Enhanced Detection of Estrogen-like Compounds by Genetically Engineered Yeast Sensor Strains.

The release of endocrine-disrupting compounds (EDCs) to the environment poses a health hazard to both humans and wildlife. EDCs can activate or inhibit endogenous endocrine functions by binding hormone receptors, leading to potentially adverse effects. Conventional analytical methods can detect EDCs at a high sensitivity and precision, but are blind to the biological activity of the detected compounds. To overcome this limitation, yeast-based bioassays have previously been developed as a pre-screening method, providing an effect-based overview of hormonal-disruptive activity within the sample prior to the application of analytical methods. These yeast biosensors express human endocrine-specific receptors, co-transfected with the relevant response element fused to the specific fluorescent protein reporter gene. We describe several molecular manipulations of the sensor/reporter circuit in a Saccharomyces cerevisiae bioreporter strain that have yielded an enhanced detection of estrogenic-like compounds. Improved responses were displayed both in liquid culture (96-well plate format) as well as in conjunction with sample separation using high-performance thin-layer chromatography (HPTLC). The latter approach allows for an assessment of the biological effect of individual sample components without the need for their chemical identification at the screening stage.

Performance upgrade of a microbial explosives' sensor strain by screening a high throughput saturation library of a transcriptional regulator.

We present a methodology for a high-throughput screening (HTS) of transcription factor libraries, based on bacterial cells and GFP fluorescence. The method is demonstrated on the Escherichia coli LysR-type transcriptional regulator YhaJ, a key element in 2,4-dinitrotuluene (DNT) detection by bacterial explosives' sensor strains. Enhancing the performance characteristics of the YhaJ transcription factor is essential for future standoff detection of buried landmines. However, conventional directed evolution methods for modifying YhaJ are limited in scope, due to the vast sequence space and the absence of efficient screening methods to select optimal transcription factor mutants. To overcome this limitation, we have constructed a focused saturation library of ca. 6.4 × 107 yhaJ variants, and have screened over 70 % of its sequence space using fluorescence-activated cell sorting (FACS). Through this screening process, we have identified YhaJ mutants exhibiting superior fluorescence responses to DNT, which were then effectively transformed into a bioluminescence-based DNT detection system. The best modified DNT reporter strain demonstrated a 7-fold lower DNT detection threshold, a 45-fold increased signal intensity, and a 40 % shorter response time compared to the parental bioreporter. The FACS-based HTS approach presented here may hold a potential for future molecular enhancement of other sensing and catalytic bioreactions.

Miniaturized bioluminescent whole-cell sensor systems.

Relying on the biological responses and activity of living cells, bioluminescent whole-cell biosensors generate an optical signal in response to the presence of target compounds. The miniaturization of low-light detectors and their integration with microfluidics have allowed the realization of portable devices for sensitive imaging and quantification of these signals. This review thus focuses on bioluminescence-based whole-cell biosensors, integrated with handheld optical detectors, with an emphasis on the use of the low-light imaging capability of modern smartphones. We highlight state-of-the-art miniaturization techniques applied to the incorporation of living cells into diverse hardware platforms, and to the sensitive recording of the biologically generated photons. The integration of sample introduction, host-cell activity maintenance, luminescence acquisition, data processing/presentation, and portability are also discussed.

Introduction of quorum sensing elements into bacterial bioreporter circuits enhances explosives' detection capabilities.

A possible solution for the standoff detection of buried landmines is based on the use of microbial bioreporters, genetically engineered to emit a remotely detectable optical signal in response to trace amounts of explosives' signature chemicals, mostly 2,4-dinitrotoluene (DNT). Previously developed DNT sensor strains were based on the fusion of a DNT-inducible gene promoter to a reporting element, either a fluorescent protein gene or a bacterial bioluminescence gene cassette. In the present study, a different approach was used: the DNT-inducible promoter activates, in Escherichia coli, the quorum-sensing luxI and luxR genes of Aliivibrio fischeri. N-Acyl homoserine lactone (AHL), synthesized by LuxI, combines with LuxR and activates the bioluminescence reporter genes. The resulting bioreporter displayed a dose-dependent luminescent signal in the presence of DNT. Performance of the sensor strain was further enhanced by manipulation of the sensing element (combining the E. coli DNT-inducible azoR and yqjF gene promoters), by replacing the luminescence gene cassette of Photorhabdus luminescens luxCDABE with A. fischeri luxCDABEG, and by introducing two mutations, eutE and ygdD, into the host strain. DNT detection sensitivity of the final bioreporter was over 340-fold higher than the original construct.

