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.

Exploring the effects of intermittent aeration on the performance of nitrifying membrane-aerated biofilm reactors.

Membrane-aerated biofilm reactors (MABRs) are an emerging technology for nutrient removal; however, a trade-off remains between their removal rate and oxygen transfer efficiency. This study compares nitrifying flow-through MABRs operated under continuous and intermittent aeration modes at mainstream wastewater ammonia levels. The intermittently-aerated MABRs maintained maximal nitrification rates, including under conditions allowing the oxygen partial pressure on the gas side of the membrane to considerably drop during the no-aeration period. Nitrous oxide emissions of all reactors were comparable and amounted to approximately 20 % of the converted ammonia. Intermittent aeration increased the transformation rate constant of atenolol, yet did not affect the removal of sulfamethoxazole. Seven additional trace organic chemicals were not biodegraded by any of the reactors. The ammonia-oxidizing bacteria in the intermittently-aerated MABRs were dominated by Nitrosospira, previously shown to be abundant at low oxygen concentrations and provide reactor stability under changing conditions. Our findings indicate that intermittently-aerated flow-through MABRs can achieve high nitrification rates and oxygen transfer efficiencies, highlighting the possible implications of air supply discontinuity on nitrous oxide emissions and trace organic chemical biotransformation.

Methanotrophic oxidation of organic micropollutants and nitrogen upcycling in a hybrid membrane biofilm reactor (hMBfR) for simultaneous O2 and CH4 supply.

Pharmaceuticals and other organic micropollutants (OMPs) present in wastewater effluents are of growing concern, as they threaten environmental and human health. Conventional biological treatments lead to limited removal of OMPs. Methanotrophic bacteria can degrade a variety of OMPs. By employing a novel bubble-free hybrid membrane biofilm bioreactor (hMBfR), we grew methanotrophic bacteria at three CH4 loading rates. Biomass productivity and CH4 loading showed a linear correlation, with a maximum productivity of 372 mg-VSS·L-1·d-1, with corresponding biomass concentration of 1117.6 ± 56.4 mg-VSS·L-1. Furthermore, the biodegradation of sulfamethoxazole and 1H-benzotriazole positively correlated with CH4 oxidation rates, with highest biodegradation kinetic constants of 3.58 L·g-1·d-1 and 5.42 L·g-1·d-1, respectively. Additionally, the hMBfR recovered nutrients as microbial proteins, with an average content 39% DW. The biofilm community was dominated by Methylomonas, while the bulk was dominated by aerobic heterotrophic bacteria. The hMBfR removed OMPs, allowing for safer water reuse while valorising CH4 and nutrients.

Distance-decay relationships partially determine diversity patterns of phyllosphere bacteria on Tamarix trees across the Sonoran Desert [corrected].

Dispersal limitation in phyllosphere communities was measured on the leaf surfaces of salt-excreting Tamarix trees, which offer unique, discrete habitats for microbial assemblages. We employed 16S rRNA gene pyrosequencing to measure bacterial community dissimilarity on leaves of spatially dispersed Tamarix specimens in sites with uniform climatic conditions across the Sonoran Desert in the Southwestern United States. Our analyses revealed diverse bacterial communities with four dominant phyla that exhibited differential effects of environmental and geographic variables. Geographical distance was the most important parameter that affected community composition, particularly that of betaproteobacteria, which displayed a statistically significant, distance-decay relationship.

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.

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.

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.

Reporter Gene Assays in Ecotoxicology.

The need for simple and rapid means for evaluating the potential toxic effects of environmental samples has prompted the development of reporter gene assays, based on tester cells (bioreporters) genetically engineered to report on sample toxicity by producing a readily quantifiable signal. Bacteria are especially suitable to serve as bioreporters owing to their fast responses, low cost, convenient preservation, ease of handling, and amenability to genetic manipulations. Various bacterial bioreporters have been introduced for general toxicity and genotoxicity assessment, and the monitoring of endocrine disrupting and dioxin-like compounds has been mostly covered by similarly engineered eukaryotic cells. Some reporter gene assays have been validated, standardized, and accredited, and many others are under constant development. Efforts are aimed at broadening detection spectra, lowering detection thresholds, and combining toxicity identification capabilities with characterization of the toxic effects. Taking advantage of bacterial robustness, attempts are also being made to incorporate bacterial bioreporters into field instrumentation for online continuous monitoring or on-site spot checks. However, key hurdles concerning test validation, cell preservation, and regulatory issues related to the use of genetically modified organisms still remain to be overcome.

High-throughput prescreening of pharmaceuticals using a genome-wide bacterial bioreporter array.

