending the activity of enzymes (catalase, peroxidase, and superoxide dismutase). Therefore, this review was written to provide a deep understanding of the mechanisms involved in eliciting their toxicity in order to highlight the necessity for development of strategies to decrease exposure to these metals, as well as to identify substances that contribute significantly to overcome their hazardous effects within the body of living organisms.
A luxCDABE-based genetically engineered bacterial bioreporter (Escherichia coli ARL1) was used to detect bioavailable ionic mercury (Hg(II)) and investigate the effects of humic acids and ethylenediaminetetraacetic acid (EDTA) on the bioavailability of mercury in E. c
oli. Results showed that the E. c
oli ARL1 bioreporter was sensitive to mercury, with a detection limit of Hg(II) of 0.5 µg/L and a linear dose/response relationship up to 2000 µg Hg(II)/L. Humic acids and EDTA decreased the Hg(II)-induced bioluminescent response of strain ARL1, suggesting that the two organic ligands reduced the bioavailability of Hg(II) via complexation with Hg(II). Compared with traditional chemical methods, the use of E. c
oli ARL1 is a cost-effective, rapid, and reliable approach for measuring aqueous mercury at very low concentrations and thus has potential for applications in field in situ monitoring.
The present study deals with the isolation, the identification, and the characterization of heavy metal-resistant bacteria selected from several polluted sources with the aim of using them as bioremediation agents of contaminated sites by heavy metals. Four protocols were used in a different way to select the maximum of isolates resistant to heavy metals. Isolates obtained, according to the fourth selection protocol by using the free nutrients medium, showed the highest values of minimum inhibitory concentration (MIC), and it can be deduced from this last result that when the bacteria are subjected to stressful conditions they develop a system that will enable them to withstand high levels of heavy metals. The study of the MIC at all resistant isolates allowed determining that the order of toxicity of the different metals was as follows: Cu > Cr > Cd > Ni > Zn > Co. These metals might exert varying effects depending on their adsorption and their complexation capacities to the bacterial cell wall. In addition, we can assume from the results of different screening protocols that the mixture of heavy metals exerts a more significant stressful effect on the life of the bacterial cell than the metal used alone. Isolates are all bacilli and cocci of gram-negative and of gram-positive bacteria. Pseudomonas aeruginosa and Staphylococcus aureus appeared as the most dominant genera. Therefore, the presence of metals in the growth medium has as an effect to conserve and to keep the character of resistance in the bacteria, contrary to its or their absence allowed the direct loss of this resistance. The study on the morphology of resistant isolates showed that the contour and the size of colonies would change in the presence of heavy metals. The heavy metal tolerant isolates were as of two types, of gram-positive and of gram-negative bacteria, and isolates of gram-negative were generally more resistant to the mixture of metals than those of gram-positive, since they could tolerate a 30 mM of heavy metal mixture. In addition, the identification of isolates selected by the mixture of metals revealed the dominance of the P. aeruginosa. The study of the growth of P. aeruginosa in the presence of increasing concentrations of metals showed a significant toxic effect of the high concentrations. Low concentrations are essential as trace elements for the bacterial growth and metabolism. Considering P. aeruginosa metal resistant strains, the quantity of total proteins synthesized appeared variable and generally an increase of the metal concentration is accompanied with an inhibition of the protein synthesis. The study of the protein profile for heavy metals resistant isolates of P. aeruginosa is mainly shown by the decrease in the level of proteins. This decrease pointed out the toxic effect of metals on the bacterial metabolism.
Trace heavy metals, such as arsenic, cadmium, lead, chromium, nickel and mercury are important
environmental pollutants, particularly in areas with high anthropogenic pressure. In addition to these
metals, copper, manganese, iron and zinc are also important trace micronutrients. The presence of trace
heavy metals in the atmosphere, soil and water, can cause serious problems to all organisms, and the
ubiquitous bioavailability of these heavy metal can result in bioaccumulation in the food chain especially
can be highly dangerous to human health. This study reviews the heavy metal contamination in several
areas of Pakistan over the past few years, particularly, to assess the heavy metal contamination in water
(ground water, surface water and waste water), soil, sediments, particulate matter and vegetables. The
listed contaminations affect the drinking water quality, ecological environment and food chain. Moreover
the toxicity induced by contaminated water, soil and vegetables poses serious threat to human health.
