Bioluminescence Reaction Research Articles

Continuous Surveillance of Bioaerosols On-Site Using an Automated Bioaerosol-Monitoring System.

Real-time on-site monitoring of bioaerosols in an air environment is important for preventing various adverse health effects including respiratory diseases and allergies caused by bioaerosols. Here, we report the development of an on-site automated bioaerosol-monitoring system (ABMS) using integrated units including a wet-cyclone bioaerosol sampler, a thermal-lysis unit for extracting adenosine triphosphate (ATP), an ATP-detection unit based on the immobilization of luciferase/luciferin for bioluminescence reactions, and a photomultiplier tube-based detector. The performance of the bioaerosol detection system was verified using Escherichia coli (E. coli) as a model source. Each unit was optimized to process ∼9.6 × 105 times the concentrated ratio of collected bioaerosol samples, using a 3 min lysis time to extract ATP, and has a detection limit of ∼375 colony-forming units (CFUs)/mL with more than 30 days of stability for the immobilized-luciferase/luciferin detection unit supported by a glass-fiber conjugation pad. After the integration of all units, the ABMS achieved E. coli bioaerosol monitoring with continuous detection at 5 min intervals and a minimum detection limit of ∼130 CFU/mair3. Furthermore, the rapid responsivity and stable operation performance of the ABMS under test-bed conditions and during a field test demonstrated that the ABMS is capable of continuously monitoring bioaerosols in real-time with high sensitivity. The monitoring system developed here with immobilization strategies for bioluminescence reactions triggered by ATP extracted from collected bioaerosol samples using a simple heat-lysis method may help establish sustainable platforms to obtain stable signals for the real-time detection of bioaerosols on-site.

PH-Dependent absorption spectrum of oxyluciferin analogues in the active site of firefly luciferase.

In the quest for the identification of the light emitter(s) responsible for the firefly bioluminescence, the study of oxyluciferin analogues with controlled chemical and electronic structures is of particular importance. In this article, we report the results of our experimental and computational investigation of the pH-dependent absorption spectra characterizing three analogues bound into the luciferase cavity, together with adenosine-monophosphate (AMP). While the analogue microscopic pKa values do not differ much from their reference values, it turns out that the AMP protonation state is analogue-dependent and never doubly-deprotonated. A careful analysis of the interactions evidences the main role of E344 glutamic acid, as well as the flexibility of the cavity which can accommodate any oxyluciferin analogue. The consideration of the absorption spectra suggests that the oxyluciferin enolate form has to be excluded from the list of the bioluminescence reaction products.

Measurement of the Promoter Activity in Escherichia coli by Using a Luciferase Reporter.

The reporter system is widely used technique for measuring promoter activity in bacterial cells. Until now, a number of reporter system have been developed, but the bioluminescent reporter constructed from the bacterial luciferase genes is one of the useful systems for measuring in vivo dynamics of gene expression. The introduced bioluciferase lux reporter enables easy, fast, and sensitive measurement of the promoter activity without cell lysis because the substrates of bioluminescent reaction are synthesized inside the bacterial cell, thereby allowing low-cost experiments. This protocol describes a high throughput technique to measure the promoter activity in Escherichia coli K-12 using the lux reporter system.

Bacterial luciferase: Molecular mechanisms and applications.

Bacterial luciferase is a flavin-dependent monooxygenase which is remarkable for its distinctive feature in transforming chemical energy to photons of visible light. The bacterial luciferase catalyzes bioluminescent reaction using reduced flavin mononucleotide, long-chain aldehyde and oxygen to yield oxidized flavin, corresponding acid, water and light at λmax around 490nm. The enzyme comprises of two non-identical α and β subunits, where α subunit is a catalytic center and β subunit is crucially required for maintaining catalytic function of the α subunit. The crystal structure with FMN bound and mutagenesis studies have assigned a number of amino acid residues that are important in coordinating critical reactions and stabilizing intermediates to attain optimum reaction efficiency. The enzyme achieves monooxygenation by generating C4a-hydroperoxyflavin intermediate that later changes its protonation status to become C4a-peroxyflavin, which is necessary for the nucleophilic attacking with aldehyde substrate. The decomposing of C4a-peroxyhemiacetal produces excited C4a-hydroxyflavin and acid product. The chemical basis regrading bioluminophore generation in Lux reaction remains an inconclusive issue. However, current data can, at least, demonstrate the involvement of electron transfer to create radical molecules which is the key step in this mechanism. Lux is a self-sufficient bioluminescent system in which all substrates can be recycled and produced by a group of enzymes from the lux operon. This makes Lux distinctively advantageous over other luciferases for reporter enzyme application. The progression of understanding of Lux catalysis is beneficial to improve light emitting efficiency in order to expand the robustness of Lux application.

