Magnetothermal Control of Temperature-Sensitive Repressors in Superparamagnetic Iron Nanoparticle-Coated Bacillus subtilis.

Superparamagnetic iron oxide nanoparticles (SPIONs) are used as contrast agents in magnetic resonance imaging (MRI) and magnetic particle imaging (MPI), and resulting images can be used to guide magnetothermal heating. Alternating magnetic fields (AMF) cause local temperature increases in regions with SPIONs, and we investigated the ability of magnetic hyperthermia to regulate temperature-sensitive repressors (TSRs) of bacterial transcription. The TSR, TlpA39, was derived from a Gram-negative bacterium and used here for thermal control of reporter gene expression in Gram-positive, Bacillus subtilis. In vitro heating of B. subtilis with TlpA39 controlling bacterial luciferase expression resulted in a 14.6-fold (12 hours; h) and 1.8-fold (1 h) increase in reporter transcripts with a 10.0-fold (12 h) and 12.1-fold (1 h) increase in bioluminescence. To develop magnetothermal control, B. subtilis cells were coated with three SPION variations. Electron microscopy coupled with energy dispersive X-ray spectroscopy revealed an external association with, and retention of, SPIONs on B. subtilis. Furthermore, using long duration AMF we demonstrated magnetothermal induction of the TSRs in SPION-coated B. subtilis with a maximum of 5.6-fold increases in bioluminescence. After intramuscular injections of SPION-coated B. subtilis, histology revealed that SPIONs remained in the same locations as the bacteria. For in vivo studies, 1 h of AMF is the maximum exposure due to anesthesia constraints. Both in vitro and in vivo, there was no change in bioluminescence after 1 h of AMF treatment. Pairing TSRs with magnetothermal energy using SPIONs for localized heating with AMF can lead to transcriptional control that expands options for targeted bacteriotherapies.

Development of Whole-Cell Biosensors for Screening of Peptidoglycan-Targeting Antibiotics in a Gram-Negative Bacterium.

There is an urgent need to develop novel antibiotics since antibiotic resistance is an increasingly serious threat to global public health. Whole-cell biosensors are one of the promising strategies for new antibiotic discovery. The peptidoglycan (PG) of the bacterial cell wall is one of the most important targets for antibiotics. However, the biosensors for the detection of PG-targeting antibiotics in Gram-negative bacteria have not been developed, mainly because of the lack of the regulatory systems that sense and respond to PG stress. Recently, we identified a novel two-component signal transduction system (PghKR) that is responsible for sensing and responding to PG damage in the Gram-negative bacterium Shewanella oneidensis. Based on this system, we developed biosensors for the detection of PG-targeting antibiotics. Using ampicillin as an inducer for PG stress and the bacterial luciferase LuxCDABE as the reporter, we found that the PghKR biosensors are specific to antibiotics targeting PG synthesis, including ß-lactams, vancomycin, and d-cycloserine. Deletion of genes encoding PG permease AmpG and ß-lactamase BlaA improves the sensitivity of the biosensors substantially. The PghKR biosensor in the background of ΔblaA is also functional on agar plates, providing a simple method for screening bacteria that produce PG-targeting antibiotics. IMPORTANCE The growing problem of antibiotic resistance in Gram-negative bacteria urgently needs new strategies so that researchers can develop novel antibiotics. Microbial whole-cell biosensors are capable of sensing various stimuli with a quantifiable output and show tremendous potential for the discovery of novel antibiotics. As the Achilles' heel of bacteria, the synthesis of the peptidoglycan (PG) is targeted by many antibiotics. However, the regulatory systems that sense and respond to PG-targeting stress in Gram-negative bacteria are reported rarely, restricting the development of biosensors for the detection of PG-targeting antibiotics. In this study, we developed a highly sensitive and specific biosensor based on a novel two-component system in the Gram-negative bacterium Shewanella oneidensis that is responsible for the sensing and responding to PG stress. Our biosensors have great potential for discovering novel antibiotics and determining the mode of action of antibiotics.

Structure-Function Relationships in Temperature Effects on Bacterial Luciferases: Nothing Is Perfect.