Enhancing DNT Detection by a Bacterial Bioreporter: Directed Evolution of the Transcriptional Activator YhaJ.

Detection of buried landmines is a dangerous and complicated task that consumes large financial resources and poses significant risks to the personnel involved. A potential alternative to conventional detection methodologies is the use of microbial bioreporters, capable of emitting an optical signal upon exposure to explosives, thus revealing to a remote detector the location of buried explosive devices. We have previously reported the design, construction, and optimization of an Escherichia coli-based bioreporter for the detection of 2,4,6-trinitrotoluene (TNT) and its accompanying impurity 2,4-dinitrotoluene (DNT). Here we describe the further enhancement of this bioreporter by the directed evolution of YhaJ, the transcriptional activator of the yqjF gene promoter, the sensing element of the bioreporter's molecular circuit. This process resulted in a 37-fold reduction of the detection threshold, as well as significant enhancements to signal intensity and response time, rendering this sensor strain more suitable for detecting the minute concentrations of DNT in the soil above buried landmines. The capability of this enhanced bioreporter to detect DNT buried in sand is demonstrated.

A bacterial bioreporter for the detection of 1,3,5-trinitro-1,3,5-triazinane (RDX).

We report the design, construction, and testing of Escherichia coli-based bioluminescent bioreporters for the detection of 1,3,5-trinitro-1,3,5-triazinane (RDX), one of the most prevalent military-grade explosives in use today. These sensor strains are based on a fusion between the promoter of either the hmp (nitric oxide dioxygenase) or the hcp (a high-affinity nitric oxide reductase) E. coli gene, to the microbial bioluminescence luxCDABEG gene cassette. Signal intensity was enhanced in ∆hmp and ∆hcp mutants, and detection sensitivity was improved when the two gene promoters were cloned in tandem. The Photobacterium leiognathi luxCDABEG reporter genes were superior to those of Aliivibrio fischeri in terms of signal intensity, but in most cases inferior in terms of detection sensitivity, due to a higher background signal. Both sensor strains were also induced by additional nitro-organic explosives, as well as by nitrate salts. Sensitive detection of RDX in a solid matrix (either LB agar or sand) was also demonstrated, with the bioreporters encapsulated in 1.5-mm calcium alginate beads. Lowest RDX concentration detected in sand was 1.67 mg/kg sand. The bioreporter strains described herein may serve as a basis for a standoff detection technology of RDX-based explosive devices, including buried landmines.

Estrogenicity of chemical mixtures revealed by a panel of bioassays.

Estrogenic compounds are widely released to surface waters and may cause adverse effects to sensitive aquatic species. Three hormones, estrone, 17ß-estradiol and 17α-ethinylestradiol, are of particular concern as they are bioactive at very low concentrations. Current analytical methods are not all sensitive enough for monitoring these substances in water and do not cover mixture effects. Bioassays could complement chemical analysis since they detect the overall effect of complex mixtures. Here, four chemical mixtures and two hormone mixtures were prepared and tested as reference materials together with two environmental water samples by eight laboratories employing nine in vitro and in vivo bioassays covering different steps involved in the estrogenic response. The reference materials included priority substances under the European Water Framework Directive, hormones and other emerging pollutants. Each substance in the mixture was present at its proposed safety limit concentration (EQS) in the European legislation. The in vitro bioassays detected the estrogenic effect of chemical mixtures even when 17ß-estradiol was not present but differences in responsiveness were observed. LiBERA was the most responsive, followed by LYES. The additive effect of the hormones was captured by ERα-CALUX, MELN, LYES and LiBERA. Particularly, all in vitro bioassays detected the estrogenic effects in environmental water samples (EEQ values in the range of 0.75-304 × EQS), although the concentrations of hormones were below the limit of quantification in analytical measurements. The present study confirms the applicability of reference materials for estrogenic effects' detection through bioassays and indicates possible methodological drawbacks of some of them that may lead to false negative/positive outcomes. The observed difference in responsiveness among bioassays - based on mixture composition - is probably due to biological differences between them, suggesting that panels of bioassays with different characteristics should be applied according to specific environmental pollution conditions.

