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.

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.

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.

Aerobic Transformation of 2,4-Dinitrotoluene by Escherichia coli and Its Implications for the Detection of Trace Explosives.

DNT (2,4-dinitrotoluene), a volatile impurity in military-grade 2,4,6-trinitrotoluene (TNT)-based explosives, is a potential tracer for the detection of buried landmines and other explosive devices. We have previously described an Escherichia coli bioreporter strain engineered to detect traces of DNT and have demonstrated that the yqjF gene promoter, the sensing element of this bioreporter, is induced not by DNT but by at least one of its transformation products. In the present study, we have characterized the initial stages of DNT biotransformation in E. coli, have identified the key metabolic products in this reductive pathway, and demonstrate that the main DNT metabolite that induces yqjF is 2,4,5-trihydroxytoluene. We further show that E. coli cannot utilize DNT as a sole carbon or nitrogen source and propose that this compound is metabolized in order to neutralize its toxicity to the cells.IMPORTANCE The information provided in this article sheds new light both on the microbial biodegradability of nitroaromatic compounds and on the metabolic capabilities of E. coli By doing so, it also clarifies the pathway leading to the previously unexplained induction of the E. coli yqjF gene by 2,4-dinitrotoluene, an impurity that accompanies 2,4,6-trinitrotoluene (TNT)-based explosives. Our improved understanding of these processes will serve to molecularly enhance the performance of a previously described microbial bioreporter of buried landmines and other explosive devices, in which the yqjF gene promoter serves as the sensing element.

Nanoscale Plasmonic V-Groove Waveguides for the Interrogation of Single Fluorescent Bacterial Cells.

We experimentally demonstrate the interrogation of an individual Escherichia coli cell using a nanoscale plasmonic V-groove waveguide. Several different configurations were studied. The first involved the excitation of the cell in a liquid environment because it flows on top of the waveguide nanocoupler, while the obtained fluorescence is coupled into the waveguide and collected at the other nanocoupler. The other two configurations involved the positioning of the bacterium within the nanoscale waveguide and its excitation in a dry environment either directly from the top or through waveguide modes. This is achieved by taking advantage of the waveguide properties not only for light guiding but also as a mechanical tool for trapping the bacteria within the V-grooves. The obtained results are supported by a set of numerical simulations, shedding more light on the mechanism of excitation. This demonstration paves the way for the construction of an efficient bioplasmonic chip for diverse cell-based sensing applications.

Microbial bioreporters of trace explosives.

Since its introduction as an explosive in the late 19th century, 2,4,6-trinitrotoluene (TNT), along with other explosive compounds, has left numerous environmental marks. One of these is widespread soil and water pollution by trace explosives in military proving grounds, manufacturing facilities, or actual battlefields. Another dramatic impact is that exerted by the millions of landmines and other explosive devices buried in large parts of the world, causing extensive loss of life, injuries, and economical damage. In this review we highlight recent advances in the design and construction of microbial bioreporters, molecularly engineered to generate a quantifiable dose-dependent signal in the presence of trace amounts of explosives. Such sensor strains may be employed for monitoring environmental pollution as well as for the remote detection of buried landmines.

Standoff detection of explosives and buried landmines using fluorescent bacterial sensor cells.

A standoff detection scheme for buried landmines and concealed explosive charges is presented. The detection procedure consists of the following: Live bacterial sensor strains, genetically engineered to produce a dose-dependent amount of green fluorescent protein (GFP) in the presence of explosives' vapors, are encapsulated and spread on the suspected area. The fluorescence produced by the bacteria in response to traces of the explosive material in their microenvironment is remotely detected by a phase-locked optoelectronic sampling system. This scheme enables fast direct access to a large minefield area, while obviating the need to endanger personnel and equipment. Moreover, the employment of phase locking detection efficiently isolates the bacterial sensors' fluorescent output from the background optical signals. This facilitates the application of bacterial sensors in an outdoor environment, where control of background illumination is not possible. Using this system, we demonstrate standoff detection of 2,4-DNT both in aqueous solution and when buried in soil, by sensor bacteria either in liquid culture or agar-immobilized, respectively, at a distance of 50 m in a realistic optically noisy environment.

Genetically engineered microorganisms for the detection of explosives' residues.