Fluorescent metal nanoclusters (NCs) have given rise to a new class of fluorescent nanomaterials for the detection of heavy metals. Here, we design a simple, rapid and highly sensitive sensing nanosystem for the detection of Hg(2+) and Cu(2+) based on fluorescence quenching of ultrasmall DNA-Ag NCs. The fluorescence intensity of DNA-Ag NCs was selectively quenched by Hg(2+) and Cu(2+), and the limit of detection (LOD) was found to be 5 nM and 10 nM, respectively. The technique was renewable employment by EDTA addition and successfully applied to detection of Hg(2+) and Cu(2+) in domestic water samples. The quantum yield (QY) of DNA-Ag NCs was significantly higher to ~30% compared to traditional water-soluble fluorescent metal NCs. The DNA-Ag NC detection system make it potentially suitable for detecting Hg(2+) and Cu(2+) and monitoring water quality in a wide range of samples regulated under the Environmental Protection Agency.
Biological communities are often structured by environmental factors even at small spatial scales. Fungi are no exception, though the patterns and mechanisms underlying their community structure are usually unknown. Previous work documented zonation in fungi under tree canopies primarily through their fruiting patterns. Here we investigate the existence of zonation patterns in fungal communities around isolated Pinus muricata trees of different ages in northern coastal California. Using a combination of ingrowth bags and pyrosequencing to target underground mycelium we found highly diverse soil fungal communities associated with single trees. Both ectomycorrhizal and non-ectomycorrhizal fungi were present in all samples, but the latter were more species rich, dominated the samples by sequence read abundance, and showed partitioning by canopy-defined zones and tree age. Soil chemistry was correlated with fungal zonation, but host root density was not. Our results indicate different guilds of fungi partition space differently and are driven by distinct environmental parameters.
Unorganized dumping of solid waste is predominant in developing countries like India and cause adverse impacts to the environment. Sources such as electronic goods, electro plating waste, painting waste, used batteries, etc., when dumped with municipal solid wastes increase the heavy metals in dumpsites. Similarly solid waste dumping without the separation of hazardous waste can raise toxic environmental effects. Slow leaching of these heavy metals under acidic environment during the degradation process leads to leachates with high metal concentrations. Since leachates are one of the potential sources of ground water pollution, monitoring heavy metal content in dumpsite can facilitate to recommend suitable remedial measures. Thus the assessment of heavy metal contents in landfill waste is a pre requisite of landfill mining, especially when the wastes are used as manures. This paper discusses the trace metal contents in fine fraction of municipal solid waste (MSW) collected from different depth levels of Perungudi dumping ground (PDG), near Chennai. The leachates were also collected and analyzed from the same sampling area. Heavy metal concentrations of these samples were compared with the water extracts prepared from the MSW fine fraction. The concentrations of As, Hg, Cr, Cd, Cu, Pb, Ni and Zn were estimated and found to be in mg/kg level in MSW while in µg/L in leachates and water extracts. In certain cases, metal contents are beyond the limits prescribed for compost by Central Pollution Control Board (CPCB). However, all values are within the acceptable limits of United States Environmental Protection Agency (USEPA) standards.
Microbial bioreporters offer excellent potentialities for the detection of the bioavailable portion of pollutants in contaminated environments, which currently cannot be easily measured. This paper describes the construction and evaluation of two microbial bioreporters designed to detect the bioavailable chromate in contaminated water samples. The developed bioreporters are based on the expression of gfp under the control of the chr promoter and the chrB regulator gene of TnOtChr determinant from Ochrobactrum tritici 5bvl1. pCHRGFP1 Escherichia coli reporter proved to be specific and sensitive, with minimum detectable concentration of 100 nM chromate and did not react with other heavy metals or chemical compounds analysed. In order to have a bioreporter able to be used under different environmental toxics, O. tritici type strain was also engineered to fluoresce in the presence of micromolar levels of chromate and showed to be as specific as the first reporter. Their applicability on environmental samples (spiked Portuguese river water) was also demonstrated using either freshly grown or cryo-preserved cells, a treatment which constitutes an operational advantage. These reporter strains can provide on-demand usability in the field and in a near future may become a powerful tool in identification of chromate-contaminated sites.
Heavy metal toxicity represents an uncommon but clinically significant medical condition, which if unrecognized or inappropriately
treated results in significant morbidity and mortality. Mercury is recognized as a potent and widely distributed toxicant
in the global environment having ability to accumulate at various levels of food chain besides possessing ability to cross
placental and blood–brain barrier. It has been seen that bacteria growing near mercury polluted sites have evolved various
means of resistance based on the expression of different genes of mer operon against different forms of mercury. Microbe based remediation/detoxification of mercury is on forefront due to low
cost and less health hazardous compared to physicochemical based strategies, which are cost intensive and hazardous to human
health. However, strategies based on the modern aspects of biological technologies employing mer operon genes in different combination are needed to be designed for exploitation in the remediation of mercury completely
from mercury contaminated environments.