Bacterial bioreporters for the detection of trace explosives: performance enhancement by DNA shuffling and random mutagenesis.

Landmines and other explosive remnants of war pose a global humanitarian problem that claims numerous casualties long after the conflict has ended. As there are no acceptable methodologies for the remote discovery of such devices, current detection practices still require the risky presence of personnel in the minefield. We have recently described bacterial sensor strains capable of reporting the existence of 2,4-dinitrotoluene (DNT) vapors in the soil above 2,4,6-trinitrotoluene (TNT)-based landmines, by generating a bioluminescent or a fluorescent signal. This may allow the identification of landmine location by remote imaging of an area over which the bacteria have been spread. In the study reported herein, we have improved the DNT-detection capabilities of these sensor strains by combining two DNT-responsive Escherichia coli gene promoters, yqjF and azoR, and subjecting them to three cycles of random mutagenesis by error-prone PCR, combined with segmentation and rearrangement ("DNA shuffling"). The activity of selected modified promoters was evaluated with the Aliivibrio fischeri and Photobacterium leiognathi luxCDABEG gene cassettes as the bioluminescent reporters, exhibiting a ten-fold background reduction that has led to a three-fold decrease in detection threshold. Signal intensity was further enhanced by modifying the ribosomal binding site of the yqjF gene promoter. The superior DNT detection capabilities on a solid matrix by the improved sensor strain were demonstrated. KEY POINTS: • Performance of microbial sensor strains for buried explosives was molecularly enhanced. • Manipulations included random mutagenesis, "DNA shuffling," and RBS reprogramming. • The re-engineered constructs exhibited superior detection of trace explosives.

An autonomous bioluminescent bacterial biosensor module for outdoor sensor networks, and its application for the detection of buried explosives.

We describe a miniaturized field-deployable biosensor module, designed to function as an element in a sensor network for standoff monitoring and mapping of environmental hazards. The module harbors live bacterial sensor cells, genetically engineered to emit a bioluminescent signal in the presence of preselected target materials, which act as its core sensing elements. The module, which detects and processes the biological signal, composes a digital record that describes its findings, and can be transmitted to a remote receiver. The module is an autonomous self-contained unit that can function either as a standalone sensor, or as a node in a sensor network. The biosensor module can potentially be used for detecting any target material to which the sensor cells were engineered to respond. The module described herein was constructed to detect the presence of buried landmines underneath its footprint. The demonstrated detection sensitivity was 0.25 mg 2,4-dinitrotoluene per Kg soil.

Coupling high-performance thin-layer chromatography with a battery of cell-based assays reveals bioactive components in wastewater and landfill leachates.

