Search Results (45)

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 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. Full article

The Escherichia coli Keio mutant collection has been a tool for assessing the role of specific genes and determining their role in E. coli physiology and uncovering novel functions. In this work, specific mutants in the DNA repair pathways and oxidative stress response were evaluated to identify the primary targets of silver nanoparticles (NPs) and their mechanism of action. The results presented in this work suggest that NPs mainly target DNA via double-strand breaks and base modifications since the recA, uvrC, mutL, and nfo mutants rendered the most susceptible phenotype, rather than involving the oxidative stress response. Concomitantly, during the establishment of the control conditions for each mutant, the katG and sodA mutants showed a hypersensitive phenotype to mitomycin C, an alkylating agent. Thus, we propose that KatG catalase plays a key role as a cellular chaperone, as reported previously for the filamentous fungus Neurospora crassa, a large subunit catalase. The Keio collection mutants may also be a key tool for assessing the resistance mechanism to metallic NPs by using their potential to identify novel pathways involved in the resistance to NPs. Full article

Medicinal chemistry is constantly searching for new approaches to develop more effective and targeted therapeutic molecules. The design of peptidomimetics is a promising emerging strategy that is aimed at developing peptides that mimic or modulate the biological activity of proteins. Among these, stapled peptides stand out for their unique ability to stabilize highly frequent helical motifs, but they have failed to be systematically reported. Here, we exploit chemically diverse helix-inducing i, i + 4 constraints—lactam, hydrocarbon, triazole, double triazole and thioether—on two distinct short sequences derived from the N-terminal peptidase domain of hACE2 upon structural characterization and in silico alanine scan. Our overall objective was to provide a sequence-independent comparison of α-helix-inducing staples using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. We identified a 9-mer lactam stapled peptide derived from the hACE2 sequence (His34-Gln42) capable of reaching its maximal helicity of 55% with antiviral activity in bioreporter- and pseudovirus-based inhibition assays. To the best of our knowledge, this study is the first comprehensive investigation comparing several cyclization methods with the goal of generating stapled peptides and correlating their secondary structures with PPI inhibitions using a highly topical model system (i.e., the interaction of SARS-CoV-2 Spike RBD with hACE2). Full article

The aim of this research is to define optimal conditions to improve the stability of gold and silver nanoparticles’ anti-zearalenone antibody conjugates for their utilisation in lateral flow immunochromatographic assay (LFIA). The Turkevich–Frens method was used to synthesise gold nanoparticles (AuNPs), which were between 10 and 110 nm in diameter. Silver nanoparticles (AgNPs) with a size distribution of 2.5 to 100 nm were synthesised using sodium borohydride as a reducing agent. The onset of AuNP and AgNP aggregation occurred at 150 mM and 80 mM NaCl concentrations, respectively. Stable Au and Ag nanoparticle–antibody conjugates were achieved at 1.2 mM of K₂CO₃ concentration, which corresponds to the pH value of ≈7. Lastly, the highest degree of conjugation between Au and Ag nanoparticles and anti-zearalenone antibodies was at 4 and 6 µg/mL of antibody concentrations. The optimisation of the conjugation conditions can contribute to better stability of nanoparticles and their antibody conjugate and can improve the reproducibility of results of bioreporter molecules in biosensing lateral flow devices. Full article

Quorum Sensing (QS) is a well-studied intercellular communication mechanism in bacteria, regulating collective behaviors such as biofilm formation, virulence, and antibiotic resistance. However, cell–cell signaling in haloarchaea remains largely unexplored. The coexistence of bacteria and archaea in various environments, coupled with the known cell–cell signaling mechanisms in both prokaryotic and eukaryotic microorganisms and the presence of cell–cell signaling mechanisms in both prokaryotic and eukaryotic microorganisms, suggests a possibility for haloarchaea to possess analogous cell–cell signaling or QS systems. Recently, N-acylhomoserine lactone (AHL)-like compounds were identified in haloarchaea; yet, their precise role—for example, persister cell formation—remains ambiguous. This study investigated the capacity of crude supernatant extract from the haloarchaeon Halorubrum saccharovorum CSM52 to stimulate bacterial AHL-dependent QS phenotypes using bioreporter strains. Our findings reveal that these crude extracts induced several AHL-dependent bioreporters and modulated pyocyanin and pyoverdine production in Pseudomonas aeruginosa. Importantly, our study suggests cross-domain communication between archaea and bacterial pathogens, providing evidence for archaea potentially influencing bacterial virulence. Using Thin Layer Chromatography overlay assays, lactonolysis, and colorimetric quantification, the bioactive compound was inferred to be a chemically modified AHL-like compound or a diketopiperazine-like molecule, potentially involved in biofilm formation in H. saccharovorum CSM52. This study offers new insights into putative QS mechanisms in haloarchaea and their potential role in interspecies communication and coordination, thereby enriching our understanding of microbial interactions in diverse environments. Full article

Bioluminescence is light emission based on the luciferin–luciferase enzymatic reaction in living organisms. Optical signals from bioluminescence (BL) reactions are available for bioanalysis and bioreporters for gene expression, in vitro, in vivo, and ex vivo bioimaging, immunoassay, and other applications. Although there are numerous bioanalysis methods based on BL signal measurements, the BL signal is measured as a relative value, and not as an absolute value. Recently, some approaches have been established to completely quantify the BL signal, resulting in, for instance, the redetermination of the quantum yield of the BL reaction and counting the photon number of the BL signal at the single-cell level. Reliable and reproducible understanding of biological events in the bioanalysis and bioreporter fields can be achieved by means of standardized absolute optical signal measurements, which is described in an International Organization for Standardization (ISO) document. Full article

Streptococcus suis LuxS/AI-2 quorum sensing system regulates biofilm formation, resulting in increased pathogenicity and drug resistance, and diminished efficacy of antibiotic treatment. The remaining peony seed cake after oil extraction is rich in monoterpenoid glycosides, which can inhibit the formation of bacterial biofilm. In this study, we investigated the effect of seven major monocomponents (suffruticosol A, suffruticosol B, suffruticosol C, paeonifloin, albiflorin, trans-ε-viniferin, gnetin H) of peony seed meal on minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of S. suis. The results showed that the MICs of the seven single components were all greater than 200 μg/mL, with no significant bacteriostatic and bactericidal advantages. Crystal violet staining and scanning electron microscope observation showed that the seven single components had a certain inhibitory effect on the biofilm formation ability of S. suis at sub-MIC concentration. Among them, the ability of paeoniflorin to inhibit biofilm was significantly higher than that of the other six single components. AI-2 signaling molecules were detected by bioreporter strain Vibrio harvey BB170. The detection results of AI-2 signal molecules found that at 1/2 MIC concentration, paeoniflorin significantly inhibited the production of S. suis AI-2 signal, and the inhibitory effect was better than that of the other six single components. In addition, molecular docking analysis revealed that paeoniflorin had a significant binding activity with LuxS protein compared with the other six single components. The present study provides evidence that paeoniflorin plays a key role in the regulation of the inhibition of S. suis LuxS/AI-2 system and biofilm formation in peony seed meal. 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