A sensor responsive to Hg2+ (pDL20) was constructed by fusing part of a narrow spectrum mer operon, including its regulatory elements, to promoterless lux genes. This was compared with another mer::lux fusion (pRB28) in order to estimate the available Hg in moss, a conventional bioindicator used for surveying Hg pollution
in terrestrial environments. Hg(II) was measured as the relative luminescence unit (RLU) emitted by E. coli cells carrying either pRB28 or pDL20. The linearity ranges of standard curves were measured by spiking HgCl2 at different nanomole levels in a phosphate buffered solution (PBS). The level of correlation between RLU and spiked HgCl2 depended on the incubation time of E. coli cells: the correlation factors (R
2) of the regression lines were highly significant only after 150 min of incubation. pDL20 detected Hg(II) concentrations in
the linear range between 0.05 nM and 0.5 nM and was approximately 27 times more sensitive than pRB28. The latter was less
sensitive and showed a different range of linearity, from 20 nM to 200 nM. Measurements of bioavailable Hg were performed
in buffered solutions leached from moss. Concentrations of Hg(II) were determined by external standard addition of HgCl2. pDL20 was found to be more reliable than pRB28 in the estimation of very low concentrations of bioavailable Hg (II). Both
sensors were unable to determine Hg(0) emitted by geothermal activities.
The assessment of the toxicants in roadside soil on regular basis has become extremely essential with the increase in awareness for the metal toxicity in the environment. The present study investigates the presence of toxic metals along National Highway (N-5), Pakistan. Averages of about 1.3 million per month of automobile vehicles ply on this route. Lead (Pb), zinc (Zn), copper (Cu), nickel (Ni), cadmium (Cd), cobalt (Co), manganese (Mn), mercury (Hg), and iron (Fe) were analyzed by atomic absorption spectrophotometry in roadside soil at the nine selected locations along the highway. Strong Pearson correlations (α = 0.05) were found between Pb and Zn (r
2 = 0.887), Fe and Mn (r
2 = 0.880), Hg and Cd (0.864), Cu and Zn (0.838), and Cu and Pb (0.814). The correlation between the elemental compositions of the main automobile components revealed vehicular traffic as the main non-point source of roadside soil pollution. Extremely high level of mercury, 144.05 mg kg − 1, was found at S5. It was revealed that the unregulated incineration and dumping sites of hazardous waste material along N-5 were also responsible for these contaminations. Multivariate analysis on the obtained data also disclosed the same interpretation. Cluster analysis of the data grouped Pb, Zn, and Cu at 85.23% similarity, whereas, Cd, Hg, and Ni were grouped at 78.75% similarity basis. The findings need swift action against the root cause of soil pollution.
Whole-cell bacterial bioreporters represent a convenient testing method for quantifying the bioavailability of contaminants in environmental samples. Despite the fact that several bioreporters have been constructed for measuring heavy metals, their application to environmental samples has remained minimal. The major drawbacks of the available bioreporters include a lack of sensitivity and specificity. Here, we report an improvement in the limit of detection of bacterial bioreporters by interfering with the natural metal homeostasis system of the host bacterium. The limit of detection of a Pseudomonas putida KT2440-based Zn/Cd/Pb-biosensor was improved by a factor of up to 45 by disrupting four main efflux transporters for Zn/Cd/Pb and thereby causing the metals to accumulate in the cell. The specificity of the bioreporter could be modified by changing the sensor element. A Zn-specific bioreporter was achieved by using the promoter of the cadA1 gene from P. putida as a sensor element. The constructed transporter-deficient P. putida reporter strain detected Zn(2+) concentrations about 50 times lower than that possible with other available Zn-bioreporters. The achieved detection limits were significantly below the permitted limit values for Zn and Pb in water and in soil, allowing for reliable detection of heavy metals in the environment.
Background:
Mycobacterium tuberculosis, the causative agent of tuberculosis, still represents a major public health threat in many countries. Bioluminescence, the production of light by luciferase-catalyzed reactions, is a versatile reporter technology with multiple applications both in vitro and in vivo. In vivo bioluminescence imaging (BLI) represents one of its most outstanding uses by allowing the non-invasive localization of luciferase-expressing cells within a live animal. Despite the extensive use of luminescent reporters in mycobacteria, the resultant luminescent strains have not been fully applied to BLI.