Over the last two decades, effect-directed analysis (EDA) gained importance as a seminal screening tool for tracking biological effects of environmental organic micro-pollutants (MPs). As EDA using high-performance liquid chromatography and bioassays is costly and time consuming, recent implementations of this approach have combined high-performance thin-layer chromatography (HPTLC) with effect-based methods (EBMs) using cell-based bioassays, enabling the detection of estrogenic, androgenic, genotoxic, photosystem II (PSII)- inhibiting, and dioxin-like sample components on a HPTLC plate. In the present study, the developed methodologies were applied as a HPTLC-based bioassay battery, to investigate toxicant elimination efficiency of wastewater treatment plants (WWTPs), and to characterize the toxic potential of landfill leachates. Activity levels detected in untreated landfill leachates, expressed as reference compound equivalence (EQ) concentration, were up to 16.8 µg ß-naphthoflavone-EQ L-1 (indicating the degree of dioxin-like activity), 1.9 µg estradiol-EQ L-1 (estrogenicity) and 8.3 µg diuron-EQ L­1 (PSII-inhibition), dropping to maximal concentrations of 47 ng ß-naphthoflavone-EQ L-1, 0.7 µg estradiol-EQ L-1 and 53.1 ng diuron-EQ L-1 following treatment. Bisphenol A (BPA) is suggested to be the main contributor to estrogenic activity, with concentrations determined by the planar yeast estrogen screen corresponding well to results from chemical analysis. In the investigated WWTP samples, a decrease of estrogenic activity of 6-100% was observed following treatment for most of the active fractions, except of a 20% increase in one fraction (Rf = 0.568). In contrast, androgenicity with concentrations up to 640 ng dihydrotestosterone-EQ L-1 was completely removed by treatment. Interestingly, genotoxic activity increased over the WWTP processes, releasing genotoxic fractions into receiving waters. We propose this combined HPTLC and EBM battery to contribute to an efficient, cheap, fast and robust screening of environmental samples; such an assay panel would allow to gain an estimate of potential biological effects for prioritization prior to substance identification, and its routine application will support an inexpensive identification of the toxicity drivers as a first tier in an EDA strategy.

Detection of buried explosives with immobilized bacterial bioreporters.

The unchecked dispersal of antipersonnel landmines since the late 19th century has resulted in large areas contaminated with these explosive devices, creating a substantial worldwide humanitarian safety risk. The main obstacle to safe and effective landmine removal is the identification of their exact location, an activity that currently requires entry of personnel into the minefields; to date, there is no commercialized technology for an efficient stand-off detection of buried landmines. In this article, we describe the optimization of a microbial sensor strain, genetically engineered for the remote detection of 2,4,6-trinitrotoloune (TNT)-based mines. This bioreporter, designed to bioluminescence in response to minute concentrations of either TNT or 2,4-dinitotoluene (DNT), was immobilized in hydrogel beads and optimized for dispersion over the minefield. Following modifications of the hydrogel matrix in which the sensor bacteria are encapsulated, as well as their genetic reporting elements, these sensor bacteria sensitively detected buried 2,4-dinitrotoluene in laboratory experiments. Encapsulated in 1.5 mm 2% alginate beads containing 1% polyacrylic acid, they also detected the location of a real metallic antipersonnel landmine under field conditions. To the best of our knowledge, this is the first report demonstrating the detection of a buried landmine with a luminescent microbial bioreporter.

Yeast-Based Fluorescent Sensors for the Simultaneous Detection of Estrogenic and Androgenic Compounds, Coupled with High-Performance Thin Layer Chromatography.

The persistence of endocrine disrupting compounds (EDCs) throughout wastewater treatment processes poses a significant health threat to humans and to the environment. The analysis of EDCs in wastewater remains a challenge for several reasons, including (a) the multitude of bioactive but partially unknown compounds, (b) the complexity of the wastewater matrix, and (c) the required analytical sensitivity. By coupling biological assays with high-performance thin-layer chromatography (HPTLC), different samples can be screened simultaneously, highlighting their active components; these may then be identified by chemical analysis. To allow the multiparallel detection of diverse endocrine disruption activities, we have constructed Saccharomyces cerevisiae-based bioreporter strains, responding to compounds with either estrogenic or androgenic activity, by the expression of green (EGFP), red (mRuby), or blue (mTagBFP2) fluorescent proteins. We demonstrate the analytical potential inherent in combining chromatographic compound separation with a direct fluorescent signal detection of EDC activities. The applicability of the system is further demonstrated by separating influent samples of wastewater treatment plants, and simultaneously quantifying estrogenic and androgenic activities of their components. The combination of a chemical separation technique with an optical yeast-based bioassay presents a potentially valuable addition to our arsenal of environmental pollution monitoring tools.

Genome-wide gene-deletion screening identifies mutations that significantly enhance explosives vapor detection by a microbial sensor.