ВЛИЯНИЕ ОСМОЛИТОВ НА БИОЛЮМИНЕСЦЕНТНУЮ РЕАКЦИЮ БАКТЕРИЙ: СТРУКТУРНО-ДИНАМИЧЕСКИЕ АСПЕКТЫ

The effects of viscous media with glycerol and sucrose (10-40%) on the kinetics of the bacterial bioluminescent reaction have been investigated by stopped-flow technique. Increment of quantum yield in media with 10% of both osmolytes was shown. Higher concentrations of glycerol, up to 30-40%, were found to reduce the efficiency of the reaction, while this effect was not observed in the media with sucrose. The molecular dynamics simulation was used to study the structure of bacterial luciferase surrounded by either water molecules solely or by mixture of water with various numbers of glycerol/sucrose molecules. It was found that both cosolvents at studied concentrations did not cause a significant change in conformation of bacterial luciferase. The calculated root mean square fluctuations for Cα-atoms of bacterial luciferase α-subunit indicated that the higher flexibility of the enzyme mobile loop could be responsible for increment of quantum yield in the presence of 10% of both osmolytes. The active site of bacterial luciferase was found to be accessible for glycerol molecules while sucrose did not enter catalytic gorge. Moreover, at 30% and 40% concentration the glycerol molecules were found to locate in the active site of bacterial luciferase throughout the whole simulation time (40 ns) and to exclude water molecules, which can explain the experimentally obtained reaction quantum yield decrease.

Atomistic Insights into Photoprotein Formation: Computational Prediction of the Properties of Coelenterazine and Oxygen Binding in Obelin.

Bioluminescence in marine systems is dominated by the use of coelenterazine for light production. The bioluminescent reaction of coelenterazine is an enzyme catalyzed oxidative decarboxylation: coelenterazine reacts with molecular oxygen to form carbon dioxide, coelenteramide, and light. One such class is the Ca2+ -regulated photoproteins. These proteins bind coelenterazine and oxygen, and trap 2-hydroperoxycoelenterazine, an intermediate along the reaction pathway. The reaction is halted until Ca2+ binding triggers the completion of the reaction. There are currently no reported experimental, atomistic descriptions of this ternary Michaelis complex. This study utilized computational techniques to develop an atomistic model of the Michaelis complex. Extensive molecular dynamics simulations were carried out to study the interactions between four tautomeric/protonation states of coelenterazine and wide-type and mutant obelin. Only minor differences in binding modes were observed across all systems. Interestingly, no basic residues were identified in the vicinity of the N7-nitrogen of coelenterazine. This observation was surprising considering that deprotonation at this position is a key mechanistic step in the proposed bioluminescent reaction. This work suggests that coelenterazine binds either as the O10H tautomer, or in the deprotonated form. Implicit ligand sampling simulations were used to identify potential O2 binding and migration pathways within obelin. A key oxygen binding site was identified close to the coelenterazine imidazopyrazinone core. The O2 binding free energy was observed to be dependent on the protonation state of coelenterazine. Taken together, the description of the obelin-coelenterazine-O2 complexes established in this study provides the basis for future computational studies of the bioluminescent mechanism. © 2019 Wiley Periodicals, Inc.

Luciferin Regeneration in Firefly Bioluminescence via Proton-Transfer-Facilitated Hydrolysis, Condensation and Chiral Inversion.