Methodology/principal findings:
One of the main obstacles to the use of bioluminescence for in vivo imaging is the achievement of reporter protein expression levels high enough to obtain a signal that can be detected externally. Therefore, as a first step in the application of this technology to the study of mycobacterial infection in vivo, we have optimised the use of firefly, Gaussia and bacterial luciferases in mycobacteria using a combination of vectors, promoters, and codon-optimised genes. We report for the first time the functional expression of the whole bacterial lux operon in Mycobacterium tuberculosis and M. smegmatis thus allowing the development of auto-luminescent mycobacteria. We demonstrate that the Gaussia luciferase is secreted from bacterial cells and that this secretion does not require a signal sequence. Finally we prove that the signal produced by recombinant mycobacteria expressing either the firefly or bacterial luciferases can be non-invasively detected in the lungs of infected mice by bioluminescence imaging.
Conclusions/significance:
While much work remains to be done, the finding that both firefly and bacterial luciferases can be detected non-invasively in live mice is an important first step to using these reporters to study the pathogenesis of M. tuberculosis and other mycobacterial species in vivo. Furthermore, the development of auto-luminescent mycobacteria has enormous ramifications for high throughput mycobacterial drug screening assays which are currently carried out either in a destructive manner using LuxAB or the firefly luciferase.
The mercury resistance (mer) operon of the gram-negative transposon Tn21 encodes not only a mercuric reductase and regulatory genes but also two inner membrane proteins (MerT and MerC) and a periplasmic protein (MerP). Although the merT, merP, and merC genes have been implicated in Hg(II) transport, the individual roles of these genes have not been established. We created in vitro precise deletion and frameshift mutations that eliminated each of the genes singly and in combination. Our results show that both merT and merP are required for Hg(II) binding but that merC is not. Both merT and merP are required for full expression of Hg(II) resistance, but loss of merP is less deleterious than loss of merT. Furthermore, mutations eliminating both merT and merP decrease resistance more than the single mutations do. In contrast, mutating merC had no effect on Hg(II) resistance. Both the merT and merP mutations increase the threshold Hg(II) concentration for induction of merA-lacZ transcriptional fusions and cause an increase in the maximal expression level. In contrast, the merC mutation had little effect on the threshold inducing concentration of Hg(II) but decreased the level of expression. Our results show that merT and merP alone are sufficient to specify a mercury transport system. The role of merC remains obscure.
The MerR metalloregulatory protein is a heavy-metal receptor that functions as the repressor and Hg(II)-responsive transcription activator of the prokaryotic mercury-resistance (mer) genes. We demonstrate that this allosterically modulated regulatory protein is sensitive to HgCl2 concentrations of 1.0 +/- 0.3 x 10(-8) M in the presence of 1.0 x 10(-3) M dithiothreitol for half-maximal induction of transcription of the mer promoter by Escherichia coli RNA polymerase in vitro. Transcription mediated by MerR increases from 10% to 90% of maximum in response to a 7-fold change in concentration of HgCl2, consistent with a threshold phenomenon known as ultrasensitivity. In addition, MerR exhibits a high degree of selectivity. Cd(II), Zn(II), Ag(I), Au(I), and Au(III) have been found to partially stimulate transcription in the presence of MerR, but concentrations at least two to three orders of magnitude greater than for Hg(II) are required. The molecular basis of the ultrasensitivity and selectivity phenomena are postulated to arise from the unusual topology of the transcription complex and a rare trigonal mercuric ion coordination environment, respectively. This mercuric ion-induced switch is to our knowledge the only known example of ultrasensitivity in a signal-responsive transcription mechanism.
Biosensors for the detection of pollutants in the environment can complement analytical methods by distinguishing bioavailable from inert, unavailable forms of contaminants. By using fusions of the well-understood Tn21 mercury resistance operon (mer) with promoterless luxCDABE from Vibrio fischeri, we have constructed and tested three biosensors for Hg(II). Bioluminescence specified by pRB28, carrying merRo/pT, by pOS14, mediating active transport of Hg(II), and by pOS15, containing an intact mer operon, was measured in rich and minimal media. The highest sensitivities were achieved in minimal medium and were 1, 0.5, and 25 nM Hg(II) for pRB28, pOS14, and pOS15, respectively. The utility of the biosensors in natural waters was demonstrated with freshwater, rain, and estuarine samples supplemented with Hg(II). mer-lux carried by pRB28 and pOS14 responded to Hg(II) in mercury-contaminated water samples collected from a freshwater pond. Semiquantitative analyses based on light emission in samples collected from the inlet (analytically determined total mercury, approximately 20 nM) and outlet (total mercury, approximately 7 nM) of the pond showed bioavailable mercury at approximately 20 and 1 to 2 nM, respectively. Thus, the biosensors described here semiquantitatively detect bioavailable inorganic mercury (at a nanomolar to micromolar concentration range) in contaminated waters.
