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The overuse and misuse of antibiotics in animal breeding for disease treatment and growth enhancement have been major drivers of the occurrence, diffusion, and accumulation of antibiotic resistance genes (ARGs) in wastewater. Strategies to combat ARG dissemination are pressingly needed for human and ecological safety. To achieve this goal, a biochar-based polymer, magnetic biochar/quaternary phosphonium salt (MBQ), was applied in livestock wastewater and displayed a high performance in bacterial deactivation and ARG decrease. Efficient antibacterial effects were achieved by both MBQ and quaternary phosphonium salt; however, the abundance and fold change of ARGs in the MBQ treatment indicated a more powerful ARG dissemination control than quaternary phosphonium salt. The application of MBQ evidently reduced the microbial diversity and may primarily be responsible for altering the ARG profiles in wastewater. Network, redundancy, and variation partitioning analyses were further employed to reveal that the microbial community and the presence of mobile genetic elements were two critical factors shaping the pattern of the antibiotic resistome in livestock wastewater. Considered together, these findings extend the application field of biochar and have important implications for reducing ARG dissemination risks in livestock wastewater.

Artificial intelligence (AI) coupled with promising machine learning (ML) techniques well known from computer science is broadly affecting many aspects of various fields including science and technology, industry, and even our day-to-day life. The ML techniques have been developed to analyze high-throughput data with a view to obtaining useful insights, categorizing, predicting, and making evidence-based decisions in novel ways, which will promote the growth of novel applications and fuel the sustainable booming of AI. This paper undertakes a comprehensive survey on the development and application of AI in different aspects of fundamental sciences, including information science, mathematics, medical science, materials science, geoscience, life science, physics, and chemistry. The challenges that each discipline of science meets, and the potentials of AI techniques to handle these challenges, are discussed in detail. Moreover, we shed light on new research trends entailing the integration of AI into each scientific discipline. The aim of this paper is to provide a broad research guideline on fundamental sciences with potential infusion of AI, to help motivate researchers to deeply understand the state-of-the-art applications of AI-based fundamental sciences, and thereby to help promote the continuous development of these fundamental sciences., Graphical abstract, Public summary • “Can machines think?” The goal of artificial intelligence (AI) is to enable machines to mimic human thoughts and behaviors, including learning, reasoning, predicting, and so on. • “Can AI do fundamental research?” AI coupled with machine learning techniques is impacting a wide range of fundamental sciences, including mathematics, medical science, physics, etc. • “How does AI accelerate fundamental research?” New research and applications are emerging rapidly with the support by AI infrastructure, including data storage, computing power, AI algorithms, and frameworks.

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 ...

Whole-cell bioreporters are living organisms and thus using them for detecting environmental contaminants would reflect biological effects of these pollutants. However, bioreporters are not widely used in field studies. Many of the bioreporter field protocols are suitable for liquid samples or include pipetting steps, which is a demanding task outside the laboratory. We present a bioreporter protocol without pipetting or sample type requirements. The protocol utilizes polyester swabs, commonly used in cleanroom technology. As an example contaminant, we used tetracycline and generated test samples with known concentrations up to the maximum tetracycline residue limit of milk set by the European Union (EU) regulation. The matrices of the test samples were Milli-Q water, milk and soil. The swabs were first dipped in the bioreporter cell cultures and then to test samples and luminescence was measured after incubation. The standard deviation of measurements from ten replicate swabs was in the same range as commonly in pipetting protocols (4-19%). The test samples with lowest tetracycline concentration (5 ng mL(-1)) were distinguished from the control samples (0 ng mL(-1) tetracycline). Our results show that swabs can be used together with luminescent whole cell bioreporters, making it possible to conduct the measurements in field conditions.

Bioluminescent Saccharomyces cerevisiae yeast- based bioreporters were used to monitor bisphenol A and other estrogenic chemicals in thermal paper samples collected mainly from Finland on two occasions in 2010/2011, and 2013. The bisphenol A-targeted (BPA-R) and the human oestrogen receptor (hERα) bioreporters were applied to ana- lyse both non-treated and extracted paper samples. Bisphenol A was readily bioavailable to the yeast bioreporters on the non-treated paper samples without any pre-treatment. Detect- ed concentrations ranged from a detection limit of 9-142 μg/g to over 20 mg/g of bisphenol A equivalents in the thermal papers. Low bisphenol A likeactivities were detected in many samples, and were considered to be caused by residual bisphenol A or other types of bisphenols, such as bisphenol S. Most of the thermal paper samples were toxic to the yeast bioreporters. The toxicity did not, however, depend on the bisphenol A concentration of the samples. The yeast bioreporters were demonstrated to be a robust and cost- efficient method to monitor thermal paper samples for their bisphenol A content and estrogenicity. Thermal paper was considered as a potential BPA source for both human expo- sure and environmental emission.

The first Saccharomyces cerevisiae yeast bioreporter for analysis of a single endocrine disrupting compound, bisphenol A (BPA), was developed. The bioreporter contains mutated human estrogen receptor α (hERα), called bisphenol A-targeted receptor (BPA-R). The BPA-R bioreporter was characterized with mixtures of estrogenic chemicals and tested with spiked influent wastewater samples. The detection limit for BPA was 4.2-fold lower (0.107 μM, i.e., 24 μg L(-1)), while that of the native hormone 17β-estradiol (E2) (1 μM, i.e., 272 μg L(-1)) was 166,000-fold higher compared to the wild type hERα bioreporter. The BPA-R bioreporter responded only to BPA in a chemical cocktail and spiked concentrated wastewater samples with high concentrations of other estrogenic chemicals. As a conclusion, wastewater and other environmental water samples can be concentrated and specifically analyzed for BPA without risk of the mixture effect caused by other estrogenic chemicals. The BPA-R bioreporter is a robust and cost-efficient choice for high-throughput monitoring of BPA and its bioavailability in complex samples.