Firefly bioluminescence is produced via luciferin enzymatic reactions in luciferase. Luciferin has to be unceasingly replenished to maintain bioluminescence. How is the luciferin reproduced after it has been exhausted? In the early 1970s, Okada proposed the hypothesis that the oxyluciferin produced by the previous bioluminescent reaction could be converted into new luciferin for the next bioluminescent reaction. To some extent, this hypothesis was evidenced by several detected intermediates. However, the detailed process and mechanism of luciferin regeneration remained largely unknown. For the first time, we investigated the entire process of luciferin regeneration in firefly bioluminescence by density functional theory calculations. This theoretical study suggests that luciferin regeneration consists of three sequential steps: the oxyluciferin produced from the last bioluminescent reaction generates 2-cyano-6-hydroxybenzothiazole (CHBT) in the luciferin regenerating enzyme (LRE) via a hydrolysis reaction; CHBT combines with L-cysteine in vivo to form L-luciferin via a condensation reaction; and L-luciferin inverts into D-luciferin in luciferase and thioesterase. The presently proposed mechanism not only supports the sporadic evidence from previous experiments but also clearly describes the complete process of luciferin regeneration. This work is of great significance for understanding the long-term flashing of fireflies without an in vitro energy supply.

A Test System for Tick-Borne Encephalitis Virus Detection Based on Bioluminescent Immunoassay

The tick-borne encephalitis virus (TBEV) is the causative agent of one of the most severe human neuroinfections. The infection transmitted by ixodid ticks is spread throughout the forest and foreststeppe zones of the temperate climatic belt of the Eurasian continent, including the Siberian region of the Russian Federation. Despite the availability of commercial analytical systems for the detection of TBEV, the task of developing approaches to a quick and reliable analysis that can be performed routinely, particularly in environmental studies, remains topical. A solid-phase bioluminescent immunoassay for determining the tick-borne encephalitis virus (TBEV) in ticks was developed. The assay is based on the hybrid protein consisting of a modified thermostable version of Renilla muelleri luciferase and a single-chain mini-antibody to protein E. This unique protein had been obtained and investigated by the authors earlier. The current study describes the expression of the hybrid protein in two different strains of recombinant E. coli cells. The optimal conditions for obtaining a highly purified protein were found. The bioluminescent reaction of the luciferase domain was triggered with the help of the stable natural form of the substrate, a Ca2+-dependent coelenterazine-binding protein, the recombinant variant of which was obtained by the authors. The conditions for production and storage of the immunoassay components (the hybrid protein, the stable form of the luciferase substrate, and activated microplates) were determined. Using the developed test system, more than 900 tick samples were analyzed for TBEV. In terms of sensitivity (89.5%) and specificity (98.9%), the proposed method is not inferior to colorimetric detection and is much simpler and faster than the latter

Two forms of substrate for the bioluminescent reaction in three species of basidiomycetes

ABSTRACTThe luminescent response of the enzymatic system of Armillaria borealis on the cold and hot extracts from cell-free culture liquids of Inonotus obliquus, Pholiota sp. and A. borealis was examined. The greatest influence on the light emission produced by the luminescent system of A. borealis was provided by the temperature at which the probes were prepared for assay. Boiling a culture liquid on water bath for a few minutes promoted a multifold increase in the luminescence. The results of luminescence assay suggest that the substance involved in the bioluminescent reaction in higher fungi is presented in culture liquids and mycelia in two forms. In one form, it is ready to interact with the enzymatic system and in the second form, it becomes accessible for the reaction after heat treatment. The pool of thermoactivated substance was found to be much large than the amount of the ready accessible one. We suggest that predecessors of hispidin, which is fungal luciferin precursor, are responsible for this phenomenon. They are not involved in bioluminescence at their original state and are converted into the substrate under the influence of high temperature.

