Search Results (23)

Wildfires that destroy residential infrastructure can generate chemically complex soil contamination; however, post-fire screening is often limited and does not directly reflect biological hazards. Herein, we integrated a multi-stress lux-based whole-cell bioreporter panel of genetically engineered Escherichia coli strains with non-targeted LC-MS profiling to obtain a mechanism-informed assessment of soils collected from a residential property impacted by the January 2025 Los Angeles wildfires. The bioreporter panel resolved heterogeneous and statistically significant stress signatures across the analyzed samples. In particular, extracts from U3–U5 produced selective suppression of the membrane and fatty acid biosynthesis bioreporters, along with reduced growth. In contrast, extract U5 induced a proteotoxic heat-shock response signature. In parallel, non-targeted LC-MS detected 1813 chemical features and enabled the putative annotation of a subset of signals consistent with combustion-derived organics and reactive electrophiles, providing a chemical context for the observed bioassay fingerprints. The integrated workflow provides mechanism-resolved hazard triage within 48 h, as implemented herein (24 h elutriate preparation plus up to 20 h microplate kinetics), supporting the prioritization of hotspots for confirmatory analysis, remediation, and risk assessment. Full article

Chemical monitoring of pollutants and hazardous materials in water supply systems traditionally depends on centralized laboratories, advanced instrumentation, and trained personnel, limiting accessibility and preventing real-time, on-site analysis. This work presents an alternative cost-effective, field-deployable approach that uses genetically engineered bioluminescent bioreporters, encapsulated in self-sufficient alginate capsules and integrated with an optoelectronic detection circuit, to detect and quantify target materials in water. We have developed a scalable single-channel prototype featuring four sensing tracks—two for sample measurement, one for clean water, and one for a standard reference solution. The latter employs the standard ratio (SR) method to ensure robust quantification, compensating for batch variability and environmental effects. System characterization showed high uniformity across tracks. Validation with nalidixic acid (NA) demonstrated reliable quantitative performance, with a blind test estimation of 5.6 mg/L for a true concentration of 5 mg/L, well within the calibration error range. Additional sensitivity testing confirmed detection of mitomycin C (MMC) at concentrations as low as 50 µg/L. Overall, the results highlight the potential of bacterial chemical sensing as a practical and scalable tool for real-time, in situ water quality monitoring networks. Full article

Crude oil contamination occurs frequently in soil; thus, on-site measurement of oil content is critical for controlling petroleum contamination, but it is challenging. Conventional chemical analysis requires complicated sample pretreatment and high-cost facilities, requiring on-site and cost-effective approaches. This study innovated a whole-cell bioreporter assay by combining Acinetobacter-hosted n-alkane and genotoxicity bioreporters to directly and simultaneously evaluate the contamination level and genotoxicities of crude oil in contaminated soils. Ultrasound pretreatment was employed to accelerate the measurement process, and the first-order release kinetic model was used to calculate crude oil content in an easy operation. The detection limit of the bioreporters was satisfactory at 0.1 mg/L, and the quantification range was 0.1–10 mg/L. The developed bioreporter assay effectively assessed the bioavailability and toxicity of crude oil in real contaminated soils and recognized distinct toxicities after soil weathering. Our findings highlight the feasibility of using the whole-cell bioreporter assay to evaluate the bioavailability and toxicity of crude oil, offering supporting data for the selection of remediation strategies. Full article

Cigarette smoking is known to be an unhealthy activity that can cause a number of human diseases, including chronic obstructive pulmonary disease (COPD) and lung cancer. It was further reported that even being exposed to secondhand cigarette smoke can affect human health. To assess the toxicity of the smoke from different cigarette brands, an artificial smoking device was developed, and three fractions designated, Filter Fraction, Smoke Fraction and Tar Fraction, were prepared from the smoke of each brand. Then, to elucidate possible effects of some of the toxins found in cigarette smoke, we investigated their effects in vitro using a bioluminescent bacterial array that comprises three bacterial strains. Using this array, we compare smoke from three cigarette brands, each with different tar and nicotine contents. GC-MS analysis showed that the cigarette smoke extracts (fractions) from different brands differed in their compositions and chemical concentrations. The results further showed that, in general, cigarette smoke triggered mainly an oxidative stress reaction in our bacterial models. The Smoke Fraction was tested for sequential smoking rounds and found to produce cumulative effects following each subsequent smoking cycle for all three cigarette brands. Finally, it was found that cigarette smoke and its specific components are toxic at various degrees with the Smoke Fraction, acting as oxidative stressors, and that this can be effectively analyzed using bioreporter panel arrays. Full article