Organotin compounds are toxic and endocrine disrupting compounds, which have been intensively used as antifouling paints for ship hulls and thus are widely spread in the environment. They are suspected to cause imposex, the formation of male characteristics in female gastropods, because of the activation of retinoid X receptor (RXR) at very low environmental concentrations. Here we report the development and optimization of a bioluminescent yeast assay for the detection of organotin compounds based on the interaction with a hybrid RXR and subsequent expression of a reporter luciferase gene. This assay is highly specific toward organotin compounds and natural ligands of the RXR. It detects tributyltin and triphenyltin in nanomolar concentrations (detection limits were found to be 30 nM and 110 nM, respectively) and allows small-scale high-throughput analyses. Furthermore it was possible to measure tributyltin directly in untreated spiked sediments. Thus, the results provided within one working day can be used for the assessment of bioavailability and mixture effect of organotin compounds in environmental samples.

Bisphenol A is an endocrine disrupting compound, which is ubiquitous in the environment due to its wide use in plastic and resin production. Seven day old cultures of the litter-decomposing fungus Stropharia coronilla removed the estrogenic activity of bisphenol A (BPA) rapidly and enduringly. Treatment of BPA with purified neutral manganese peroxidase (MnP) from this fungus also resulted in 100% reduction of estrogenic activity, as analyzed using a bioluminescent yeast assay, and in the formation of polymeric compounds. In cultures of Stropharia rugosoannulata, estrogenic activity also quickly disappeared but temporarily re-emerged in the further course of cultivation. LC–MS analysis of the extracted estrogenic culture liquid revealed [M−H]− ions with m/z values of 219 and 235. We hypothesize that these compounds are ring fission products of BPA, which still exhibit one intact hydroxyphenyl group to interact with estrogen receptors displayed by the yeast.

A bacterial biosensor method for the selective determination of a bioavailable organomercurial compound, methylmercury, is presented. A recombinant luminescent whole-cell bacterial strain responding to total mercury content in samples was used. The bacterial cells were freeze-dried and used as robust, reagent-like compounds, without batch-to-batch variations. In this bacteria-based sensing method, luciferase is used as a reporter, which requires no substrate additions, therefore allowing homogenous, real-time monitoring of the reporter gene expression. A noninducible, constitutively light-producing control bacterial strain was included in parallel for determining the overall cytotoxicity of the samples. The specificity of the total mercury sensor Escherichia coli MC1061 (pmerRBlux) bacterial resistance system toward methylmercury is due to a coexpressed specific enzyme, organomercurial lyase. This enzyme mediates the cleavage of the carbon–mercury bond of methylmercury to yield mercury ions, which induce the reporter genes and produce a self-luminescent cell. The selective analysis of methylmercury with the total mercury strain is achieved by specifically chelating the inorganic mercury species from the sample using an optimized concentration of EDTA as a chelating agent. After the treatment with the chelating agent, a cross-reactivity of 0.2% with ionic mercury was observed at nonphysiological ionic mercury concentrations (100 nM). The assay was optimized to be performed in 3 h but results can already be read after 1 h incubation. Total mercury strain E. coli MC1061 (pmerRBlux) has been shown to be highly sensitive and capable of determining methylmercury at a subnanomolar level in optimized assay conditions with a very high dynamic range of two decades. The limit of detection of 75 ng/l (300 pM) allows measurement of methylmercury even from natural samples.

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.

The gene cluster pbrTRABCD from Cupriavidus metallidurans CH34 is thought to encode a unique, specific resistance mechanism for lead. However, the exact functions of these genes are unknown. In this study we examine the metal specificity and functions of pbrABCD by expressing these genes in different combinations and comparing their ability to restore Pb(2+), Zn(2+) and Cd(2+) resistance in a metal-sensitive C. metallidurans strain DN440. We show that lead resistance in C. metallidurans is achieved through the cooperation of the Zn/Cd/Pb-translocating ATPase PbrA and the undecaprenyl pyrophosphate phosphatase PbrB. While PbrA non-specifically exported Pb(2+), Zn(2+) and Cd(2+), a specific increase in lead resistance was observed when PbrA and PbrB were coexpressed. As a model of action for PbrA and PbrB we propose a mechanism where Pb(2+) is exported from the cytoplasm by PbrA and then sequestered as a phosphate salt with the inorganic phosphate produced by PbrB. Similar operons containing genes for heavy metal translocating ATPases and phosphatases were found in several different bacterial species, suggesting that lead detoxification through active efflux and sequestration is a common lead-resistance mechanism.

Recently, we presented a simple method for generating biological functional protein-based nanoparticles that are ready for use as label agents in bioaffinity assays (Jääskeläinen et al., 2007 Small 3:1362-1367). In this process, the particle shell (ferritin protein) and binding molecules are conjugated via genetic fusion, and particles with binding capacity are produced in a single bacterial cultivation. Production is combined with simple, non-chromatographic purification during which Europium ions are introduced into particles to serve as marker agents. Denaturation-refolding has previously performed by means of pH changes. Here, we test urea as an alternative agent for denaturation, and examine techniques to improve refolding of the functional particles. Three different types of binding molecules were employed in our experiments: biotin carboxyl carrier protein (a small protein with 87 amino acids), single chain antibody fragment (a complex binding protein) and calmodulin-binding peptide (27 amino acids). Urea was successfully utilized to generate functional particles with inherent binding activity and label function. Additionally, particle yield was effectively optimized by analyzing various refolding and bacterial production conditions. Our results clearly demonstrate that this simple biological method of producing functional ferritin-based particles is flexible, and different types of binding moieties can be applied by adjusting the production conditions.