The evaluation of temperature effects on the structure and function of enzymes is necessary to understand the mechanisms underlying their adaptation to a constantly changing environment. In the current study, we investigated the influence of temperature variation on the activity, structural dynamics, thermal inactivation and denaturation of Photobacterium leiognathi and Vibrio harveyi luciferases belonging to different subfamilies, as well as the role of sucrose in maintaining the enzymes functioning and stability. We used the stopped-flow technique, differential scanning calorimetry and molecular dynamics to study the activity, inactivation rate, denaturation and structural features of the enzymes under various temperatures. It was found that P. leiognathi luciferase resembles the properties of cold-adapted enzymes with high activity in a narrow temperature range and slightly lower thermal stability than V. harveyi luciferase, which is less active, but more thermostable. Differences in activity at the studied temperatures can be associated with the peculiarities of the mobile loop conformational changes. The presence of sucrose does not provide an advantage in activity but increases the stability of the enzymes. Differential scanning calorimetry experiments showed that luciferases probably follow different denaturation schemes.

QM/MM Molecular Modeling Reveals Mechanism Insights into Flavin Peroxide Formation in Bacterial Luciferase.

Bacterial luciferase (Lux) catalyzes oxidation of reduced flavin mononucleotide (FMN) and aldehyde to form oxidized FMN and carboxylic acid via molecular oxygen with concomitant light generation. The enzyme is useful for various detection applications in biomedical experiments. Upon reacting with oxygen, the reduced FMN generates C4a-peroxy-FMN (FMNH-C4a-OO-) as a reactive intermediate, which is required for light generation. However, the mechanism and control of FMNH-C4a-OO- formation are not clear. This work investigated the reaction of FMNH-C4a-OO- formation in Lux using QM/MM methods. The B3LYP/6-31G*/CHARMM27 calculations indicate that Lux controls the formation of FMNH-C4a-OO- via the conserved His44 residue. The steps in intermediate formation are found to be as follows: (i) H+ reacts with O2 to generate +OOH. (ii) +OOH attacks C4a of FMNH- to generate FMNH-C4a-OOH. (iii) H+ is transferred from FMNH-C4a-OOH to His44 to generate FMNH-C4a-OO- while His44 stabilizes FMNH-C4a-OO- by forming a hydrogen bond to an oxygen atom. This controlling key mechanism for driving the change from FMNH-C4a-OOH to the FMNH-C4a-OO- adduct is confirmed because FMNH-C4a-OO- is more stable than FMNH-C4a-OOH in the luciferase active site.

Golden Gate Assembly of Aerobic and Anaerobic Microbial Bioreporters.

Microbial bioreporters provide direct insight into cellular processes by producing a quantifiable signal dictated by reporter gene expression. The core of a bioreporter is a genetic circuit in which a reporter gene (or operon) is fused to promoter and regulatory sequences that govern its expression. In this study, we develop a system for constructing novel Escherichia coli bioreporters based on Golden Gate assembly, a synthetic biology approach for the rapid and seamless fusion of DNA fragments. Gene circuits are generated by fusing promoter and reporter sequences encoding yellow fluorescent protein, mCherry, bacterial luciferase, and an anaerobically active flavin-based fluorescent protein. We address a barrier to the implementation of Golden Gate assembly by designing a series of compatible destination vectors that can accommodate the assemblies. We validate the approach by measuring the activity of constitutive bioreporters and mercury and arsenic biosensors in quantitative exposure assays. We also demonstrate anaerobic quantification of mercury and arsenic in biosensors that produce flavin-based fluorescent protein, highlighting the expanding range of redox conditions that can be examined by microbial bioreporters. IMPORTANCE Microbial bioreporters are versatile genetic tools with wide-ranging applications, particularly in the field of environmental toxicology. For example, biosensors that produce a signal output in the presence of a specific analyte offer less costly alternatives to analytical methods for the detection of environmental toxins such as mercury and arsenic. Biosensors of specific toxins can also be used to test hypotheses regarding mechanisms of uptake, toxicity, and biotransformation. In this study, we develop an assembly platform that uses a synthetic biology technique to streamline construction of novel Escherichia coli bioreporters that produce fluorescent or luminescent signals either constitutively or in response to mercury and arsenic exposure. Beyond the synthesis of novel biosensors, our assembly platform can be adapted for numerous applications, including labeling bacteria for fluorescence microscopy, developing gene expression systems, and modifying bacterial genomes.

Bacterial Luciferases from Vibrio harveyi and Photobacterium leiognathi Demonstrate Different Conformational Stability as Detected by Time-Resolved Fluorescence Spectroscopy.

