Mechanistic Insights into the Bacterial Luciferase-based Bioluminescence Resonance Energy Transfer Luminescence: The Role of Protein Complex Dimer.

Bacterial bioluminescence holds significant potential in the realm of optical imaging due to the inherent advantages of bioluminescence and ease of operation. However, its practical utility is hindered by its low light intensity. The fusion of bacterial luciferase with a highly fluorescent protein has been demonstrated to significantly enhance autonomous luminescence. Nevertheless, the underlying mechanism behind this enhancement remains unclear, and there is a dearth of research investigating the mechanistic aspects of bioluminescence resonance energy transfer (BRET) luminescence, whether it occurs naturally or can be achieved through experimental means. In this study, we investigated the phenomenon of bacterial luciferase-based BRET luminescence employing a range of computational techniques, including structural modeling, molecular docking, molecular dynamics simulations, as well as combined quantum mechanics and molecular mechanics calculations. The theoretical findings suggest that the BRET luminescence occurs through resonance energy transfer between the excited bioluminophore and the ground chromophore within the protein complex dimer. The proposed mechanism of the protein complex dimer offers a microscopic understanding of the intriguing BRET phenomenon and has the potential to inspire further practical applications in the field of optical imaging.

Coupling of NAD(P)H:FMN-oxidoreductase and luciferase from luminous bacteria in a viscous medium: Finding the weakest link in the chain.

The study aims at revealing the mechanisms of the viscous medium effects on the kinetic features of NAD(P)H:FMN-oxidoreductase from luminous bacteria (Red), which are exhibited in a single enzyme assay and in coupling with bacterial luciferase (BLuc). Different concentrations of glycerol and sucrose were used to vary the medium viscosity. The activity of Red, alone and in the presence of BLuc, was analyzed, as well as BLuc activity in the presence of Red, whereas in the absence of BLuc, the Red activity was suppressed in viscous medium, and in the presence of BLuc, the increase in Red activity was observed at low glycerol concentrations (5-20 wt%). The interaction of glycerol and sucrose with Red substrates FMN and NADH was studied using absorption spectroscopy and molecular dynamics. Glycerol was found to form hydrogen bonds with the phosphate groups of the substrates, unlike sucrose. A mechanism for the activation of Red in the presence of BLuc in glycerol solutions through the acceleration of FMN reoxidation was proposed. Thus, it was concluded that, under the conditions used, the weakest link of the coupled enzyme system BLuc-Red in viscous medium is the FMN concentration, which depends on Red activity and the medium viscosity.

Enhanced brightness of bacterial luciferase by bioluminescence resonance energy transfer.

Using the lux operon (luxCDABE) of bacterial bioluminescence system as an autonomous luminous reporter has been demonstrated in bacteria, plant and mammalian cells. However, applications of bacterial bioluminescence-based imaging have been limited because of its low brightness. Here, we engineered the bacterial luciferase (heterodimer of luxA and luxB) by fusion with Venus, a bright variant of yellow fluorescent protein, to induce bioluminescence resonance energy transfer (BRET). By using decanal as an externally added substrate, color change and ten-times enhancement of brightness was achieved in Escherichia coli when circularly permuted Venus was fused to the C-terminus of luxB. Expression of the Venus-fused luciferase in human embryonic kidney cell lines (HEK293T) or in Nicotiana benthamiana leaves together with the substrate biosynthesis-related genes (luxC, luxD and luxE) enhanced the autonomous bioluminescence. We believe the improved luciferase will forge the way towards the potential development of autobioluminescent reporter system allowing spatiotemporal imaging in live cells.

Use of Bacterial Luciferase as a Reporter Gene in Eukaryotic Systems.

Reporter gene assays are powerful tools for monitoring dynamic molecular changes and for evaluating the responses that occur at the genetic elements within cells in response to exogenous molecules. In general, various protein systems can be used as reporter genes, including luciferases. Here, the present protocol introduces a unique reporter gene system for monitoring molecular events in cells using bacterial luciferase (lux), which can generate blue-green light suitable for gene reporter applications with the highest cost performance. The protocol also guides the assay conditions and necessary components for using of lux gene (lux) as a eukaryotic reporter system. The lux system can be applied to monitor variety of molecular events inside mammalian cellular systems.

Protonation status and control mechanism of flavin-oxygen intermediates in the reaction of bacterial luciferase.