According to in silico analysis, the genome of Pseudomonas putida KT2440 encodes at least four Zn/Cd/Pb efflux transporters—two P-type ATPases (CadA1 and CadA2) and two czc chemiosmotic transporters (CzcCBA1 and CzcCBA2). In this study we showed that all these transporters are functional, but under laboratory conditions only two of them were involved in the mediation of heavy metal resistance in P. putida KT2440. CadA2 conferred Cd 2+ and Pb 2+ resistance, whereas CzcCBA1 was involved in export of Zn 2+ , Cd 2+ , and possibly Pb 2+ . CadA1, although nonfunctional in P. putida , improved Zn 2+ resistance and slightly improved Cd 2+ resistance when it was expressed in Escherichia coli . CzcCBA2 contributed to Zn resistance of a czcA1 -defective P. putida strain or when the CzcA2 subunit was overexpressed in a transporter-deficient strain. It seemed that CzcA2 could complex with CzcC1 and CzcB1 subunits and therefore complement the loss of CzcA1. The CzcCBA2 transporter itself, however, did not function. Expression of cadA1 , cadA2 , and czcCBA1 was induced by heavy metals, and the expression levels were dependent on the growth medium and growth phase. Expression of cadA2 and czcCBA1 was nonspecific; both genes were induced by Zn 2+ , Cd 2+ , Pb 2+ , Ni 2+ , Co 2+ , and Hg 2+ . On the other hand, remarkably, expression of cadA1 was induced only by Zn 2+ . Possible roles of distinct but simultaneously functioning transporters are discussed.

Nanoparticles are increasingly used as labels for analytical purposes. In general, nanoparticles need to be functionalized with binding molecules (mostly antibodies or fragments thereof) and label substances using a multistep process that requires several manufacturing and purification steps. Here, we present a biological method of producing functionalized nanoparticles for effective use as label agents in a bioaffinity assay. The particles are based on the globular protein shell of human ferritin. A single chain Fv fragment (scFv) of an antibody is used as the binding moiety and Eu3+ ions as the label substance. Conventional chemical conjugation of the particle and antibody fragment is replaced with genetic fusion between the ferritin subunit and scFv genes. The material, for example, the fusion construct is produced in a single bacterial culture as insoluble forms that are easily purified by centrifugations. The subunits are solubilized and self-assembled, and label ions are introduced by shifting the pH. The functionality of these particles is demonstrated with a bioaffinity assay. This method of producing nanoparticles with inherent antigen binding activity presents several possibilities for the simple production of specific, functional nanoparticles. Production is fast, economical, and environmentally sustainable, making the system advantageous, particularly in applications requiring large quantities of specific nanoparticles.

Release rates of total copper in artificial seawater (without organic matter) from anti-fouling paints of different active copper compounds range from 0.5 to 75 μg cm-2 day-1. Approximately 80% of ...

Fibre-optic biosensors for Hg and As were developed by attaching alginate-immobilised recombinant luminescent Hg- and As-sensor bacteria onto optical fibres. The optimised biosensors (consisting of seven layers of fibre-attached bacteria pre-grown till mid-logarithmic growth phase) enabled quantification of environmentally relevant concentrations of the target analytes: 2.6 μg l −1 of Hg(II) and 141 μg l −1 of As(V) or 18 μg l −1 of As(III). The highest viability and sensitivity for target analyte was obtained when fibre tips were stored in CaCl 2 solution at −80 °C. Applicability of the fibre-optic biosensors in parallel to the respective non-immobilised sensors was assessed on 10 natural soil and sediment samples from Aznalcollar mining area (Spain). On the average 0.2% of the total Hg and 0.87% of the total As proved bioavailable to fibre-attached bacteria. Interestingly, about 20-fold more Hg and 4-fold more As was available to non-immobilised sensor bacteria indicating the importance of direct cell contact (possible only for non-immobilised cells) for enhanced bioavailability of these metals in solid samples.

A whole-cell recombinant bacterial sensor for the detection of phenolic compounds was constructed and used for the analysis of bioavailable phenols in natural samples. The sensor Pseudomonas fluorescens OS8(pDNdmpRlux) contains luxCDABE operon as a reporter under the control of phenol-inducible Po promoter from Pseudomonas sp. CF600. Expression of lux genes from the Po promoter, and thus the production of bioluminescence is controlled by the transcriptional activator DmpR, which initiates transcription in the presence of phenolic compounds. To take into account possible quenching (turbidity, toxicity) and/or stimulating effects of the environmental samples on the bacterial luminescence, control bacteria comparable to the sensors but lacking the phenol recognising elements were constructed and used in parallel in assays. The sensor bacteria were inducible with phenol, methylphenols, 2,3-, 2,4-, 2,6- and 3,4-dimethylphenol, resorcinol and 5-methylresorcinol but not with 2,5-dimethylresorcinol. The detection limits for different phenols varied from 0.03 mg/l (2-methylphenol) to 42.7 mg/l (5-methylresorcinol), being 0.08 mg/l for phenol, the most abundant phenolic contaminant in the environment. Different phenolic compounds had an additive effect on the inducibility of the sensor. The constructed sensor bacteria were applied on groundwaters and semi-coke leachates to estimate the bioavailable fraction of phenols. The sensor-determined amount of phenols in different samples varied from 6% to 95% of total phenol content depending on the nature of the sample. As the phenol-recognising unit in the sensor originates from a natural phenol biodegradation pathway, the sensor-determined amount of phenols corresponds to the biodegradable amount of phenolic pollutants in the samples and therefore this sensor could be used to estimate the natural biodegradation potential of phenolic compounds in the complex environmental mixtures and matrixes.