A fast and efficient stabilization of firefly luciferase on MIL-53(Al) via surface adsorption mechanism

Stabilization and immobilization on a proper support is essential in processing and production of enzymes. Luciferase is an unstable, thermally sensitive, and light-emitting enzyme that requires stabilization for efficiency and productivity in bio-luminescence reactions. In this work, a known and well-porous framework, MIL-53(Al), was used, for the first time, to support the immobilization of firefly luciferase. Interestingly, all the stability values were improved through the immobilization process, and the content of the loading enzyme on MIL-53(Al) was found to be significant. The results obtained indicated that the stabilization of firefly luciferase on MIL-53(Al) proceeded through the adsorption method. MIL-53(Al) was also characterized by the FT-IR, X-ray diffraction, FE-SEM, TEM, BET, and TGA techniques in order to determine the carboxylic groups and symmetric/asymmetric CO2, percentage of crystallinity and its purity, morphology, size, and shape of the MIL-53(Al) particles, and surface-to-area ratio and thermal gravimetry of support.

Reaction mechanism of the bioluminescent protein mnemiopsin1 revealed by X-ray crystallography and QM/MM simulations

Bioluminescence of a variety of marine organisms, mostly cnidarians and ctenophores, is carried out by Ca2+-dependent photoproteins. The mechanism of light emission operates via the same reaction in both animal families. Despite numerous studies on the ctenophore photoprotein family, the detailed catalytic mechanism and arrangement of amino acid residues surrounding the chromophore in this family are a mystery. Here, we report the crystal structure of Cd2+-loaded apo-mnemiopsin1, a member of the ctenophore family, at 2.15 Å resolution and used quantum mechanics/molecular mechanics (QM/MM) to investigate its reaction mechanism. The simulations suggested that an Asp-156-Arg-39-Tyr-202 triad creates a hydrogen-bonded network to facilitate the transfer of a proton from the 2-hydroperoxy group of the chromophore coelenterazine to bulk solvent. We identified a water molecule in the coelenteramide-binding cavity that forms a hydrogen bond with the amide nitrogen atom of coelenteramide, which, in turn, is hydrogen-bonded via another water molecule to Tyr-131. This observation supports the hypothesis that the function of the coelenteramide-bound water molecule is to catalyze the 2-hydroperoxycoelenterazine decarboxylation reaction by protonation of a dioxetanone anion, thereby triggering the bioluminescence reaction in the ctenophore photoprotein family.

Comparing bacterial, fungal, and human cell concentrations with rapid adenosine triphosphate measurements for indicating microbial surface contamination

The goal of this study was to test for associations between adenosine triphosphate (ATP) content and microbial concentrations on desk surfaces in school classrooms. ATP bioluminescence and quantitative polymerase chain reaction (qPCR) techniques were employed to measure total bacterial, fungal, and human cell concentrations on 66 high-traffic desks spread across 9 schools: 3 in Connecticut (CT) and 6 in Oklahoma (OK). In CT, 6 samples were taken from each desk, 1 precleaning and 5 postcleaning (after 30 minutes, 1 day, 3 days, 7 days, and 21 days). In OK, samples were taken immediately before and after cleaning each desk. Based on simple linear regression analyses, ATP values were good predictors of microbial concentrations (r = 0.8, P = .003) in both CT school postcleaning samples and OK pre- and postcleaning samples (r = 0.7, P = .00002). However, biomass reductions measured after cleaning were 1.5-2 times greater when measured by ATP than by qPCR (P = .007). Overall, ATP bioluminescence measurements correlate with qPCR-based surface measurements on school desks but may overestimate the physical removal of bacteria and fungi due to cleaning.

Mechanistic insights into the efficient intramolecular chemiexcitation of dioxetanones from TD‐DFT and multireference calculations

AbstractDespite decades of research, it is still not clear what is the mechanism behind the efficient chemiexcitation of dioxetanones in chemiluminescent and bioluminescent reactions. In fact, long‐standing theories (charge transfer‐initiated luminescence and chemically induced electron‐exchange luminescence) have been demonstrated to not be able to explain this phenomenon. Herein, a theoretical approach using reliable and up‐to‐date methodology was used to address this problem, by focusing on model dioxetanones. Time‐dependent (TD)‐Density functional theory (DFT) and multireference complete‐active‐space second‐order perturbation theory (CASPT2) calculations provided evidence that points to efficient intramolecular chemiexcitation being the result of the reacting molecules having access to a long zone of the Potential energy surface (PES), within the biradicalar region, where S0 and S1 are degenerate. Molecules with inefficient chemiexcitation are unable to reach this zone of degeneracy. Our main finding is that access to the region of degeneracy appears to be given due to increased interaction between the keto and CO2 moieties, as supported by the use of the activation strain model and Born‐Oppenheimer molecular dynamics, which extends the biradical region by delaying the rupture of the peroxide ring. Increased interaction derives from attractive electrostatic interactions between the moieties of dioxetanone. Thus, we hypothesize that efficient chemiexcitation results not only from electron/charge transfer and subsequent charge annihilation but is instead based on the degree of interaction between the keto and CO2 moieties, which controls the access to a region of degeneracy between the ground and excited states.