β-lactams are an important family of antibiotics that are prone to undergo resistance inhibition though the production of β-lactamases by some microorganisms. To combat this resistance and preserve the efficacy of β-lactam antibiotics, we developed a strategy for the discovery of such β-lactamase inhibitors. When combined with β-lactams, these inhibitors allow the antibiotics to be effective and prevent resistance. To date, the development of such combinatory drugs is limited due to the complexity of screening for new β-lactamase inhibitors. Therefore, to facilitate this development, it was essential to find sensitive assays to effectively screen for lactamase inhibitory compounds. To this end, a novel bioassay utilizing bioluminescent indicator bacteria as bioreporters was developed. The assay was first optimized using commercial antibiotics together with known β-lactamase inhibitors. Using this bioassay, we then screened for novel natural β-lactamase inhibitors derived from coral-associated fungi. We showed that the fungus Penicillium spinulosum, originating from the coral Pocillopora sp. from the Gulf of Aqaba Eilat, produced compounds with anti-β-lactamase activity. We further demonstrated that the bioreporter bacteria used here responded to the combined antibiotics and β-lactamase inhibitors in a concentration-dependent manner, indicating their usefulness for β-lactamase-inhibiting compound discovery. Future structural identification will promote the validation of this assay’s usefulness. Full article

Sediments are key players in the optimum functioning of ecosystems; however, they also represent the largest known repository of harmful contaminants. The vast variety of these sediment-associated contaminants may exert harmful effects on marine communities and can impair ecosystem functioning. Whole-cell biosensors are a rapid and biologically relevant tool for assessing environmental toxicity. Therefore, in this study, we developed a bioassay-based toxicity measurement system using genetically modified bacteria to create a whole-cell optical biosensor. Briefly, reporter bacteria were integrated and immobilized using a calcium alginate matrix on fiber-optic tips connected to a photon counter placed inside a light-proof, portable case. The calcium alginate matrix acts as a semi-permeable membrane that protects the reporter-encapsulated optical fiber tips and allows the inward passage of toxicant(s) to induce a dose-dependent response in the bioreporter. The samples were tested by directly submerging the fiber tip with immobilized bacteria into vials containing either water or suspended sediment samples, and the subsequent bioluminescent responses were acquired. In addition to bioavailable sediment toxicity assessments, conventional chemical methods, such as liquid chromatography–mass spectroscopy (LC-MS) and inductively coupled plasma optical emission spectroscopy (ICP-OES), were used for comprehensive evaluation. The results demonstrated the efficacy of the biosensor in detecting various toxicity levels corresponding to identified contaminants, highlighting its potential integration into environmental monitoring frameworks for enhanced sediment and water quality assessments. Despite its utility, this study notes the system’s operational challenges in field conditions, recommending future enhancements for improved portability and usability in remote locations. Full article

Soil and sediment contamination with heavy metals (HMs) is a critical environmental issue, posing significant risks to both ecosystems and human health. Whole-cell bioreporter (WCB) technology offers a promising alternative to traditional detection techniques due to its ability to rapidly assess the bioavailability of pollutants. Specifically, lights-on WCBs quantify pollutant bioavailability by measuring bioluminescence or fluorescence in response to pollutant exposure, demonstrating comparable accuracy to traditional methods for quantitative pollutant detection. However, when applied to soil and sediment, the signal intensity directly measured by WCBs is often attenuated due to interference from solid particles, leading to the underestimation of bioavailability. Currently, no standardized method exists to correct for this signal attenuation. This review provides a critical analysis of the benefits and limitations of traditional detection methods and WCB technology in assessing HM bioavailability in soil and sediment. Based on the approaches used to address WCB signal attenuation, correction methods are categorized into four types: the assumed negligible method, the non-inducible luminescent control method, the addition of a standard to a reference soil, and a pre-exposure bioreporter. We provide a comprehensive analysis of each method’s applicability, benefits, and limitations. Lastly, potential future directions for advancing WCB technology are proposed. This review seeks to establish a theoretical foundation for researchers and environmental professionals utilizing WCB technology for pollutant bioavailability assessment in soil and sediment. Full article