We assessed the applicability of multi-strain bacterial bioreporter bioassays to drug screening. To this end, we investigated the reactions of a panel of 15 luminescent recombinant Escherichia coli bacterial bioreporters to a library of 420 pharmaceuticals. The panel included bacterial bioreporters associated with oxidative stress, DNA damage, heat shock, and efflux of excess metals. Eighty nine drugs elicited a response from at least one of the panel members and formed distinctive clusters, some of which contained closely related drugs. In addition, we tested a group of selected nine drugs against a collection of about 2000 different fluorescent transcriptional reporters that covers the great majority of gene promoters in E. coli. The sets of induced genes were in accord with the in vitro toxicity of the tested drugs, as reflected by the response patterns of the 15-member panel, and provided more insights into their toxicity mechanisms. Facilitated by microplates and robotic systems, all assays were conducted in high-throughput. Our results thus suggest that multi-strain assemblages of bacterial bioreporters have the potential for playing a significant role in drug development alongside current in vitro toxicity tests.

Broad spectrum detection and "barcoding" of water pollutants by a genome-wide bacterial sensor array.

An approach for the rapid detection and classification of a broad spectrum of water pollutants, based on a genome-wide reporter bacterial live cell array, is proposed and demonstrated. An array of ca. 2000 Escherichia coli fluorescent transcriptional reporters was exposed to 25 toxic compounds as well as to unpolluted water, and its responses were recorded after 3 h. The 25 toxic compounds represented 5 pollutant classes: genotoxicants, metals, detergents, alcohols, and monoaromatic hydrocarbons. Identifying unique gene expression patterns, a nearest neighbour-based model detected pollutant presence and predicted class attribution with an estimated accuracy of 87%. Sensitivity and positive predictive values varied among classes, being higher for pollutant classes that were defined by mode of action than for those defined by structure only. Sensitivity for unpolluted water was 0.90 and the positive predictive value was 0.79. All pollutant classes induced the transcription of a statistically significant proportion of membrane associated genes; in addition, the sets of genes responsive to genotoxicants, detergents and alcohols were enriched with genes involved in DNA repair, iron utilization and the translation machinery, respectively. Following further development, a methodology of the type described herein may be suitable for integration in water monitoring schemes in conjunction with existing analytical and biological detection techniques.

Whole-cell biochips for online water monitoring.

Chip-integrated luminescent recombinant reporter bacteria were combined with fluidics and light detection systems to form a real-time water biomonitor. The biomonitor was exposed to a continuous water flow for up to ten days, in the course of which it was challenged with spikes of both model toxic compounds and toxic environmental samples. All simulated contamination events were reported within 0.5-2.5 h. Furthermore, the response pattern of the reporter bacteria was indicative of the nature of the contaminating chemicals. Efforts were aimed at improving signal quality and at the development of an alarm management software. Following further research, a device of the proposed design could be implemented in monitoring networks as an early warning system against water pollution by toxic chemicals.

Microbial sensor cell arrays.

Motivated by the advantages endowed by high-throughput analysis, researchers have succeeded in incorporating multiple reporter cells into a single platform; the technology now allows the simultaneous scrutiny of a large collection of sensor strains. We review current aspects in cell array technology with emphasis on microbial sensor arrays. We consider various techniques for patterning live cells on solid surfaces, describe different array-based applications and devices, and highlight recent efforts for live cell storage. We review mathematical approaches for deciphering the data emanating from bioreporter collections, and discuss the future of single cell arrays. Innovative technologies for cell patterning, preservation and interpretation are continuously being developed; when they all mature, cell arrays may become an efficient analytical tool, in a scope resembling that of DNA microarray biochips.

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.

Negative regulation of σ(70)-driven promoters by σ(70).

The Escherichia coli yjbEFGH operon, encoding genes involved in exopolysaccharide production, has previously been shown to be induced by osmotic stress and to be negatively regulated by σ(38). Promoter analysis suggested that like most E. coli genes, its transcription is driven by the housekeeping sigma factor σ(70). Indeed, manipulation of any of the other five alternative sigma factors did not affect its induction by osmotic stress. Surprisingly, when assayed in a strain expressing low levels of σ(70), yjbEFGH induction in response to osmotic stress was higher than in a strain expressing normal levels of σ(70). Similar phenomena were observed in the σ(70)-driven promoters of sulA, uvrA, recA, fecI, entC and lacZ, the transcription of which is directly controlled by a repressor protein (LexA, Fur and LacI), but not in promoters of the housekeeping genes ftsA and ftsY, or in σ(38)-driven treA promoter. Since transcription factors are generally present in the cell in low numbers, we hypothesize that a decrease in σ(70), that drives the expression of lexA, fur and lacI as well, further diminishes their number in the cell and thus de-represses the induction of genes which are subjected to their repression.