At the EILATox-Oregon Workshop, nine luminescent whole-cell bacterial sensors were used for the determination of bioavailable metals in blind samples (17 synthetic and 3 environmental). A non-inducible luminescent control strain was used to determine sample matrix effects and bacterial toxicity. Whole-cell bacterial sensors capable of determining arsenic, inorganic mercury and its organic derivatives, cadmium, lead or copper were used in suspensions and a bacterial sensor for the detection of inorganic mercury was immobilized onto fibre-optic tips using calcium alginate. Bioavailable amounts of metals were estimated using calibration plots, that were constructed to determine the range of metals giving rise to a linear relationship between luminescence and the amount of metals present in the standard solutions. EILATox-Oregon sample 5, which contained 74 mg l−1 of Hg, gave a significant response with both formats of the mercury sensor. The bioavailable amounts of mercury according to the measurement of bacterial sensor in suspension and immobilized onto a fibre-optic tip were 76 and 93 mg l−1, respectively. The bacterial sensor for arsenic and copper showed a response with sample 6 (58 mg l−1 of As) and sample 8 (400 mg l−1 of metham sodium), respectively. This study showed that the bacterial sensors in suspension or immobilized onto optical fibres are capable of quantifying bioavailable metals from unknown samples. The measurement protocol of bacterial sensors is simple and possible to perform in the field. Moreover, the samples do not need any pretreatment before analysis. Construction and characterization of the strain for the detection of bioavailable copper are described. Copyright © 2004 John Wiley & Sons, Ltd.

Recombinant luminescent bacterial sensors for the detection of zinc [MC1061(pzntRluc)] and chromate [AE104(pchrBluc)] were constructed. The sensors carry firefly luciferase gene as a reporter under the control of zinc-inducible regulatory unit from Zn resistance system in chromosomal DNA of Escherichia coli or chromate-inducible unit from Ralstonia metallidurans CH34 megaplasmid pMOL28. The response of the sensors was calibrated using Zn 2+ , Cr 2 O 7 2− and CrO 4 2− , respectively. The detection limit of the zinc sensor for zinc was 40 μM (2.6 mg l −1 of Zn) and that of chromate sensor for dichromate 30 nM (1.6 μg l −1 of Cr) and for chromate 50 nM (2.6 μg l −1 of Cr), respectively. The specificity of the two above-mentioned sensors constructed in this study and a mercury-inducible bacterial sensor MC1061(pmerBR BS luc) constructed by us earlier [Anal. Chem. 73 (2001) 5168] was determined by using different heavy metal compounds. The zinc and mercury sensors were not completely specific to the target metals. The zinc sensor was co-inducible with cadmium and mercury and the mercury sensor with cadmium. The chromate sensor was inducible not only by chromate but also with Cr 3+ . The bacterial sensors constructed were used for the estimation of bioavailable fraction of heavy metals in soils spiked with different amounts of zinc, cadmium, mercury and chromate. Both soil–water suspensions and soil–water extracts were analyzed. The results showed that the majority of heavy metals remained adsorbed to soil particles: only 0.6% of Cd, 1.3% of Hg, 2% of Zn and 46% of Cr (VI) were available to the bacterial sensors in soil–water extracts. However, when the soil–water suspensions were analyzed, approximately 20 times more cadmium and 30 times more mercury (12 and 40%, respectively) was available for the sensor bacteria whereas the available fractions of zinc and chromate in soil–water extract and suspension were similar. Thus, this study showed that in case of cadmium and mercury (but not zinc and chromium) the particle-bound metals were also partially bioavailable.

We have generated microbial sensors for analyzing the presence of various metals or metalloids by recombinant DNA technology. The strains are based on strictly regulated promoters controlling the expression of the firefly luciferase gene in microbial cells. The regulator-reporter constructs are located in shuttle plasmids capable of replicating in gram-negative or -positive microbial organisms. The sensors developed are real-time indicators of metal responsive gene expression giving results in approximately 30 min, with optimal induction times ranging from 60 to 240 min. We describe here the performance of these metal sensing bacteria for the assessment of different water samples spiked with lead, arsenic, mercury or cadmium. We show that these bacteria are sensitive detectors of metal bioavailability, which is difficult or even impossible to measure by traditional analytical chemistry methods. All measurements were done using freeze-dried bacteria, which makes these sensors reagent-like and also easy to use in field conditions.

The suitability of a luminescent bacterial sensor strain Escherichia coli MC1061(pTOO11) [Virta, M.; Lampinen, J.; Karp, M. Anal Chem 1995, 67, 667-669] for the measuring of mercury from sediment samples was evaluated. The sensor strain is based on the control of expression of a reporter gene, firefly luciferase, by a mercury sensitive regulation unit. The sensor responds to mercury by increased luminescence as a consequence of increased production of the reporter protein luciferase. The method is simple to perform since the luminescence is recorded with a portable luminometer and the sensor bacteria are freeze-dried. The results obtained from river sediment samples were compared with the total mercury content of the samples, which was measured by atomic absorption spectrometry and Leco® Mercury analyzer and the modified photobacteria luminescence inhibition test (Lappalainen, J.; Juvonen, R.; Vaajasaari, K.; Karp, M. Chemosphere 1999, 38, 1069-1083). The correlation between the bacterial sensor results with the total mercury content, ranging from 0.01 mg/kg to 16 mg/kg, was significant with 32 samples tested (R 2 up to 0.8115). There was no correlation between the total mercury content and toxicity measured with Vibrio fischeri in this sample panel.

A sensor plasmid was constructed by inserting the regulation unit from the cadA determinant of plasmid pI258 to control the expression of firefly luciferase. The resulting sensor plasmid pTOO24 is capable of replicating in Gram-positive and Gram-negative bacteria. The expression of the reporter gene as a function of added extracellular heavy metals was studied in Staphylococcus aureus strain RN4220 and Bacillus subtilis strain BR151. Strain RN4220(pTOO24) mainly responded to cadmium, lead and antimony, the lowest detectable concentrations being 10 nM, 33 nM and 1 nM respectively. Strain BR151(pTOO24) responded to cadmium, antimony, zinc and tin at concentrations starting from 3.3 nM, 33 nM, 1 microM, and 100 microM, respectively. The luminescence ratios between induced and uninduced cells, the induction coefficients, of strains RN4220(pTOO24) and BR151(pTOO24) were 23-50 and about 5, respectively. These results were obtained with only 2-3 h incubation times. Freeze-drying of the sensor strains had only moderate effects on the performance with respect to sensitivity or induction coefficients.