Bacterial luciferase catalyzes a bioluminescent reaction by oxidizing long-chain aldehydes to acids using reduced FMN and oxygen as co-substrates. Although a flavin C4a-peroxide anion is postulated to be the intermediate reacting with aldehyde prior to light liberation, no clear identification of the protonation status of this intermediate has been reported. Here, transient kinetics, pH variation, and site-directed mutagenesis were employed to probe the protonation state of the flavin C4a-hydroperoxide in bacterial luciferase. The first observed intermediate, with a λmax of 385 nm, transformed to an intermediate with a λmax of 375 nm. Spectra of the first observed intermediate were pH-dependent, with a λmax of 385 nm at pH < 8.5 and 375 at pH > 9, correlating with a pKa of 7.7-8.1. These data are consistent with the first observed flavin C4a intermediate at pH < 8.5 being the protonated flavin C4a-hydroperoxide, which loses a proton to become an active flavin C4a-peroxide. Stopped-flow studies of His44Ala, His44Asp, and His44Asn variants showed only a single intermediate with a λmax of 385 nm at all pH values, and none of these variants generate light. These data indicate that His44 variants only form a flavin C4a-hydroperoxide, but not an active flavin C4a-peroxide, indicating an essential role for His44 in deprotonating the flavin C4a-hydroperoxide and initiating chemical catalysis. We also investigated the function of the adjacent His45; stopped-flow data and molecular dynamics simulations identify the role of this residue in binding reduced FMN.

A Minimized Chemoenzymatic Cascade for Bacterial Luciferase in Bioreporter Applications.

Bacterial luciferase (Lux) catalyzes a bioluminescence reaction by using long-chain aldehyde, reduced flavin and molecular oxygen as substrates. The reaction can be applied in reporter gene systems for biomolecular detection in both prokaryotic and eukaryotic organisms. Because reduced flavin is unstable under aerobic conditions, another enzyme, flavin reductase, is needed to supply reduced flavin to the Lux-catalyzed reaction. To create a minimized cascade for Lux that would have greater ease of use, a chemoenzymatic reaction with a biomimetic nicotinamide (BNAH) was used in place of the flavin reductase reaction in the Lux system. The results showed that the minimized cascade reaction can be applied to monitor bioluminescence of the Lux reporter in eukaryotic cells effectively, and that it can achieve higher efficiencies than the system with flavin reductase. This development is useful for future applications as high-throughput detection tools for drug screening applications.

Kinetics of the thermal inactivation and the refolding of bacterial luciferases in Bacillus subtilis and in Escherichia coli differ.

Here we present a study of the thermal inactivation and the refolding of the proteins in Gram positive Bacillus subtilis. To enable use of bacterial luciferases as the models for protein thermal inactivation and refolding in B. subtilis cells, we developed a variety of bright luminescent B. subtilis strains which express luxAB genes encoding luciferases of differing thermolability. The kinetics of the thermal inactivation and the refolding of luciferases from Photorhabdus luminescens and Photobacterium leiognathi were compared in Gram negative and Gram positive bacteria. In B. subtilis cells, these luciferases are substantially more thermostable than in Escherichia coli. Thermal inactivation of the thermostable luciferase P. luminescens in B. subtilis at 48.5°Ð¡ behaves as a first-order reaction. In E.coli, the first order rate constant (Kt) of the thermal inactivation of luciferase in E. coli exceeds that observed in B. subtilis cells 2.9 times. Incubation time dependence curves for the thermal inactivation of the thermolabile luciferase of P. leiognathi luciferase in the cells of E. coli and B. subtilis may be described by first and third order kinetics, respectively. Here we shown that the levels and the rates of refolding of thermally inactivated luciferases in B. subtilis cells are substantially lower that that observed in E. coli. In dnaK-negative strains of B. subtilis, both the rates of thermal inactivation and the efficiency of refolding are similar to that observed in wild-type strains. These experiments point that the role that DnaKJE plays in thermostability of luciferases may be limited to bacterial species resembling E. coli.

Bacterial glycosyltransferase-mediated cell-surface chemoenzymatic glycan modification.