Microbial cell arrays.

The coming of age of whole-cell biosensors, combined with the continuing advances in array technologies, has prepared the ground for the next step in the evolution of both disciplines - the whole cell array. In the present chapter, we highlight the state-of-the-art in the different disciplines essential for a functional bacterial array. These include the genetic engineering of the biological components, their immobilization in different polymers, technologies for live cell deposition and patterning on different types of solid surfaces, and cellular viability maintenance. Also reviewed are the types of signals emitted by the reporter cell arrays, some of the transduction methodologies for reading these signals, and the mathematical approaches proposed for their analysis. Finally, we review some of the potential applications for bacterial cell arrays, and list the future needs for their maturation: a richer arsenal of high-performance reporter strains, better methodologies for their incorporation into hardware platforms, design of appropriate detection circuits, the continuing development of dedicated algorithms for multiplex signal analysis, and - most importantly - enhanced long term maintenance of viability and activity on the fabricated biochips.

Optical modeling of bioluminescence in whole cell biosensors.

Bioluminescence-based whole cell biosensors are devices that can be very useful for environmental monitoring applications. The advantages of these devices are that they can be produced as a single-chip, low-power, rugged, inexpensive component, and can be deployed in a variety of non-laboratory settings. However, such biosensors encounter inherent problems in overall system light collection efficiency. The light emitted from the bioluminescent microbial cells is isotropic and passes through various media before it reaches the photon detectors. We studied the bioluminescence distribution and propagation in microbial whole cell biochips. Optical emission and detection were modeled and simulated using an optical ray tracing method. Light emission, transfer and detection were simulated and optimized with respect to two fundamental system parameters: system geometry and bacterial concentration. Optimization elucidated some of the optical aspects of the biochip, e.g. detector radius values between 300 and 750 microm, and bacterial fixation radius values between 800 and 1200 microm. Understanding theses aspects may establish a basis for future optimization of similar chips.

Toxicant identification by a luminescent bacterial bioreporter panel: application of pattern classification algorithms.

Genetically engineered microorganisms, tailored to respond by a dose-dependent signal to the presence of toxic chemicals, are a potentially useful tool for environmental monitoring. One manifestation of this approach is based on a panel of luminescent bacterial bioreporters, harboring fusions of the luxCDABE operon to various stress-responsive gene promoters. Such sensors can report by a dose-dependent luminescent signal on the stress sensed by the cells and thus on the presence of toxic compound(s), but they lack the ability to identify the chemicals involved. Here, we demonstrate how the use of a panel of such sensors might offer a solution to this drawback. Five selected Escherichia coli reporter strains harboring fusions of selected gene promoters (grpE, nhoA, oraA, lacZ, and mipA) to luxCDABE were exposed to five model toxicants and to a toxicant-free control in a 40-repetition format. Each of the six treatments activated different promoters to different extents, producing its own unique fingerprint. Two machine learning schemes were challenged with the obtained data set: Bayesian decision theory and the nonparametric nearest-neighbor technique. The Bayesian classifiers performed better and were able to identify the sample's contents within 30 min with an error rate estimate that did not exceed 3% at a 95% confidence level and with zero false negatives. Performance in tap water and wastewater samples was similar. Given the coming of age of whole-cell sensing devices, pattern classification algorithms such as the ones described here offer a step toward the incorporation of reporter cells into future biosensor formats, including whole-cell arrays.

Microbial whole-cell arrays.

The coming of age of whole-cell biosensors, combined with the continuing advances in array technologies, has prepared the ground for the next step in the evolution of both disciplines - the whole-cell array. In the present review, we highlight the state-of-the-art in the different disciplines essential for a functional bacterial array. These include the genetic engineering of the biological components, their immobilization in different polymers, technologies for live cell deposition and patterning on different types of solid surfaces, and cellular viability maintenance. Also reviewed are the types of signals emitted by the reporter cell arrays, some of the transduction methodologies for reading these signals and the mathematical approaches proposed for their analysis. Finally, we review some of the potential applications for bacterial cell arrays, and list the future needs for their maturation: a richer arsenal of high-performance reporter strains, better methodologies for their incorporation into hardware platforms, design of appropriate detection circuits, the continuing development of dedicated algorithms for multiplex signal analysis and - most importantly - enhanced long-term maintenance of viability and activity on the fabricated biochips.