In this article, we present a new bioluminescent test system for the screening of chemical compounds with an inhibitory effect on protein synthesis. The test is based on the measurement of real-time in vivo light production by Escherichia coli strains expressing different luciferase genes. The eukaryotic lucGR gene from Pyrophorus plagiophthalamus was found to be the best of three types of luciferase genes tested. Chemicals with known inhibitory effects on protein synthesis were used as test chemicals together with some general toxicants. The incubation of a test chemical with cells was performed either prior to or after the induction of protein synthesis, and the difference in the results of the two methods distinguishes the possible influence on protein synthesis from direct metabolic inhibition. Using lyophilized bacteria, the test is performed in less than an hour without any bacterial cultivation, which makes the test suitable for rapid and sensitive screening of chemicals or environmental samples. Compared with the standardized 50% inhibitory concentration calculation method of the bioluminescent cytotoxicity test, the more direct approach of calculation developed in this study proved to be more convenient than and as reliable as the standard method.

Microbes in sediments below fish farming cages are exposed to a variety of compounds used in aquaculture, such as antifoulants, antimicrobials and mercury. Here, we assessed the abundance of the mercury resistance gene merA in the sediment of four fish farms and four pristine sites using quantitative PCR, and at three fish farms and two pristine sites by constructing clone libraries and sequencing merA amplicons. The abundance of merA was consistently greater at fish farm sites than at pristine sites, and the merA phylotypes were different between fish farm and pristine sites. Mercury concentrations were too low to cause selection pressure and did not differ significantly among samples. The primers designed specifically for this study were capable of detecting two new potential merA clades.

Whole-cell bioluminescent (BL) bioreporter technology is a useful analytical tool for developing biosensors for environmental toxicology and preclinical studies. However, when applied to real samples, several methodological problems prevent it from being widely used. Here, we propose a methodological approach for improving its analytical performance with complex matrix. We developed bioluminescent Escherichia coli and Saccharomyces cerevisiae bioreporters for copper ion detection. In the same cell, we introduced two firefly luciferases requiring the same luciferin substrate emitting at different wavelengths. The expression of one was copper ion specific. The other, constitutively expressed, was used as a cell viability internal control. Engineered BL cells were characterized using the noninvasive gravitational field-flow fractionation (GrFFF) technique. Homogeneous cell population was isolated. Cells were then immobilized in a polymeric matrix improving cell responsiveness. The bioassay was performed in 384-well black polystyrene microtiter plates directly on the sample. After 2 h of incubation at 37 °C and the addition of the luciferin, we measured the emitted light. These dual-color bioreporters showed more robustness and a wider dynamic range than bioassays based on the same strains with a single reporter gene and that uses a separate cell strain as BL control. The internal correction allowed to accurately evaluate the copper content even in simulated toxic samples, where reduced cell viability was observed. Homogenous cells isolated by GrFFF showed improvement in method reproducibility, particularly for yeast cells. The applicability of these bioreporters to real samples was demonstrated in tap water and wastewater treatment plant effluent samples spiked with copper and other metal ions.

Whole-cell bioreporters are living microorganisms that produce a specific, quantifiable output in response to target chemicals. Typically, whole-cell bioreporters combine a sensor element for the substance of interest and a reporter element coding for an easily detectable protein. The sensor element is responsible for recognizing the presence of an analyte. In the case of metal bioreporters, the sensor element consists of a DNA promoter region for a metal-binding transcription factor fused to a promoterless reporter gene that encodes a signal-producing protein. In this review, we provide an overview of specific whole-cell bioreporters for heavy metals. Because the sensing of metals by bioreporter microorganisms is usually based on heavy metal resistance/homeostasis mechanisms, the basis of these mechanisms will also be discussed. The goal here is not to present a comprehensive summary of individual metal-specific bioreporters that have been constructed, but rather to express views on the theory and applications of metal-specific bioreporters and identify some directions for future research and development.

Baker's yeast, Saccharomyces cerevisiae, is the simplest and most well-known representative of eukaryotic cells and thus a convenient model organism for evaluating toxic effects in human cells and tissues. Yeast cell sensors are easy to maintain with short generation times, which makes the analytical method of assessing antifungal toxicity cheap and less-time consuming. In this work, the toxicity of test compounds was assessed in bioassays based on bioluminescence inhibition and on traditional growth inhibition on agar plates. The model organism in both tests was a modified S. cerevisiae sensor strain that produces light when provided with D-luciferin in an insect luciferase reporter gene activity assay. The bioluminescence assay showed toxic effects for yeast cell sensor of 5,6-benzoflavone, rapamycin, nystatin and cycloheximide at concentrations of nM to mu M. In addition, arsenic compounds, cadmium chloride, copper sulfate and lead acetate were shown to be potent non-specific inhibitors of the reporter organism described here. The results from a yeast agar diffusion assay correlated with the bioluminescence assay results. Baker's yeast, Saccharomyces cerevisiae, is the simplest and most well-known representative of eukaryotic cells and thus a convenient model organism for evaluating toxic effects in human cells and tissues. Yeast cell sensors are easy to maintain with short generation times, which makes the analytical method of assessing antifungal toxicity cheap and less-time consuming. In this work, the toxicity of test compounds was assessed in bioassays based on bioluminescence inhibition and on traditional growth inhibition on agar plates. The model organism in both tests was a modified S. cerevisiae sensor strain that produces light when provided with D-luciferin in an insect luciferase reporter gene activity assay. The bioluminescence assay showed toxic effects for yeast cell sensor of 5,6-benzoflavone, rapamycin, nystatin and cycloheximide at concentrations of nM to mu M. In addition, arsenic compounds, cadmium chloride, copper sulfate and lead acetate were shown to be potent non-specific inhibitors of the reporter organism described here. The results from a yeast agar diffusion assay correlated with the bioluminescence assay results. Baker's yeast, Saccharomyces cerevisiae, is the simplest and most well-known representative of eukaryotic cells and thus a convenient model organism for evaluating toxic effects in human cells and tissues. Yeast cell sensors are easy to maintain with short generation times, which makes the analytical method of assessing antifungal toxicity cheap and less-time consuming. In this work, the toxicity of test compounds was assessed in bioassays based on bioluminescence inhibition and on traditional growth inhibition on agar plates. The model organism in both tests was a modified S. cerevisiae sensor strain that produces light when provided with D-luciferin in an insect luciferase reporter gene activity assay. The bioluminescence assay showed toxic effects for yeast cell sensor of 5,6-benzoflavone, rapamycin, nystatin and cycloheximide at concentrations of nM to mu M. In addition, arsenic compounds, cadmium chloride, copper sulfate and lead acetate were shown to be potent non-specific inhibitors of the reporter organism described here. The results from a yeast agar diffusion assay correlated with the bioluminescence assay results.