This review attempts to summarize my three decades-long involvement in, and contribution to, the design, construction and testing of bioluminescent microbial sensor strains (bioreporters). With the understanding that such a document cannot be completely free of bias, the review focuses on studies from my own lab only, with almost no coverage of the parallel progress made by others in similar fields. This admittedly subjective approach by no way detracts from the achievements of countless excellent researchers who are not mentioned here, and whose contributions to the field are at least as important as that of my own. The review covers basic aspects of microbial sensor design, and then progresses to describe approaches to performance improvement of sensor strains aimed at the detection of either specific chemicals, groups of chemicals sharing similar characteristics, or global effects, such as toxicity and genotoxicity. The need for integration of live sensor cells into a compatible hardware platform is highlighted, as is the importance of long-term maintenance of the cells’ viability and activity. The use of multi-member sensors’ panels is presented as a means for enhancing the detection spectrum and sample “fingerprinting”, along with a list of different purposes to which such sensors have been put to use. Full article

This study presents a rapid and comprehensive method for screening mushroom extracts for the putative discovery of bioactive molecules, including those exhibiting antimicrobial activity. This approach utilizes a panel of bioluminescent bacteria, whose light production is a sensitive indicator of various cellular effects triggered by the extracts, including disruption of bacterial communication (quorum sensing), protein and DNA damage, fatty acid metabolism alterations, and oxidative stress induction. The bioassay’s strength is its ability to efficiently analyze a large number of extracts simultaneously while also assessing several different mechanisms of toxicity, significantly reducing screening time. All samples analyzed exhibited more than one cellular effect, as indicated by the reporter bacteria. Four samples (C. cornucopioides, F. fomentarius, I. obliquus, and M. giganteus) displayed the highest number (six) of possible mechanisms of antibacterial activity. Additionally, combining extraction and purification protocols with a bioluminescent bacterial panel enables simultaneous improvement of the desired antimicrobial properties of the extracts. The presented approach offers a valuable tool for uncovering the diverse antimicrobial mechanisms of mushroom extracts. Full article

There is currently a critical need for understanding how the response and activity of whole-cell bacterial reporters positioned in a complex biological or environmental matrix are impacted by the physicochemical properties of their micro-environment. Accordingly, a comprehensive analysis of the bioluminescence response of Cd(II)-inducible PzntA-luxCDABE Escherichia coli biosensors embedded in silica-based hydrogels is reported to decipher how metal bioavailability, cell photoactivity and ensuing light bioproduction are impacted by the hydrogel environment and the associated matrix effects. The analysis includes the account of (i) Cd speciation and accumulation in the host hydrogels, in connection with their reactivity and electrostatic properties, and (ii) the reduced bioavailability of resources for the biosensors confined (deep) inside the hydrogels. The measurements of the bioluminescence response of the Cd(II) inducible-lux biosensors in both hydrogels and free-floating cell suspensions are completed by those of the constitutive rrnB P1-luxCDABE E. coli so as to probe cell metabolic activity in these two situations. The approach contributes to unraveling the connections between the electrostatic hydrogel charge, the nutrient/metal bioavailabilities and the resulting Cd-triggered bioluminescence output. Biosensors are hosted in hydrogels with thickness varying between 0 mm (the free-floating cell situation) and 1.6 mm, and are exposed to total Cd concentrations from 0 to 400 nM. The partitioning of bioavailable metals at the hydrogel/solution interface following intertwined metal speciation, diffusion and Boltzmann electrostatic accumulation is addressed by stripping chronopotentiometry. In turn, we detail how the bioluminescence maxima generated by the Cd-responsive cells under all tested Cd concentration and hydrogel thickness conditions collapse remarkably well on a single plot featuring the dependence of bioluminescence on free Cd concentration at the individual cell level. Overall, the construction of this master curve integrates the contributions of key and often overlooked processes that govern the bioavailability properties of metals in 3D matrices. Accordingly, the work opens perspectives for quantitative and mechanistic monitoring of metals by biosensors in environmental systems like biofilms or sediments. Full article

The continuous emergence of new illegal compounds, particularly psychoactive chemicals, poses significant challenges for current drug detection methods. Developing new protocols and kits for each new drug requires substantial time, effort, and dedicated manpower. Whole-cell bacterial bioreporters have been proven capable of detecting diverse hazardous compounds in both laboratory and field settings, identifying not only single compounds but also chemical families. We present the development of a microbial bioreporter for the detection of cocaine, the nervous system stimulant that is the second-most widely used illegal drug in the US. Escherichia coli was transformed with a plasmid containing a bacterial luxCDABEG bioluminescence gene cassette, activated by a cocaine-responsive signaling cascade. The engineered bioreporter is demonstrated to be a sensitive and specific first-generation detection system for cocaine, with detection thresholds of 17 ± 8 μg/L and 130 ± 50 μg/L in a buffer solution and in urine, respectively. Further improvement of the sensor’s performance was achieved by altering the nucleotide sequence of the PBen gene promoter, the construct’s sensing element, using accelerated site-directed evolution. The applicability of ready-to-use paper strips with immobilized bioreporter cells was demonstrated for cocaine detection in aqueous solutions. Full article