The manufacture and use of explosives throughout the past century has resulted in the extensive pollution of soils and groundwater, and the widespread interment of landmines imposes a major humanitarian risk and prevents civil development of large areas. As most current landmine detection technologies require actual presence at the surveyed areas, thus posing a significant risk to personnel, diverse research efforts are aimed at the development of remote detection solutions. One possible means proposed to fulfill this objective is the use of microbial bioreporters: genetically engineered microorganisms "tailored" to generate an optical signal in the presence of explosives' vapors. The use of such sensor bacteria will allow to pinpoint the locations of explosive devices in a minefield. While no study has yet resulted in a commercially operational system, significant progress has been made in the design and construction of explosives-sensing bacterial strains. In this article we review the attempts to construct microbial bioreporters for the detection of explosives, and analyze the steps that need to be undertaken for this strategy to be applicable for landmine detection.

Detection of 2,4-dinitrotoluene and 2,4,6-trinitrotoluene by an Escherichia coli bioreporter: performance enhancement by directed evolution.

The use of bacterial bioreporters for the detection of buried landmines and other explosive devices has been promoted for over 25 years, and several bacterial sensor strains capable of detecting traces of either 2,4,6-trinitrotoluene (2,4,6-TNT) or 2,4-dinitrotoluene (2,4-DNT) have since been reported. In all cases, however, detection sensitivity failed to reach the levels required to reliably sense the minute concentrations of 2,4-DNT vapors expected to exist in the soil above buried landmines. Here, we report on the application of a directed evolution process to enhance the performance of a previously described E. coli-based bioreporter harboring a plasmid-borne genetic fusion between the yqjF gene promoter and either luxCDABE or gfp genes, generating bioluminescent or fluorescent signals, respectively, in the presence of either 2,4,6-TNT or its main "signature" compound, 2,4-DNT. We have performed four sequential rounds of random mutagenesis to the yqjF promoter region, yielding a fourth-generation sensor that displayed significantly improved 2,4-DNT detection characteristics compared to the wild-type and to previous generations. Luminescence intensity in the presence of aqueous 2,4-DNT increased over 3000-fold, response ratios were improved over 50-fold, detection threshold was reduced by 75 %, and response time was cut down to half. These features were manifested also upon exposure to 2,4-DNT vapors or to 2,4-DNT and 2,4,6-TNT buried in sand. An analysis of the point mutations accumulated in the course of this process indicated that the major contributors to these effects were manipulations of the -35 element of the yqjF gene promoter.

Water pollutant monitoring by a whole cell array through lens-free detection on CCD.

Environmental contamination has become a serious problem to human and environmental health, as exposure to a wide range of possible contaminants continuously increases due to industrial and agricultural activities. Whole cell sensors have been proposed as a powerful tool to detect class-specific toxicants based upon their biological activity and bioavailability. We demonstrated a robust toxicant detection platform based on a bioluminescence whole cell sensor array biochip (LumiChip). LumiChip harbors an integrated temperature control and a 16-member sensor array, as well as a simple but highly efficient luminescence collection setup. On LumiChip, samples were infused in an oxygen-permeable microfluidic flow channel to reach the sensor array. Time-lapse changes in bioluminescence emitted by the array members were measured on a single window-removed linear charge-coupled device (CCD) commonly used in commercial industrial process control or in barcode readers. Removal of the protective window on the linear CCD allowed lens-free direct interfacing of LumiChip to the CCD surface for measurement with high light collection efficiency. Bioluminescence induced by simulated contamination events was detected within 15 to 45 minutes. The portable LumiSense system utilizing the linear CCD in combination with the miniaturized LumiChip is a promising potential platform for on-site environmental monitoring of toxicant contamination.

A miniature porous aluminum oxide-based flow-cell for online water quality monitoring using bacterial sensor cells.