Changes in chemical speciation of copper and the capacity of concrete pavement to retain copper in runoff water from external buildings have been investigated at urban field conditions, and in parallel laboratory experiments simulating outdoor scenarios. The research study showed the concrete surface to form a copper rich surface layer ( approximately 50 microm thick) upon exposure, and a high capacity to significantly reduce the bioavailable fraction of released copper (20-95%). The retention capacity of copper varied between 5 and 20% during single runoff events in the laboratory, and between 10 and 40% of the total copper release during single natural runoff events. The capacity to retain and reduce the bioavailable fraction of non-retained copper increased with increasing wetness of the concrete surfaces, increasing pH of the runoff water and decreasing flow rates. Bioassay testing with bacterial and yeast bioreporters showed the bioavailable fraction of non-retained copper to be significantly lower than the total copper concentration in the runoff water, between 22 and 40% for bacteria and between 8 and 31% for yeast. The application of generated data to simulate a fictive outdoor scenario, suggests a significant reduction of bioavailable and total copper to background values during environmental entry as a result of dilution, and the interaction with solid surfaces, organic matter and complexing agents already in the drainage system.

A scheme of bioassay-directed analysis has been developed which combines a yeast assay screening for estrogenic activity with a liquid chromatographic-mass spectrometric (LC-MS/MS) chemical analysis, chromatographic fractionation, solid phase extraction and freeze-drying. The test scheme was applied on effluent samples collected from a municipal sewage treatment plant. The aim was to determine the substances responsible for main portion of the estrogenic activity in the samples and to compare the efficiency of different procedures for isolation and concentration of estogenicity. LC-MS/MS analyses were used for the quantification of 17beta-estradiol, estrone, estriol and 17alpha-ethinylestradiol, and the measured concentrations compared with the activities found in the yeast assay. Following conversion of the concentrations measured by LC-MS/MS to 17beta-estradiol equivalents it was concluded that freeze-drying, solid phase extraction and the chemical analysis gave comparable activities. Since estrone was the major estrogen in the effluent, this estrogen was also the major contributor to the estrogenic activity in the effluent. The estrogenic activity was equivalent to 4-7 ng/L of 17beta-estradiol. The yeast assay results from the tests of the chromatographic fractions showed that the major activity resides in the fraction where estrone, 17beta-estradiol and 17alpha-ethinylestradiol eluted. The activity of this fraction was substantially higher than the activity of the original wastewater sample. The reason for this could in part be explained by an inhibition of activity occurring in the original water sample.

In the construction of luminescent yeast cell based fibre-optic biosensors, we demonstrate a novel approach for estrogenic endocrine disrupting chemical (EDC) biodetection by entrapping genetically modified Saccharomyces cerevisiae cells, containing the estrogen receptor alpha-mediated expression of the luc reporter gene, in hydrogel matrices based on calcium alginate or PVA. In order to insure a significant signal, an optimal immobilization ratio of 1:2 alginate 3% (w/v): 5 x 10(6) [cells/ml], respectively, was used with the highest 17-beta-estradiol (beta-E2) induction factor after 2.5 h of incubation with 10[nM] beta-E2. It was shown that biocompatible alginate beads, 4.27-4.55 x 10(5) [CFU/bead], which were characterized by a detection limit of 0.08[microg l(-1)] and an EC50 of 0.64[microg l(-1)] for beta-E2, retained their viability for luminescence measurements after 1 month of storage at -80 degrees C slow freeze condition, and thus repeated cell cultivations were not required. The assay reproducibility for each tested EDC, represented by the coefficients of variation (CV), ranged from 4.35 to 18.47%. An alternative immobilization method, based on a room temperature partial drying of polyvinyl alcohol (PVA) solution (LentiKat Liquid) and cell suspension mix, was investigated with only a slightly lower detection limit for beta-E2 than that reported with alginate beads. Alginate yeast based hydrogels may also be applicable to the analysis of environmental water samples since the trend of detected estrogenic activities with alginate beads roughly correlated with LC-MS-MS analytical results.

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The Flash test, which can be used to measure the toxicity from clear, turbid and coloured samples with identical protocol, was used in the EILATox-Oregon Workshop. During the workshop, 17 synthetic samples and three environmental samples were tested. In the Flash method the photobacteria Vibrio fischeri are initially dispensed on top of the sample and the light output of the bioluminescence reaction is recorded at a rate of several readings per second. The change in the light production was measured at two points: 30 s and 30 min after exposure at room temperature. Toxicity calculations were done by using either the peak height of the sample or the peak height of the control sample (maximum signal at 0–2 s). When the peak height of the sample is used as a reference, the colour and turbidity effect of solid particles are taken into account throughout the whole measurement period. In the EILATox-Oregon Workshop toxicity was seen in samples 1 (chlordimeform), 4 (phosdrin), 5 (HgCl2), 6 (sodium arsenite), 8 (metham sodium), 12 (p-chlorophenol), 14 (sodium selenite) and 17 (MNNG). Only samples 1, 4 and 12 showed different ec50 values between different calculations of toxicity. The ec50 values were calculated and compared with the ec50 values of Microtox derived from the literature. The Flash test will not replace the standardized photobacteria test, but is the advanced version of it and is needed when the sample matrix is complex and when speed and low costs are required. Copyright © 2004 John Wiley & Sons, Ltd.