The transposon Tn21 and a group of closely related transposons (the Tn21 family) are involved in the global dissemination of antibiotic resistance determinants in gram-negative facultative bacteria. The molecular basis for their involvement is carriage by the Tn21 family of a mobile DNA element (the integron) encoding a site-specific system for the acquisition of multiple antibiotic resistance genes. The paradigm example, Tn21, also carries genes for its own transposition and a mercury resistance (mer) operon. We have compiled the entire 19,671-bp sequence of Tn21 and assessed the possible origins and functions of the genes it contains. Our assessment adds molecular detail to previous models of the evolution of Tn21 and is consistent with the insertion of the integron In2 into an ancestral Tn501-like mer transposon. Codon usage analysis indicates distinct host origins for the ancestral mer operon, the integron, and the gene cassette and two insertion sequences which lie within the integron. The sole gene of unknown function in the integron, orf5, resembles a puromycin-modifying enzyme from an antibiotic producing bacterium. A possible seventh gene in the mer operon (merE), perhaps with a role in Hg(II) transport, lies in the junction between the integron and the mer operon. Analysis of the region interrupted by insertion of the integron suggests that the putative transposition regulator, tnpM, is the C-terminal vestige of a tyrosine kinase sensor present in the ancestral mer transposon. The extensive dissemination of the Tn21 family may have resulted from the fortuitous association of a genetic element for accumulating multiple antibiotic resistances (the integron) with one conferring resistance to a toxic metal at a time when clinical, agricultural, and industrial practices were rapidly increasing the exposure to both types of selective agents. The compendium offered here will provide a reference point for ongoing observations of related elements in multiply resistant strains emerging worldwide.
Mercury (Hg) bioavailability to bacteria in marine systems is the first step towards its bioamplification in food webs. These systems exhibit high salinity and ionic strength which will both alter Hg speciation and properties of the bacteria cell walls. The role of Hg speciation on Hg bioavailability in marine systems has not been teased apart from that of ionic strength on cell wall properties, however. We developed and optimized a whole-cell Hg bioreporter capable of functioning under aerobic and anaerobic conditions and exhibiting no physiological limitations of signal production to changes in ionic strength. We show that ionic strength controls the bioavailability of Hg species, regardless of their charge, possibly by altering properties of the bacterial cell wall. The unexpected anaerobic bioavailability of negatively charged halocomplexes may help explain Hg methylation in marine systems such as the oxygen-deficient zone in the oceanic water column, sea ice or polar snow.
Whole-cell biosensors are still the method of choice when measuring bioavailable mercury, though their implementation in environmental monitoring is limited by low sensitivity, lack of portability and use of environmentally irrelevant bacteria. To address these issues, we have engineered a new luminescence-based whole-cell mercury biosensor, as part of a standalone fully automated portable device. Our method allows the incorporation of any environmentally relevant bacterial cell, which has been modified to translate the concentration of biologically available mercury into a dose-dependent luminescent signal. The use of environmentally relevant bacteria, Pseudomonas putida for fresh waters and Allivibrio fischeri for salt waters, demonstrated that environmental samples will not exhibit toxic effects, when appropriate microorganisms are implemented. Additionally, by assuring efficient aeration of the medium and thus sufficient oxygenation of sensor cells during generation of the luminescence signal, we obtained a clear dose dependent response and observed an increased sensitivity of the method up to 100-times (the LOD was determined to be as low as ∼10 ng L⁻¹). Finally, using our automated device, we demonstrated that in the environment the biologically available fraction of mercury can (i) represent an important part of the total mercury content (40–70 %) and (ii) it can correspond to the changes of total mercury content, which results in higher bioavailability of mercury closer to the source of mercury contamination.