We present a generic quantitative chemical sensing methodology for assessing the concentration of a target material (TM) in an aqueous solution by using bioluminescent microbial bioreporters as the core sensing elements. Such bioreporters, genetically engineered to respond to the presence of a TM in their microenvironment by emitting bioluminescence, have previously been mostly designed to report the presence or absence of the TM in the sample. We extend this methodology to also assess the TM concentration, by exploiting the dose-dependency of the TM-induced luminescence. To overcome luminescence intensity variations due to bacterial batch differences and the ambient temperature, simultaneous measurements were carried out on sample solutions containing known concentrations of the TM. A “standard ratio” parameter, defined as the ratio between the two measurements, is shown to be independent of the bacterial batch and the temperature, and hence provides the conceptual basis for a generic quantitative chemical sensing methodology. Assessment of 2,4-dinitrotoluene (DNT) concentration in solutions is demonstrated with an accuracy of 2.5% over a wide dynamic range. Full article

Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that remains a prevalent clinical and environmental challenge. Quorum-sensing (QS) molecules are effective biomarkers in pinpointing the presence of P. aeruginosa. This study aimed to develop a convenient-to-use, whole-cell biosensor using P. aeruginosa reporters individually encapsulated within alginate-poly-L-lysine (alginate-PLL) microbeads to specifically detect the presence of bacterial autoinducers. The PLL-reinforced microbeads were prepared using a two-step method involving ionic cross-linking and subsequent coating with thin layers of PLL. The alginate-PLL beads showed good stability in the presence of a known cation scavenger (sodium citrate), which typically limits the widespread applications of calcium alginate. In media containing synthetic autoinducers—such as N-(3-oxo dodecanoyl) homoserine lactone (3-oxo-C₁₂-HSL) and N-butanoyl-L-homoserine lactone (C₄-HSL), or the cell-free supernatants of planktonic or the flow-cell biofilm effluent of wild P. aeruginosa (PAO1)—the encapsulated bacteria enabled a dose-dependent detection of the presence of these QS molecules. The prepared bioreporter beads remained stable during prolonged storage at 4 and −80 °C and were ready for on-the-spot sensing without the need for recovery. The proof-of-concept, optical fiber-based, and whole-cell biosensor developed here demonstrates the practicality of the encapsulated bioreporter for bacterial detection based on specific QS molecules. Full article

The availability of new bioluminescent proteins with tuned properties, both in terms of emission wavelength, kinetics and protein stability, is highly valuable in the bioanalytical field, with the potential to improve the sensitivity and analytical performance of the currently used methods for ATP detection, whole-cell biosensors, and viability assays among others. We present a new luciferase mutant, called BgLuc, suitable for developing whole-cell biosensors and in vitro biosensors characterized by a bioluminescence maximum of 548 nm, narrow emission bandwidth, favorable kinetic properties, and excellent pH- and thermo-stabilities at 37 and 45 °C and pH from 5.0 to 8.0. We assessed the suitability of this new luciferase for whole-cell biosensing with a cell-based bioreporter assay for Nuclear Factor-kappa B (NF-kB) signal transduction pathway using 2D and 3D human embryonic kidney (HEK293T) cells, and for ATP detection with the purified enzyme. In both cases the luciferase showed suitable for sensitive detection of the target analytes, with better or similar performance than the commercial counterparts. Full article

Creating unique microenvironments, hyphal surfaces and their surroundings allow for spatially distinct microbial interactions and functions at the microscale. Using a microfluidic system and pH-sensitive whole-cell bioreporters (Synechocystis sp. PCC6803) attached to hyphae, we spatially resolved the pH along surfaces of growing hyphae of the basidiomycete Coprinopsis cinerea. Time-lapse microscopy analysis of ratiometric fluorescence signals of >2400 individual bioreporters revealed an overall pH drop from 6.3 ± 0.4 (n = 2441) to 5.0 ± 0.3 (n = 2497) within 7 h after pH bioreporter loading to hyphal surfaces. The pH along hyphal surfaces varied significantly (p < 0.05), with pH at hyphal tips being on average ~0.8 pH units lower than at more mature hyphal parts near the entrance of the microfluidic observation chamber. Our data represent the first dynamic in vitro analysis of surface pH along growing hyphae at the micrometre scale. Such knowledge may improve our understanding of spatial, pH-dependent hyphal processes, such as the degradation of organic matter or mineral weathering. Full article