Protein-based fluorescent bioassay for low-dose gamma radiation exposures.

The study suggests an application of a coelenteramide-containing fluorescent protein (CLM-CFP) as a simplest bioassay for gamma radiation exposures. "Discharged obelin," a product of the bioluminescence reaction of the marine coelenterate Obelia longissima, was used as a representative of the CLM-CFP group. The bioassay is based on a simple enzymatic reaction-photochemical proton transfer in the coelenteramide-apoprotein complex. Components of this reaction differ in fluorescence color, providing, by this, an evaluation of the proton transfer efficiency in the photochemical process. This efficiency depends on the microenvironment of the coelenteramide within the protein complex, and, hence, can evaluate a destructive ability of gamma radiation. The CLM-CFP samples were exposed to gamma radiation (137Cs, 2mGy/h) for 7 and 16days at 20°C and 5°C, respectively. As a result, two fluorescence characteristics (overall fluorescence intensity and contributions of color components to the fluorescence spectra) were identified as bioassay parameters. Both parameters demonstrated high sensitivity of the CLM-CFP-based bioassay to the low-dose gamma radiation exposure (up to 100mGy). Higher temperature (20°C) enhanced the response of CLM-CFP to gamma radiation. This new bioassay can provide fluorescent multicolor assessment of protein destruction in cells and physiological liquids under exposure to low doses of gamma radiation. Graphical abstract ᅟ.

Exploring Bioluminescence Function of the Ca2+ -regulated Photoproteins with Site-directed Mutagenesis.

Site-directed mutagenesis is a powerful tool to investigate the structure-function relationship of proteins and a function of certain amino acid residues in catalytic conversion of substrates during enzymatic reactions. Hence, it is not surprising that this approach was repeatedly applied to elucidate the role of certain amino acid residues in various aspects of photoprotein bioluminescence, mostly for aequorin and obelin, and to design mutant photoproteins with altered properties (modified calcium affinity, faster or slower bioluminescence kinetics, different emission color) which would either allow the development of novel bioluminescent assays or improvement of characteristics of the already existing ones. This information, however, is scattered over different articles. In this review, we systematize the findings that were made using site-directed mutagenesis studies regarding the impact of various amino acid residues on bioluminescence of hydromedusan Ca2+ -regulated photoproteins. All key residues that have been identified are pinpointed, and their influence on different aspects of photoprotein functioning such as active photoprotein complex formation, bioluminescence reaction, calcium response and light emitter formation is discussed.

2-S-cysteinylhydroquinone is an intermediate for the firefly luciferin biosynthesis that occurs in the pupal stage of the Japanese firefly, Luciola lateralis

Firefly luciferin is a natural product that is well-known to function as the substrate of the bioluminescence reaction in luminous beetles. However, the details of the biosynthetic system are still unclear. In this study, we showed by LC-MS/MS analysis that stable isotope-labeled 2-S-cysteinylhydroquinone was incorporated into firefly luciferin in living firefly specimens. Comparison of the incorporation efficiency among the developmental stages suggested that firefly luciferin is biosynthesized predominantly in the pupal stage. We also accomplished the in vitro biosynthesis of firefly luciferin using 2-S-cysteinylhydroquinone and the crude buffer extract of firefly pupae, suggesting the presence of a biosynthetic enzyme in the pupal extract.

Disposable luciferase-based microfluidic chip for rapid assay of water pollution.