Twenty urban soil samples, with a wide range of Pb (14-5323 mg/kg) and Cu (8-12987 mg/kg), were used to compare the operational speciation of a five-step sequential leach with the bioavailability determined with bioluminescent Pb (RN4220(pTOO24)) and Cu (MC1061(pSLcueR/pDNPcopAluc)) specific bacterial biosensors and a Cu specific yeast sensor. The bioavailable Pb concentrations were all similar or lower than the first sequential leach step (1M NaOAc). In contrast, in some samples the bioavailable concentrations of Cu clearly exceeded even the second sequential leach step (0.1 M Na4P2O7). With the yeast sensor 12/20 samples were below detection, however, the yeast sensor was capable of detecting all high Cu concentrations. The biosensors used in this study are not capable of detecting the natural soil concentrations of Pb and Cu in the studied area.

Estrogen receptor (ER) is a ligand-activated transcriptional factor, able to dimerize after activation and to bind specific DNA sequences (estrogen response elements), thus activating gene target transcription. Since ER homo- and hetero-dimerization (giving a-a and a-b isoforms) is a fundamental step for receptor activation, we developed an assay for detecting compounds that induce human ERa homo-dimerization based on bioluminescence resonance energy transfer (BRET). BRET is a non-radiative energy transfer, occurring between a luminescent donor and a fluorescent acceptor, that strictly depends on the closeness between the two proteins and can therefore be used for studying protein-protein interactions. We cloned ERa coding sequence in frame with either a variant of the green fluorescent protein (enhanced yellow fluorescent protein, EYFP) or Renilla luciferase (RLuc). Upon ERa homo-dimerization, BRET process takes place in the presence of the RLuc substrate coelenterazine resulting in EYFP emission at its characteristic wavelength. The ER alpha-Rluc and ER alpha-EYFP fusion proteins were cloned, then the occurrence of BRET in the presence of ER alpha activators was assayed both in vivo, within cells, and in vitro, with purified fusion proteins.

A new bioluminescence resonance energy transfer (BRET) homogeneous assay to evaluate the presence of estrogen-like compounds has been developed and optimized. The assay is based on the direct evaluation of estrogen alphareceptor (ERalpha) homodimerization as a result of estrogen-like compound binding. ERalpha monomer was genetically fused either to Renilla luciferase (Rluc) or to enhanced yellow fluorescent protein (EYFP). In the presence of estrogens, ERalpha dimerization brings Rluc and EYFP molecules close enough for an energy transfer. An in vitro BRET assay was first developed using purified fusion proteins (ERalpha-Rluc and ERalpha-EYFP) expressed in Escherichia coli to evaluate and optimize the analytical performances of the assay in the presence of 17-beta estradiol. The "in vivo" BRET quantitative assay was then developed by coexpressing the two fusion proteins in live HepG2 cells. The assay can be performed in 96-well microplate format with a 30-min incubation and allows detection with adequate accuracy and precision of as low as 1 nM of 17-beta estradiol. This new "in vivo" BRET assay allows evaluating the estrogen-like activity and synthetic xenoestrogens from biological and environmental samples.

A new biosensor for the determination of bioavailable mercury is presented. It utilizes firefly luciferase gene as a reporter under the control of the mercury-inducible mer promoter from transposon Tn21 and Escherichia coli MC1061 as a host organism. The luminescence-based sensor was evaluated for the selectivity and sensitivity of the detection of mercury. The lowest detectable concentration of mercury was 0.1 tM. Cadmium, zinc, cobalt, copper and manganese ions did not interfere with the measurement even at millionfold concentrations compared to mercury. The results were in agreement with the results obtained by a mercury-independent luciferase expressing strain which acted as a control strain measuring total toxicity. The measurement of the bioavailability of metals is quite difficult with traditional analytical methods. The bioavailability of a metal is a very important factor in the determination of a metal toxicity,' and therefore, the detection of bioavailable metals is of great interest. Biosensors provide a promising way to assess bioavailability of different substances in the environment2 Microbes offer a cornucopia for the construction of biosensors for monitoring the status of the environment. Numerous unspecific sensors that react to any kind of toxic substance have been developed.2 A novel approach for a microbial biosensor is to connect a strictly regulated promoter to a sensitive reporter gene. The most interesting promoters for environmental analysis are found in bacteria that survive in environments contaminated by, for example, heavy metals or organic compounds. The ability of the bacteria to survive in a contaminated environment is usually based on a genetically encoded resistance system, the expression of which is regulated very preci~ely.~ Some biosensors using the promoter-reporter gene concept have been developed, for example, for the detection of mercws5 and xenobiotic com pound^.^^^ However, these approaches have been limited by the lack of sensitivity (parts per trillion, pptr) required for a biosensor that is intended to be used as an early warning system.

Environmental hazard of heavy metals in soils depends to a large extent on their bioavailability. The approach used in this study enables the determination of bioavailable metals in solid-phase samples. Two recombinant bacterial sensors, one responding specifically to cadmium and the other to lead and cadmium by increase of luminescence (firefly luciferase was used as a reporter) were used to determine the bioavailability of these metals in soil–water suspensions (a contact assay) and respective particle-free extracts. Fifty agricultural soils sampled near zinc and lead smelters in the Northern France containing up to (mg/kg) 20.1 of Cd, 1050 of Pb and 1390 of Zn were analysed. As the soil matrix interferes with the assay, recombinant luminescent control bacteria lacking the metal recognizing protein and corresponding promoter (thus, being not metal-inducible) but otherwise comparable to the sensor bacteria (the same host bacterium and plasmid encoding luciferase) were used in parallel to take into account the possible quenching and/or stimulating effects of the sample on the luminescence of the sensor bacteria. Both, chemical and sensor analysis showed that only μg/l levels of metals were extracted from the soil into the water phase (0.1% of the total Cd, 0.07% of Pb and 0.5% of Zn). However, 115-fold more Cd and 40-fold more Pb proved bioavailable if the sensor bacteria were incubated in soil suspensions (i.e., in the contact assay). The bioavailability of metals in different soils varied (depending probably on soil type) ranging from 0.5% to 56% for cadmium and from 0.2% to 8.6% for lead.