Chemoenzymatic modification of cell-surface glycan structures has emerged as a complementary approach to metabolic oligosaccharide engineering. Here, we identify Pasteurella multocida α2-3-sialyltransferase M144D mutant, Photobacterium damsela α2-6-sialyltransferase, and Helicobacter mustelae α1-2-fucosyltransferase, as efficient tools for live-cell glycan modification. Combining these enzymes with Helicobacter pylori α1-3-fucosyltransferase, we develop a host-cell-based assay to probe glycan-mediated influenza A virus (IAV) infection including wild-type and mutant strains of H1N1 and H3N2 subtypes. At high NeuAcα2-6-Gal levels, the IAV-induced host-cell death is positively correlated with haemagglutinin (HA) binding affinity to NeuAcα2-6-Gal. Remarkably, an increment of host-cell-surface sialyl Lewis X (sLeX) exacerbates the killing by several wild-type IAV strains and a previously engineered mutant HK68-MTA. Structural alignment of HAs from HK68 and HK68-MTA suggests formation of a putative hydrogen bond between Trp222 of HA-HK68-MTA and the C-4 hydroxyl group of the α1-3-linked fucose of sLeX, which may account for the enhanced host cell killing of that mutant.

The Sensitized Bioluminescence Mechanism of Bacterial Luciferase.

After more than one-half century of investigations, the mechanism of bioluminescence from the FMNH2 assisted oxygen oxidation of an aliphatic aldehyde on bacterial luciferase continues to resist elucidation. There are many types of luciferase from species of bioluminescent bacteria originating from both marine and terrestrial habitats. The luciferases all have close sequence homology, and in vitro, a highly efficient light generation is obtained from these natural metabolites as substrates. Sufficient exothermicity equivalent to the energy of a blue photon is available in the chemical oxidation of the aldehyde to the corresponding carboxylic acid, and a luciferase-bound FMNH-OOH is a key player. A high energy species, the source of the exothermicity, is unknown except that it is not a luciferin cyclic peroxide, a dioxetanone, as identified in the pathway of the firefly and the marine bioluminescence systems. Besides these natural substrates, variable bioluminescence properties are found using other reactants such as flavin analogs or aldehydes, but results also depend on the luciferase type. Some rationalization of the mechanism has resulted from spatial structure determination, NMR of intermediates and dynamic optical spectroscopy. The overall light path appears to fall into the sensitized class of chemiluminescence mechanism, distinct from the dioxetanone types.

Expression of genes encoding the luciferase from Photobacterium leiognathi in Escherichia coli Rosetta (DE3) and its application in NADH detection.

Cloning of genes encoding the luciferase from Photobacterium leiognathi YL in Escherichia coli Rosetta (DE3) was performed successfully and the expressed forms of lux AB were purified to homogeneity. Experimental measurements revealed that luciferase from Photobacterium leiognathi YL has good thermal stability and a high residual activity at extreme pH values, which are extremely important for its various ecological, industrial and medical applications. Furthermore, we made a first attempt for quantitative detection of NADH by recombinant E. coli Rosetta (DE3) coupled enzyme system. A good linear relationship between luminescence intensity and NADH with low (1-12 nmol/L) and high (10-500 nmol/L) concentration was observed, whose standard curve was y = 772.97× + 4041.1, R2  = 0.9884 and y = 1710× + 4.99 × 105 , R2  = 0.9727, respectively. Our results demonstrate a high sensitivity of recombinant E. coli coupled enzyme system to NADH on the basis of high soluble expression of recombinant luciferase and continuous and stable expression of some NAD(P)H-dependent flavin mononucleotide (FMN) reductases.

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.

Strongly enhanced bacterial bioluminescence with the ilux operon for single-cell imaging.

Bioluminescence imaging of single cells is often complicated by the requirement of exogenous luciferins that can be poorly cell-permeable or produce high background signal. Bacterial bioluminescence is unique in that it uses reduced flavin mononucleotide as a luciferin, which is abundant in all cells, making this system purely genetically encodable by the lux operon. Unfortunately, the use of bacterial bioluminescence has been limited by its low brightness compared with other luciferases. Here, we report the generation of an improved lux operon named ilux with an approximately sevenfold increased brightness when expressed in Escherichia coli; ilux can be used to image single E. coli cells with enhanced spatiotemporal resolution over several days. In addition, since only metabolically active cells produce bioluminescent signal, we show that ilux can be used to observe the effect of different antibiotics on cell viability on the single-cell level.

Re-engineering of Bacterial Luciferase; For New Aspects of Bioluminescence.