The use of live bacterial reporters as sensing entities in whole-cell biosensors allows the investigation of the biological effects of a tested sample, as well as the bioavailability of its components. Here we present a proof of concept for a new design for online continuous water monitoring flow-cell biosensor, incorporating recombinant reporter bacteria, engineered to generate an optical signal (fluorescent or bioluminescent) in the presence of the target compound(s). At the heart of the flow-cell is a disposable chip made of porous aluminum oxide (PAO), which retains the sensor microorganisms on its rigid planar surface, while its high porosity allows an undisturbed access both to the sample and to essential nutrients. The ability of the bacterial reporters to detect model toxic chemicals was first demonstrated using a "naked" PAO chip placed on solid agar, and later in a chip encased in a specially designed flow-through configuration which enables continuous on-line monitoring. The applicability of the PAO chip to simultaneous online detection of diverse groups of chemicals was demonstrated by the incorporation of a 6-member sensor array into the flow-through chip. The selective response of the array was also confirmed in spiked municipal wastewater effluents. Sensing activity was retained by the bacteria after 12-weeks storage of freeze-dried biochips, demonstrating the biochip potential as a simple minimal maintenance "plug-in" cartridge. This low-cost and easy to handle PAO-based flow-cell biosensor may serve as a basis for a future platform for water quality monitoring.

Molecular manipulations for enhancing luminescent bioreporters performance in the detection of toxic chemicals.

Microbial whole-cell bioreporters are genetically modified microorganisms that produce a quantifiable output in response to the presence of toxic chemicals or other stress factors. These bioreporters harbor a genetic fusion between a sensing element (usually a gene regulatory element responsive to the target) and a reporter element, the product of which may be quantitatively monitored either by its presence or by its activity. In this chapter we review genetic manipulations undertaken in order to improve bioluminescent bioreporter performance by increasing luminescent output, lowering the limit of detection, and shortening the response time. We describe molecular manipulations applied to all aspects of whole-cell bioreporters: the host strain, the expression system, the sensing element, and the reporter element. The molecular construction of whole-cell luminescent bioreporters, harboring fusions of gene promoter elements to reporter genes, has been around for over three decades; in most cases, these two genetic elements are combined "as is." This chapter outlines diverse molecular manipulations for enhancing the performance of such sensors.

Escherichia [corrected] coli ribose binding protein based bioreporters revisited.

Bioreporter bacteria, i.e., strains engineered to respond to chemical exposure by production of reporter proteins, have attracted wide interest because of their potential to offer cheap and simple alternative analytics for specified compounds or conditions. Bioreporter construction has mostly exploited the natural variation of sensory proteins, but it has been proposed that computational design of new substrate binding properties could lead to completely novel detection specificities at very low affinities. Here we reconstruct a bioreporter system based on the native Escherichia coli ribose binding protein RbsB and one of its computationally designed variants, reported to be capable of binding 2,4,6-trinitrotoluene (TNT). Our results show in vivo reporter induction at 50 nM ribose, and a 125 nM affinity constant for in vitro ribose binding to RbsB. In contrast, the purified published TNT-binding variant did not bind TNT nor did TNT cause induction of the E. coli reporter system.

Mixtures of chemical pollutants at European legislation safety concentrations: how safe are they?

The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant, and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on 11 organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity, iii) immobilization in the crustacean Daphnia magna, iv) fish embryo toxicity, v) impaired frog embryo development, and vi) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentrations.

Escherichia coli bioreporters for the detection of 2,4-dinitrotoluene and 2,4,6-trinitrotoluene.

The primary explosive found in most land mines, 2,4,6-trinitrotoluene (2,4,6-TNT), is often accompanied by 2,4-dinitrotoluene (2,4-DNT) and 1,3-dinitrobenzene (1,3-DNB) impurities. The latter two compounds, being more volatile, have been reported to slowly leak through land mine covers and permeate the soil under which they are located, thus serving as potential indicators for buried land mines. We report on the construction of genetically engineered Escherichia coli bioreporter strains for the detection of these compounds, based on a genetic fusion between two gene promoters, yqjF and ybiJ, to either the green fluorescent protein gene GFPmut2 or to Photorhabdus luminescens bioluminescence luxCDABE genes. These two gene promoters were identified by exposing to 2,4-DNT a comprehensive library of about 2,000 E. coli reporter strains, each harboring a different E. coli gene promoter controlling a fluorescent protein reporter gene. Both reporter strains detected 2,4-DNT in an aqueous solution as well as in vapor form or when buried in soil. Performance of the yqjF-based sensor was significantly improved in terms of detection threshold, response time, and signal intensity, following two rounds of random mutagenesis in the promoter region. Both yqjF-based and ybiJ-based reporters were also induced by 2,4,6-TNT and 1,3-DNB. It was further demonstrated that both 2,4,6-TNT and 2,4-DNT are metabolized by E. coli and that the actual induction of both yqjF and ybiJ is caused by yet unidentified degradation products. This is the first demonstration of an E. coli whole-cell sensor strain for 2,4-DNT and 2,4,6-TNT, constructed using its own endogenous sensing elements.