Here we report two Enterobacter cloacae ST231 isolates co-producing KPC-3 and NDM-1, causing lethal infections in a tertiary hospital in China. The blaNDM-1-harboring plasmids carry IncA/C2 and IncR replicons, showing a mosaic plasmid structure, and the blaNDM-1 is harbored on a novel class I integron-like element. The blaKPC-3 is located on a Tn3-ΔblaTEM-1-blaKPC-3-ΔTn1722 element, flanked by two 9-bp direct repeat sequences, and harbored on a novel IncX6 plasmid.
Cell wall envelopes treated with sodium hydroxide and spray-dried were used as mercury sorbents. The sorbent having sorption capacity 17.7 ± 0.1 μmol/g determined was employed for preconcentration of mercury containing 1-10 ng/L. After preconcentration, bioavailable mercury was detected in samples of soil, stream, and tap water via induction of bioluminescence of E. coli ARL1. Iron and manganese at concentrations of tenth microgram per liter interfered bioluminescence detection of mercury. In tap water was detected semiquantitatively 0.127 ± 0.1 nmol/L by the induction of bioluminescence of E. coli ARL1 in medium with tryptone after preconcentration using a method of standard addition.
A whole-cell bacterial reporter was used to probe the bioavailability of mercury in the presence of a strong metal chelator, ethylenediaminetetraacetic acid (EDTA). Strain ARL1 was constructed by inserting a merR::luxCDABE fusion into the chromosome of Escherichia coli. The response of the bioreporter to Hg-II was monitored as a function of added EDTA. In parallel, square-wave voltammetry (SWV) measurements and thermodynamic calculations using MINEQL were performed to study the chemical speciation of mercury. The amount of electro-labile Hg-II measured by SWV was similar to the amount of non-complexed Hg-II predicted from equilibrium calculations. In contrast, the bioavailable fraction measured by the bioreporter was greater than the fraction predicted by either equilibrium calculation or electrochemical analysis. These results suggest that conventional chemical measurements and equilibrium calculations are not necessarily good proxies for predicting the bioavailable metal fraction. Additional factors such as kinetic effects or biological ligand competition must be considered.
The characteristics of bacteria take up mercury into cells via membrane potential-dependent sequence-divergent members of the mercuric ion (Mer) superfamily, i.e., a periplasmic mercuric ion scavenging protein (MerP) and one or more inner membrane-spanning proteins (MerC, MerE, MerF, and MerT), which transport mercuric ions into the cytoplasm, have been applied in engineering of bioreactor used for mercurial bioremediation. We engineered bacteria to express MerC, MerE, MerF, or MerT with or without MerP to clarify their individual role and potential in transport of mercurial.By immunoblot analysis using specific polyclonal antibody, the proteins encoded by merC, merE, merF, merT or merP, were certainly expressed and identified in the membrane fraction. Bacteria expressing MerC, MerE, MerF or MerT in the absence of MerP transported significantly more C6H5Hg(I) and Hg(II) across bacterial membrane than their isogenic strain. In vivo expression of MerP in the presence of all the transporters did not cause apparent difference to the C6H5Hg(I) transport, but gives a apparently higher Hg(II) transport than that did by MerE, MerF or MerT but not by MerC. Among the four transporters studied, MerC showed more potential to transport Hg(II) across bacterial membrane than MerE, MerF and MerT. Together these findings, we demonstrated for the first time that in addition to MerE and MerT, MerF and MerC are broad-spectrum mercury transporters that mediate both Hg(II) and phenylmercury transport into cells. Our results suggested that MerC is the most efficient tool for designing mercurial bioremediation systems, because MerC is sufficient for mercurial transport into cells.
Bacterial reduction of Hg(2+) to Hg(0) , mediated by the mercuric reductase (MerA), is important in the biogeochemical cycling of Hg in temperate environments. Little is known about the occurrence and diversity of merA in the Arctic. Seven merA determinants were identified amongst bacterial isolates from High Arctic snow, freshwater and sea-ice brine. Three determinants in Bacteriodetes, Firmicutes and Actinobacteria showed < 92% (amino acid) sequence similarity to known merA, while one merA homologue in Alphaproteobacteria and 3 homologues from Betaproteobacteria and Gammaproteobacteria were > 99% similar to known merA's. Phylogenetic analysis showed the Bacteroidetes merA to be part of an early lineage in the mer phylogeny, whereas the Betaproteobacteria and Gammaproteobacteria merA appeared to have evolved recently. Several isolates, in which merA was not detected, were able to reduce Hg(2+) , suggesting presence of unidentified merA genes. About 25% of the isolates contained plasmids, two of which encoded mer-operons. One plasmid was a broad-host-range IncP-α plasmid. No known incompability group could be assigned to the others. The presence of conjugative plasmids, and an incongruent distribution of merA within the taxonomic groups, suggests horizontal transfer of merA as a likely mechanism for High Arctic microbial communities to adapt to changing mercury concentration. This article is protected by copyright. All rights reserved.