The bioavailable arsenic (As) content of contaminated soils was determined by joint analyses of acid-soluble, total water-soluble, and biovailable As by using a luminescent bacterial sensor, Escherichia coli MC1061(pTOO31). According to the results of this study, a significant positive correlation was found between the concentration of total water-soluble As and the bioavailability of As. However, the bioavailability of As in soil varied between sampling sites and was not equal when compared to the concentration of total water-soluble As; bioavailable As was 3 to 77% of total water-soluble As in soil. Our experiments also showed that aging and sequestration of As occurs in contaminated soils and As compounds thus become progressively less bioavailable with time. As a consequence, the bioavailability and toxicity of As should be considered when evaluating the ecological risks of contaminated soils.

Mercury and its organic compounds, especially methylmercury, are hazardous compounds that concentrate in biota via biomagnification and cause severe neurological disorders in animals. In this paper, a recombinant whole-cell bacterial sensor for the detection of the organic compounds of mercury was constructed. The sensor carries firefly luciferase gene as a reporter under the control of the mercury-inducible regulatory part of broad spectrum mer operon from pDU1358. In addition, a gene-encoding organomercurial lyase (an enzyme necessary for cleavage of the mercury-carbon bond) was coexpressed in the sensor strain. The sensitivity of the sensor was evaluated on some environmentally important organomercurial compounds. The lowest detectable concentrations were 0.2 nM (50 ng/L), 1 nM (0.34 microg/L), and 10 microM (2.3 mg/L) for methylmercury chloride, phenylmercury acetate, and dimethylmercury, respectively. The sensor responded also to inorganic mercury and, therefore, using the sensor described here together with sensor bacteria responding only to inorganic mercury, it should be possible to characterize the mercury contamination, for example, in environmental samples.

We have generated new sensors for the specific detection and studies of bioavailability of metals by engineering Pseudomonas fluorescens with reporter gene systems. One broad host range mercury (pTPT11) and two arsenite (pTPT21 and pTPT31) sensor plasmids that express metal presence by luminescence phenotype were constructed and transferred into Escherichia coli DH5a and Pseudomonas fluorescens OS8. The maximal induction was reached after 2 h of incubation in metal solutions at room temperature (22 degrees C). In optimized conditions the half maximal velocity of reaction was achieved at acidic pH using a d-luciferin substrate concentration that was nearly sixfold lower for P. fluorescens OS8 than for E. coli DH5a. When using a luciferin concentration (150 mM) that was optimal for E. coli the luminescence declined rapidly in the case of Pseudomonas, for which the substrate level 25 mM gave a stable reading between about 20 min and 3 h. The ability of the strain OS8 to quantitatively detect specific heavy metals in spiked soil and soil extracts is as good, or even better in being a real-time reporter system, than that of a traditional chemical analysis. The Pseudomonas strain used is an isolate from pine rhizosphere in oil and heavy metal contaminated soil. It is also a good humus soil colonizer and is therefore a good candidate for measuring soil heavy metal bioavailability.

Luminescent bacterial strains for the measurement of bioavailable arsenite and antimony were constructed. The expression of firefly luciferase was controlled by the regulatory unit of the ars operon of Staphylococcus aureus plasmid pI258 in recombinant plasmid pTOO21, with S. aureus RN4220, Bacillus subtilis BR151, and Escherichia coli MC1061 as host strains. Strain RN4220(pTOO21) was found to be the most sensitive for metal detection responding to arsenite, antimonite, and cadmium, the lowest detectable concentrations being 100, 33, and 330 nM, respectively. Strains BR151(pTOO21) and MC1061(pTOO21) responded to arsenite, arsenate, antimonite, and cadmium, the lowest detectable concentrations being 3.3 and 330 microM and 330 and 330 nM with BR151(pTOO21), respectively, and 3.3, 33, 3.3, and 33 microM with MC1061(pTOO21), respectively. In the absence of the mentioned ions, the expression of luciferase was repressed and only a small amount of background light was emitted. Other ions did not notably interfere with the measurement in any of the strains tested. Freeze-drying of the cells did not decrease the sensitivity of the detection of arsenite; however, the induction coefficients were somewhat lower.

The antimicrobial activities of two nitric oxide-releasing compounds against Escherichia coli were investigated by using recombinant E. coli cloned with a luciferase gene from Pyrophorus plagiophthalamus. Since luciferase uses intracellular ATP to generate visible light which can be measured from living cells in real time, we wanted to compare the extent to which cell viability parallels light emission. Results from luminescence measurements and CFU counts were in good agreement, and the decrease in light emission was shown to provide a rapid and more sensitive indication of cytotoxicity.

A panel of luminescent Saccharomyces cerevisiae cell-based nuclear receptor assays, consisting of human estrogen receptors α and β, androgen receptor, and aryl hydrocarbon receptor, was miniaturized from the standard 96-well microplate format to high-throughput 384- and 1536-well microplate formats. In these assays, firefly luciferase lacking the peroxisome targeting sequence was used as a reporter and D-luciferin substrate was pre-mixed with the yeast cells before the incubation step, eliminating cell lysis and substrate addition steps, and allowing multiple readings at any desired time point. All of the assays were highly functional in the 384-well format, and most functioned well in the 1536-well format. The detection limit of the estrogen receptor α assay was even lower in the miniaturized microplate formats than in the original 96-well format. The panel of yeast-cell-based nuclear receptor assays can be used for high-throughput chemical testing and environmental monitoring of potential endocrine-disrupting activity of compounds and samples.