Simultaneous quantification of the fluorescent responses of an ensemble of bacterial sensors.

Bacterial bioreporters are genetically engineered microbial strains capable of detecting specific chemicals, groups of chemicals or global biological effects such as toxicity or genotoxicity. A scheme for simultaneous selective detection of the fluorescent signals emitted by a bacterial biosensor array, able to detect four different types of toxicants, using a single photodetector (photomultiplier) is presented. The underlying principle of the scheme is to convert the spatially distributed signals from all the elements in the array to temporally distributed frequency multiplexed signals at the output of the photodetector. Experimental proof of this concept is demonstrated in a four-channel system, in which low power (a few tens of picowatts) fluorescent signals produced by the bacterial sensors are measured, while maintaining a wide dynamic range of detection (more than 3 orders of magnitude). Simultaneous monitoring of concentrations down to a few mg/l of different chemicals in a liquid sample is demonstrated.

A bacterial reporter panel for the detection and classification of antibiotic substances.

The ever-growing use of pharmaceutical compounds, including antibacterial substances, poses a substantial pollution load on the environment. Such compounds can compromise water quality, contaminate soils, livestock and crops, enhance resistance of microorganisms to antibiotic substances, and hamper human health. We report the construction of a novel panel of genetically engineered Escherichia coli reporter strains for the detection and classification of antibiotic substances. Each of these strains harbours a plasmid that carries a fusion of a selected gene promoter to bioluminescence (luxCDABE) reporter genes and an alternative tryptophan auxotrophy-based non-antibiotic selection system. The bioreporter panel was tested for sensitivity and responsiveness to diverse antibiotic substances by monitoring bioluminescence as a function of time and of antibiotic concentrations. All of the tested antibiotics were detected by the panel, which displayed different response patterns for each substance. These unique responses were analysed by several algorithms that enabled clustering the compounds according to their functional properties, and allowed the classification of unknown antibiotic substances with a high degree of accuracy and confidence.

Online monitoring of water toxicity by use of bioluminescent reporter bacterial biochips.

We describe a flow-through biosensor for online continuous water toxicity monitoring. At the heart of the device are disposable modular biochips incorporating agar-immobilized bioluminescent recombinant reporter bacteria, the responses of which are probed by single-photon avalanche diode detectors. To demonstrate the biosensor capabilities, we equipped it with biochips harboring both inducible and constitutive reporter strains and exposed it to a continuous water flow for up to 10 days. During these periods we challenged the biosensor with 2-h pulses of water spiked with model compounds representing different classes of potential water pollutants, as well as with a sample of industrial wastewater. The biosensor reporter panel detected all simulated contamination events within 0.5-2.5 h, and its response was indicative of the nature of the contaminating chemicals. We believe that a biosensor of the proposed design can be integrated into future water safety and security networks, as part of an early warning system against accidental or intentional water pollution by toxic chemicals.

Microbial genotoxicity bioreporters based on sulA activation.

A bacterial genotoxicity reporter strain was constructed in which the tightly controlled strong promoter of the Escherichia coli SOS response gene sulA was fused to the alkaline phosphatase-coding phoA reporter gene. The bioreporter responded in a dose-dependent manner to three model DNA-damaging agents-hydrogen peroxide, nalidixic acid (NA), and mitomycin C (MMC)-detected 30-60 min after exposure. Detection thresholds were 0.15 µM for MMC, 7.5 µM for nalidixic acid, and approximately 50 µM for hydrogen peroxide. A similar response to NA was observed when the bioreporter was integrated into a specially designed, portable electrochemical detection platform. Reporter sensitivity was further enhanced by single and double knockout mutations that enhanced cell membrane permeability (rfaE) and inhibited DNA damage repair mechanisms (umuD, uvrA). The rfaE mutants displayed a five- and tenfold increase in sensitivity to MMC and NA, respectively, while the uvrA mutation was advantageous in the detection of hydrogen peroxide. A similar sensitivity was displayed by the double rfaE/uvrA mutant when challenged with the pre-genotoxic agents 2-amino-3-methylimidazo[4,5-f]quinoline and 2-aminoanthracene following metabolic activation with an S9 mammalian liver fraction.