Genetically engineered microbial biosensors, capable of detecting traces of explosives residues above buried military ordnance and emitting an optical signal in response, may potentially serve for the standoff detection of buried landmines. A promising candidate for such an application is a previously reported Escherichia coli-based reporter strain that employs the yqjF gene promoter as its sensing element; however, for this sensor to be able to detect actual landmines reliably, it was necessary for its detection sensitivity and signal intensity to be enhanced. In this study, a high-throughput approach was employed to screen the effects of individual gene deletions on yqjF activation by 2,4-dinitrotoluene (DNT). Several genes were identified, the deletion of which elicited a significant enhancement of yqjF induction by DNT. The most promising of these mutations were introduced into the sensor strain, individually or in pairs, yielding a considerable increase in signal intensity and a lowering of the detection threshold. A strain harboring two of the identified mutations, ygdD and eutE, appears to be the most sensitive microbial biosensor currently described for the detection of traces of landmine explosives.

An optical detection module-based biosensor using fortified bacterial beads for soil toxicity assessment.

An optical biosensor module for soil contamination assessment is presented, employing bioluminescent bacterial bioreporters encapsulated in poly-dopamine (PD)-coated alginate microbeads. The PD-coated beads displayed improved mechanical strength and stability, but somewhat delayed responses to the inducing toxicant. Using toluene as a model soil contaminant, two bioluminescent reporter strains were employed for its detection in the ambient light-blocking, temperature-controlled biosensor module. Bioluminescence of strain TV1061 (harboring an inducible grpE::luxCDABE fusion) increased and that of strain GC2 (harboring a constitutive lac::luxCDABE fusion) decreased in the presence of increasing toluene concentrations. In the former case, a maximal effect was observed in the presence of 1% toluene. This simple optical detection biosensor module may potentially be utilized for monitoring soil contamination from areas suspected of chemical pollution such petrochemical industrial zones or petrol stations.

Detection and Quantification of Photosystem II Inhibitors Using the Freshwater Alga Desmodesmus subspicatus in Combination with High-Performance Thin-Layer Chromatography.

We present a novel tool for detecting and monitoring photosystem II (PSII) inhibitors, using the freshwater alga Desmodesmus subspicatus, in environmental samples fractionated by high-performance thin-layer chromatography (HPTLC). After chromatographic separation of a sample on a HPTLC plate, the algal suspension is sprayed homogeneously on the plate, and PSII-inhibition by specific sample components is detected based on changes in fluorescence yield, viewed by a maxi Imaging-Pulse-Amplitude-Modulation fluorometer. Dose-dependent responses to the PSII-inhibitor herbicides atrazine and diuron, frequently detected in water bodies, are demonstrated without and with chromatographic separation. The limits of quantification for atrazine and diuron with chromatographic separation were 1.94 ng and 99 pg, respectively, allowing the detection of environmentally relevant concentrations of these herbicides. The developed method was also employed to analyze sample extracts collected during a passive sampling campaign in surface waters. The obtained data correlated well with results from LC-MS/MS chemical analysis but also revealed unknown PSII-inhibiting activities. The proposed methodology represents a rapid and sensitive screening tool for the simultaneous effect-based detection of PSII-inhibitors in environmental samples.

A Smartphone-Based Whole-Cell Array Sensor for Detection of Antibiotics in Milk.

We present an integral smartphone-based whole-cell biosensor, LumiCellSense (LCS), which incorporates a 16-well biochip with an oxygen permeable coating, harboring bioluminescent Escherichia coli bioreporter cells, a macro lens, a lens barrel, a metal heater tray, and a temperature controller, enclosed in a light-impermeable case. The luminescence emitted by the bioreporter cells in response to the presence of the target chemicals is imaged by the phone's camera, and a dedicated phone-embedded application, LCS_Logger, is employed to calculate photon emission intensity and plot it in real time on the device's screen. An alert is automatically given when light intensity increases above the baseline, indicating the presence of the target. We demonstrate the efficacy of this system by the detection of residues of an antibiotic, ciprofloxacin (CIP), in whole milk, with a detection threshold of 7.2 ng/mL. This value is below the allowed maximum as defined by European Union regulations.