Results are described for optimizing the rapid determination of precious metals in geological samples (principally of ophiolitic origin, including chromitites) using an aqua regia[HCl + HNO3(3 + 1)] leach followed by inductively coupled plasma mass spectrometry. Little improvement in extraction efficiency is afforded by aqua regia attack for longer than 30 min and heating the extraction mixture appears to have a detrimental effect on the extraction efficiency of the precious metals. The procedure was evaluated by analysis of a suite of standard reference materials and some independently analysed ophiolitic rock samples. Generally, Au and Pd are quantitatively extracted, Pt, Rh, Ru and Os are extracted to a lower but significant extent (20–40% recovery) and lr is poorly extracted (typically 1–10% recovery). The insolubility of selected platinum group element minerals in aqua regia is considered to be the dominant effect for the non-quantitative recoveries.
Mercury and its compounds are distributed widely across the earth. Many of the chemical forms of mercury are toxic to all living organisms. However, bacteria have evolved mechanisms of resistance to several of these different chemical forms, and play a major role in the global cycling of mercury in the natural environment. Five mechanisms of resistance to mercury compounds have been identified, of which resistance to inorganic mercury (HgR) is the best understood, both in terms of the mechanisms of resistance to mercury and of resistances to heavy metals in general. Resistance to inorganic mercury is encoded by the genes of the mer operon, and can be located on transposons, plasmids and the bacterial chromosome. Such systems have a worldwide geographical distribution, and furthermore, are found across a wide range of both Gram-negative and Gram-positive bacteria from both natural and clinical environments. The presence of mer genes in bacteria from sediment cores suggest that mer is an ancient system. Analysis of DNA sequences from mer operons and genes has revealed genetic variation both in operon structure and between individual genes from different mer operons, whilst analysis of bacteria which are sensitive to inorganic mercury has identified a number of vestigial non-functional operons. It is hypothesised that mer, due to its ubiquity with respect to geographical location, environment and species range, is an ancient system, and that ancient bacteria carried genes conferring resistance to mercury in response to increased levels of mercury in natural environments, perhaps resulting from volcanic activity. Models for the evolution of both a basic mer operon and for the Tn21-related family of mer operons and transposons are suggested. The study of evolution in bacteria has recently become dominated by the generation of phylogenies based on 16S rRNA genes. However, it is important not to underestimate the roles of horizontal gene transfer and recombinational events in evolution. In this respect mer is a suitable system for evaluating phylogenetic methods which incorporate the effects of horizontal gene transfer. In addition, the mer operon provides a model system in the study of environmental microbiology which is useful both as an example of a genotype which is responsive to environmental pressures and as a generic tool for the development of new methodology for the analysis of bacterial communities in natural environments.
The bacterial transposon Tn501 carries, in addition to the genetic information for its own transposition, the genes of the mer operon (in the order merRTPAD), which code for resistance to Hg2+ ions. The basis for the resistance to Hg2+ is the enzymatic reduction of Hg2+ to Hg0 by mercuric reductase, the product of the Tn501 merA gene. We show here that deletion of the merT and merP genes from Tn501 leads to almost complete loss of the resistance phenotype, even if mercuric reductase is still present in the cytoplasm. Expression of the merT and merP genes in the absence of mercuric reductase gives a mercury-supersensitive phenotype. Mercurydependent induction of transcription of the mer operon can occur in the absence of the merT and merP gene products. However, this induction is reduced by the presence of mercuric reductase in the cell. We conclude that one or both of the merT and merP genes of Tn501 are required for the expression of the mercury-resistance phenotype. They may in addition have a role in maximising the induction of the mer operon in the presence of Hg2+ ions. This is consistent with their proposed role in transport of Hg2+ into the cytoplasm.
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.