Bacterial luminescence is the end-product of biochemical reactions catalyzed by the luciferase enzyme. Nowadays, this fascinating phenomenon has been widely used as reporter and/or sensors to detect a variety of biological and environmental processes. The enhancement or diversification of the luciferase activities will increase the versatility of bacterial luminescence. Here, to establish the strategy for luciferase engineering, we summarized the identity and relevant roles of key amino acid residues modulating luciferase in Vibrio harveyi, a model luminous bacterium. The current opinions on crystal structures and the critical amino acid residues involved in the substrate binding sites and unstructured loop have been delineated. Based on these, the potential target residues and/or parameters for enzyme engineering were also suggested in limited scale. In conclusion, even though the accurate knowledge on the bacterial luciferase is yet to be reported, the structure-guided site-directed mutagenesis approaches targeting the regulatory amino acids will provide a useful platform to re-engineer the bacterial luciferase in the future.

Development of a luminescent mutagenicity test for high-throughput screening of aquatic samples.

The Salmonella reversion based Ames test is the most widely used method for mutagenicity testing. For rapid toxicity assessment of e.g. water samples and for effect-directed analysis, however, the Ames test suffers from lack of throughput and is regarded as a laborious, time consuming method. To achieve faster analysis, with increased throughput, a (downscaled) luminescent derivative of the Ames Salmonella/microsome fluctuation test has been developed through expression of the Photorhabdus luminescens luciferase in the Salmonella TA98 and TA100 strains. The applicability of this test is demonstrated by analysis of environmentally relevant compounds, a suspended particulate matter extract and an industrial effluent sample. Use of the luminescent reporter reduced the required detection time from 48 to 28h with a specificity of 84% for responses reported in the literature to a set of 14 mutagens as compared to 72% in the unmodified fluctuation test. Testing of the same compounds in a downscaled luminescent format resulted in an 88% similarity with the response found in the regular luminescent format. The increase in throughput, faster analysis and potential for real-time bacterial quantification that luminescence provides, allows future application in the high-throughput screening of large numbers of samples or sample fractions, as required in effect-directed analysis in order to accelerate the identification of (novel) mutagens.

Ketones 2-heptanone, 2-nonanone, and 2-undecanone inhibit DnaK-dependent refolding of heat-inactivated bacterial luciferases in Escherichia coli cells lacking small chaperon IbpB.

Many bacteria, fungi, and plants produce volatile organic compounds (VOCs) emitted to the environment. Bacterial VOCs play an important role in interactions between microorganisms and in bacterial-plant interactions. Here, we show that such VOCs as ketones 2-heptanone, 2-nonanone, and 2-undecanone inhibit the DnaKJE-ClpB bichaperone dependent refolding of heat-inactivated bacterial luciferases. The inhibitory activity of ketones had highest effect in Escherichia coli ibpB::kan cells lacking small chaperone IbpB. Effect of ketones activity increased in the series: 2-pentanone, 2-undecanone, 2-heptanone, and 2-nonanone. These observations can be explained by the interaction of ketones with hydrophobic segments of heat-inactivated substrates and the competition with the chaperones IbpAB. If the small chaperone IbpB is absent in E. coli cells, the ketones block the hydrophobic segments of the polypeptides and inhibit the action of the bichaperone system. These results are consistent with the data on inhibitory effects of VOCs on survival of bacteria. It can be suggested that the inhibitory activity of the ketones indicated is associated with different ability of these substances to interact with hydrophobic segments in proteins.

Evidence for the generation of myristylated FMN by bacterial luciferase.

The genes responsible for the light production in bioluminescent bacteria are present as an operon, luxCDABEG. Many strains of Photobacteria carry an additional gene, termed luxF. X-ray crystallographic analysis of LuxF revealed the presence of four molecules of a flavin derivative, i.e. 6-(3'-(R)-myristyl) flavin adenine mononucleotide (myrFMN) non-covalently bound to the homodimer. In the present study, we exploited the binding of myrFMN to recombinant apo-LuxF to explore the occurrence of myrFMN in various bioluminescent bacteria. MyrFMN was detected in all bacterial strains tested including Vibrio and Aliivibrio indicating that it is more widely occurring in bioluminescent bacteria than previously assumed. We also show that apo-LuxF captures myrFMN and thereby relieves the inhibitory effect on luciferase activity. Thus our results provide support for the hypothesis that LuxF acts as a scavenger of myrFMN in bioluminescent bacteria. However, the source of myrFMN remained obscure. To address this issue, we established a cofactor regeneration enzyme-catalyzed cascade reaction that supports luciferase activity in vitro for up to 3 days. This approach enabled us to unambiguously demonstrate that myrFMN is generated in the bacterial bioluminescent reaction. Based on this finding we postulate a reaction mechanism for myrFMN generation that is based on the luciferase reaction.