Combination of yeast-based in vitro screens with high-performance thin-layer chromatography as a novel tool for the detection of hormonal and dioxin-like compounds.

The combination of classic in vitro bioassays with high-performance thin-layer chromatography (HPTLC) is a promising technique to directly link chemical analysis of contaminants to their potential adverse biological effects. With respect to endocrine disruption, much work is focused on estrogenicity. While a direct combination of HPTLC and the yeast estrogen screen is already developed, it is well accepted that further endocrine effects are relevant for monitoring environmental wellbeing. Here we show that non-estrogenic specific biological endpoints, (partly) related to the endocrine system, can also be addressed by combining respective yeast reporter gene assays with HPTLC to support effect-directed analysis (EDA). These are: androgenicity (YAS), thyroidogenicity (YTS), dioxin-like effects (YDS), effects on the vitamin D (YVS) and the retinoic acid receptor (YRaS). A proof of principle is demonstrated within this study by the characterization of dose-dependent responses to different model compounds for the respective receptors with and without chromatographic development of the HPTLC-plate. Limits of quantification (LOQ) for several model compounds were determined, e.g. 37 pg for testosterone (p-YAS), 0.476 ng for ß-naphthoflavone (p-YDS) and 1.02 ng for calcipotriol hydrate (p-YVS) with chromatographic development. The LOQ for p-YTS and p-YRaS were 10.16 pg for 3,3',5-triiodothyroacetic acid (p-YTS) and 0.41 pg for tamibarotene (p-YRaS), without chromatographic separation. Furthermore, we challenged the developed methodology using environmental samples, demonstrating an elimination efficiency of androgenic activity from municipal wastewater by a wastewater treatment plant between 99.4 and 100%. We anticipate our methodology to substantially broaden the spectrum of specific endpoints combined with HPTLC for an efficient and robust screening of environmental samples to guide a subsequent in-depth EDA.

The involvement of superoxide radicals in medium pressure UV derived inactivation.

Today, two types of lamp systems dominate the UV disinfection industry: low-pressure (LP) UV lamps and medium-pressure (MP) polychromatic lamps. Both lamp types have their advantages and disadvantages in microorganism inactivation, with LP lamps being cheaper, having longer life, and working at lower temperature, hence reducing fouling, and MP lamps showing better inactivation per germicidal dose for certain microorganisms. Bacterium-based biosensors were used to compare LP and MP irradiation. These biosensors were Escherichia coli bacteria carrying the lux operon genes under the control of different stress-responding promoters, where activation of the specific promoter is manifested as bioluminescence. MP irradiation, considerably more than LP irradiation, resulted in activation of the superoxide dismutase expression, indicating the formation of superoxide radicals inside the cells. Accordingly, pre-exposure (immunization) of the bacteria to an activator that produces superoxide radicals resulted in lower inactivation and increased resistance to MP irradiation, but not to LP irradiation. This study shows that the difference in germicidal efficiency may result from the production of intracellular superoxide radicals by MP irradiation, at wavelengths other than 254 nm, as emitted by LP lamps.

Coupling High-Performance Thin-Layer Chromatography with Bacterial Genotoxicity Bioreporters.

We present an innovative technological platform for monitoring the direct genotoxicity of individual components in complex environmental samples, based on bioluminescent Escherichia coli genotoxicity bioreporters, sprayed onto the surface of a high-performance thin-layer chromatography (HPTLC) plate. These sensor strains harbor plasmid-borne fusions of selected gene promoters of the E. coli SOS DNA repair system to the Photorhabdus luminescens luxABCDE gene cassette, and mark by increased luminescence the presence of potentially DNA-damaging sample components separated on the plate. We demonstrate an "on plate" quantifiable dose-dependent response to several model genotoxicants (without metabolic activation). We further demonstrate the applicability of the system by identifying as genotoxic specific components of HPTLC-separated influent and effluent samples of wastewater treatment plants, thereby alleviating the need for a comprehensive chemical analysis of the sample.