Polar regions are subject to contamination by mercury (Hg) transported from lower latitudes, severely impacting human and animal health. Atmospheric Mercury Depletion Events (AMDEs) are an episodic process by which Hg is transferred from the atmospheric reservoir to arctic snowpacks. The fate of Hg deposited during these events is the subject of numerous studies, but its speciation remains unclear, especially in terms of environmentally relevant forms such as bioavailable mercury (BioHg). Here, using a bacterial mer-lux biosensor, we report the fraction of newly deposited Hg at the surface and at the bottom of the snowpack that is bioavailable. Snow samples were collected over a two-month arctic field campaign in 2008. In surface snow, BioHg is related to atmospheric Hg deposition and snow fall events were shown to contribute to higher proportions of BioHg than AMDEs. Based on our data, AMDEs represent a potential source of 20 t.y(-1) of BioHg, while wet and dry deposition pathways may provide 135-225 t.y(-1) of BioHg to Arctic surfaces.
A single-use Hg(II) patch biosensor has been developed consisting of 1.25-cm diameter patches of two acrylic vinyl acetate copolymer layers coated on polyester. The top layer copolymer was 47 microm thick whereas the bottom layer of copolymer plus E. coli cells was 30 microm thick. The immobilized E. coli HB101 cells harbored a mer-lux plasmid construct and produced a detectable light signal when exposed to Hg(II). The immobilized-cell Hg(II) biosensor had a sensitivity similar to that of suspended cells but a significantly larger detection range. The levels of mercury detected by the patches ranged from 0.1 nM to 10 000 nM HgCl2 in pyruvate buffer, and luciferase induction as a function of Hg(II) concentration was sigmoidal. Luciferase activity was detected in immobilized cells for more than 78 h after exposure of the cells to HgCl2. Addition of 1 mM D-cysteine to the pyruvate buffer increased luciferase induction more than 100-fold in the immobilized cell patches and 3.5-fold in a comparable suspension culture. The copolymer patches with immobilized cells were stable at -20 degrees C for at least 3 months, and the Hg(II)-induced luciferase activity after storage was similar to that of samples assayed immediately after coating. Patches stored desiccated at room temperature for 2 weeks showed lower mercury-induced luciferase activity when compared to freshly prepared patches, but they still had a considerable detection range of 1 to 10 000 nM HgCl2.
We describe MUSCLE, a new computer program for creating multiple alignments of protein sequences. Elements of the algorithm
include fast distance estimation using kmer counting, progressive alignment using a new profile function we call the log‐expectation score, and refinement using tree‐dependent
restricted partitioning. The speed and accuracy of MUSCLE are compared with T‐Coffee, MAFFT and CLUSTALW on four test sets
of reference alignments: BAliBASE, SABmark, SMART and a new benchmark, PREFAB. MUSCLE achieves the highest, or joint highest,
rank in accuracy on each of these sets. Without refinement, MUSCLE achieves average accuracy statistically indistinguishable
from T‐Coffee and MAFFT, and is the fastest of the tested methods for large numbers of sequences, aligning 5000 sequences
of average length 350 in 7 min on a current desktop computer. The MUSCLE program, source code and PREFAB test data are freely
available at http://www.drive5. com/muscle.
The bacterial and archaeal assemblages at two offshore sites located in polar (Greenland Sea; depth: 50 and 2000 m) and Mediterranean (Ionian Sea; depth 50 and 3000 m) waters were studied by PCR amplification and sequencing of the last 450-500 bp of the 16S rRNA gene. A total of 1621 sequences, together with alignable 16S rRNA gene fragments from the Sargasso Sea metagenome database, were analysed to ascertain variations associated with geographical location and depth. The Ionian 50 m sample appeared to be the most diverse and also had remarkable differences in terms of the prokaryotic groups retrieved; surprisingly, however, many similarities were found at the level of large-scale diversity between the Sargasso database fragments and the Greenland 50 m sample. Most sequences with more than 97% sequence similarity, a value often taken as indicative of species delimitation, were only found at a single location/depth; nevertheless, a few examples of cosmopolitan sequences were found in all samples. Depth was also an important factor and, although both deep-water samples had overall similarities, there were important differences that could be due to the warmer waters at depth of the Mediterranean Sea.
Molecular Cloning: A Laboratory Manual
- M.R. Green
- J. Sambrook
Characterization of mercury resistant and growth promoting Enterobacter sp. from rhizosphere to use as a biofertilizer
- Mobeen N.
Heavy metals and human health: mechanistic insight into toxicity and counter defense system of antioxidants
- A. Jan
- M. Azam
- K. Siddiqui
- A. Ali
- I. Choi
- Q. Haq
Genome sequencing revealed chromium and other heavy metal resistance genes in E. cloacae B2‐Dha
- A. Rahman
- B. Olsson
- J. Jass
- N. Nawani
- S. Ghosh
- A. Mandal