In the present study, we demonstrate the use of a disposable luciferase-based microfluidic bioassay chip for environmental monitoring and methods for fabrication. The designed microfluidic system includes a chamber with immobilized enzymes of bioluminescent bacteria Photobacterium leiognathi and Vibrio fischeri and their substrates, which dissolve after the introduction of the water sample and thus activate bioluminescent reactions. Limits of detection for copper (II) sulfate, 1,3-dihydroxybenzene and 1,4-benzoquinone for the proposed microfluidic biosensor measured 3μM, 15mM, and 2μM respectively, and these values are higher or close to the level of conventional environmental biosensors based on lyophilized bacteria. Approaches for entrapment of enzymes on poly(methyl methacrylate) (PMMA) plates using a gelatin scaffold and solvent bonding of PMMA chip plates under room temperature were suggested. The proposed microfluidic system may be used with some available luminometers and future portable luminescence readers.

Luciferase of the Japanese syllid polychaete Odontosyllis umdecimdonta

Odontosyllis undecimdonta is a marine syllid polychaete that produces bright internal and exuded bioluminescence. Despite over fifty years of biochemical investigation into Odontosyllis bioluminescence, the light-emitting small molecule substrate and catalyzing luciferase protein have remained a mystery. Here we describe the discovery of a bioluminescent protein fraction from O. undecimdonta, the identification of the luciferase using peptide and RNA sequencing, and the in vitro reconstruction of the bioluminescence reaction using highly purified O. undecimdonta luciferin and recombinant luciferase. Lastly, we found no identifiably homologous proteins in publicly available datasets. This suggests that the syllid polychaetes contain an evolutionarily unique luciferase among all characterized luminous taxa.

Bacterial Luciferase Gene Cassette as a Real-time Bioreporter for Infection Model and Drug Evaluation.

The bacterial luciferase gene cassette (lux) is an ideal bioreporter for real-time monitoring of the dynamics of bacteria because it is a fully autonomous, substrate-free bioluminescent reporter system available in a prokaryotic or eukaryotic host background. The lux operon is emerging as a powerful bioreporter for the study of a wide range of biological processes such as gene function, drug discovery and development, cellular trafficking, protein-protein interactions, and especially tumorigenesis and cancer treatment. Furthermore, the use of a high signal to noise bioluminescent bioreporter is quickly replacing traditional fluorescent bioreporter because of the lack of endogenous bioluminescent reactions in living animals. This review briefly describes how the lux operon is used for bioluminescence imaging. Current advances in bioluminescence bacteria development are summarized, focusing on their construction strategy and applications in bacterial infection and antibiotic treatment. Different construction methods of lux-expressing cell lines are also discussed. Taken together, this review provides valuable guidelines toward the development of an ideal bioluminescent bacteria or cell lines to evaluate the efficacy of a drug.

Optimized application of the secreted Nano-luciferase reporter system using an affinity purification strategy.

Secreted Nano-luciferase (secNluc) is a newly engineered secreted luciferase that possesses advantages of high structural stability, long half-life, and glow-type kinetics together with high light emission intensity, and thus would become one of the most valuable tools for bioluminescence assays. However, like other secreted luciferases, secNluc has to mix with the components in the conditioned medium surrounding test cells, or in the biological samples such as blood or urine after being secreted. These components may interfere with secNluc-catalyzed bioluminescence reactions and thus limit the application of the secNluc reporter system. In this study, we first examined the effects of three factors, pH, serum and residual reagents, on secNluc-catalyzed bioluminescence reactions, finding that these factors could interfere with bioluminescence reactions and result in background signal. To resolve these problems, we applied a simple affinity purification strategy in which secNluc was fused with a FLAG-tag, and anti-FLAG magnetic beads were used to catch and transfer the fusion protein to PBST, an optimal buffer for secNluc-catalyzed bioluminescence reactions that was identified in this study. The results indicated that this strategy could not only negate the interferences from serum or residual reagents and enhance the stability of light emission but also greatly increase signal intensity through enzyme enrichment. This strategy may contribute to biomedical studies that utilize secNluc and other secreted luciferases, especially those requiring superior sensitivity, low background noise and high reproducibility.