Electrochemical Control of Bioluminescence by Blocking the Adsorption of the Bacterial Luciferase Using a Mercaptobipyridine Self-assembled Monolayer.

An N-butyl-N'-(4-mercaptobutyl)-4,4'-bipyridinium (4BMBP) was modified on a gold electrode to improve the electrochemical control of the bacterial luciferase (BL) luminescence system. The 4BMBP-modified gold electrode (4BMBP/Au) was able to prevent the adsorption of BL on the electrode surface, and enhanced the electrochemical regeneration rate of the reduced flavin mononucleotide (FMNH2), which is one of the substrates of the BL luminescence reaction. By using the 4BMBP/Au, the luminescence intensity increased by about 27% compared to that of a bare gold electrode (bare Au). Moreover, the modified electrode improved the time required for analysis because the modified layer prevented BL adsorption. Even without a refreshing procedure for each measurement, a constant luminescence intensity could be observed, and the analysis time was reduced to half (about 10 min) for one sample. The 4BMBP/Au is not only useful to control of the BL luminescence system, but also for electrochemical measurements in the presence of proteins.

Synthesis of α,ß-unsaturated aldehydes as potential substrates for bacterial luciferases.

Bacterial luciferase catalyzes the monooxygenation of long-chain aldehydes such as tetradecanal to the corresponding acid accompanied by light emission with a maximum at 490nm. In this study even numbered aldehydes with eight, ten, twelve and fourteen carbon atoms were compared with analogs having a double bond at the α,ß-position. These α,ß-unsaturated aldehydes were synthesized in three steps and were examined as potential substrates in vitro. The luciferase of Photobacterium leiognathi was found to convert these analogs and showed a reduced but significant bioluminescence activity compared to tetradecanal. This study showed the trend that aldehydes, both saturated and unsaturated, with longer chain lengths had higher activity in terms of bioluminescence than shorter chain lengths. The maximal light intensity of (E)-tetradec-2-enal was approximately half with luciferase of P. leiognathi, compared to tetradecanal. Luciferases of Vibrio harveyi and Aliivibrio fisheri accepted these newly synthesized substrates but light emission dropped drastically compared to saturated aldehydes. The onset and the decay rate of bioluminescence were much slower, when using unsaturated substrates, indicating a kinetic effect. As a result the duration of the light emission is doubled. These results suggest that the substrate scope of bacterial luciferases is broader than previously reported.

A bioluminescent arsenite biosensor designed for inline water analyzer.

Whole-cell biosensors based on the reporter gene system can offer rapid detection of trace levels of organic or metallic compounds in water. They are well characterized in laboratory conditions, but their transfer into technological devices for the surveillance of water networks remains at a conceptual level. The development of a semi-autonomous inline water analyzer stumbles across the conservation of the bacterial biosensors over a period of time compatible with the autonomy requested by the end-user while maintaining a satisfactory sensitivity, specificity, and time response. We focused here on assessing the effect of lyophilization on two biosensors based on the reporter gene system and hosted in Escherichia coli. The reporter gene used here is the entire bacterial luciferase lux operon (luxCDABE) for an autonomous bioluminescence emission without the need to add any substrate. In the cell-survival biosensor that is used to determine the overall fitness of the bacteria when mixed with the water sample, lux expression is driven by a constitutive E. coli promoter PrpoD. In the arsenite biosensor, the arsenite-inducible promoter P ars involved in arsenite resistance in E. coli controls lux expression. Evaluation of the shelf life of these lyophilized biosensors kept at 4 °C over a year evidenced that about 40 % of the lyophilized cells can be revived in such storage conditions. The performances of the lyophilized biosensor after 7 months in storage are maintained, with a detection limit of 0.2 µM arsenite for a response in about an hour with good reproducibility. These results pave the way to the use in tandem of both biosensors (one for general toxicity and one for arsenite contamination) as consumables of an autonomous analyzer in the field.