Detecting the folding/unfolding pathways of biological macromolecules is one of the urgent problems of molecular biophysics. The unfolding of bacterial luciferase from Vibrio harveyi is well-studied, unlike that of Photobacterium leiognathi, despite the fact that both of them are actively used as a reporter system. The aim of this study was to compare the conformational transitions of these luciferases from two different protein subfamilies during equilibrium unfolding with urea. Intrinsic steady-state and time-resolved fluorescence spectra and circular dichroism spectra were used to determine the stages of the protein unfolding. Molecular dynamics methods were applied to find the differences in the surroundings of tryptophans in both luciferases. We found that the unfolding pathway is the same for the studied luciferases. However, the results obtained indicate more stable tertiary and secondary structures of P. leiognathi luciferase as compared to enzyme from V. harveyi during the last stage of denaturation, including the unfolding of individual subunits. The distinctions in fluorescence of the two proteins are associated with differences in the structure of the C-terminal domain of α-subunits, which causes different quenching of tryptophan emissions. The time-resolved fluorescence technique proved to be a more effective method for studying protein unfolding than steady-state methods.

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.

A Whole-Cell Bacterial Biosensor for Blood Markers Detection in Urine.

The early detection of blood in urine (hematuria) can play a crucial role in the treatment of serious diseases (e.g., infections, kidney disease, schistosomiasis, and cancer). Therefore, the development of low-cost portable biosensors for blood detection in urine has become necessary. Here, we designed an ultrasensitive whole-cell bacterial biosensor interfaced with an optoelectronic measurement module for heme detection in urine. Heme is a red blood cells (RBCs) component that is liberated from lysed cells. The bacterial biosensor includes Escherichia coli cells carrying a heme-sensitive synthetic promoter integrated with a luciferase reporter (luxCDABE) from Photorhabdus luminescens. To improve the bacterial biosensor performance, we re-engineered the genetic structure of luxCDABE operon by splitting it into two parts (luxCDE and luxAB). The luxCDE genes were regulated by the heme-sensitive promoter, and the luxAB genes were regulated by either constitutive or inducible promoters. We examined the genetic circuit's performance in synthetic urine diluent supplied with heme and in human urine supplied with lysed blood. Finally, we interfaced the bacterial biosensor with a light detection setup based on a commercial optical measurement single-photon avalanche photodiode (SPAD). The whole-cell biosensor was tested in human urine with lysed blood, demonstrating a low-cost, portable, and easy-to-use hematuria detection with an ON-to-OFF ratio of 6.5-fold for blood levels from 5 × 104 to 5 × 105 RBC per mL of human urine.

QM/MM Investigation of the Spectroscopic Properties of the Fluorophore of Bacterial Luciferase.

We employ replica-exchange molecular dynamics (REMD) and a hybrid ab initio multiconfigurational quantum mechanics/molecular mechanics (QM/MM) approach to model the absorption and fluorescence properties of bacterial luciferin-luciferase. Specifically, we employ complete active space perturbation theory (CASPT2) and study the effect of active space, basis set, and IPEA shift on the computed energies. We discuss the effect of the protein environment on the fluorophore's excited-state potential energy surface and the role that the protein plays in enhancing the fluorescence quantum yield in bacterial bioluminescence.

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.

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 ß subunits, where α subunit is a catalytic center and ß 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.

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.

Improving Estrogenic Compound Screening Efficiency by Using Self-Modulating, Continuously Bioluminescent Human Cell Bioreporters Expressing a Synthetic Luciferase.

A synthetic bacterial luciferase-based autobioluminescent bioreporter, HEK293ERE/Gal4-Lux, was developed in a human embryonic kidney (HEK293) cell line for the surveillance of chemicals displaying endocrine disrupting activity. Unlike alternative luminescent reporters, this bioreporter generates bioluminescence autonomously without requiring an external light-activating chemical substrate or cellular destruction. The bioreporter's performance was validated against a library of 76 agonistic and antagonistic estrogenic endocrine disruptor chemicals and demonstrated reproducible half maximal effective concentration (EC50) values meeting the U.S. Environmental Protection Agency (EPA) guidelines for Tier 1 endocrine disrupting chemical screening assays. For model compounds, such as the estrogen receptor (ER) agonist 17ß-estradiol, HEK293ERE/Gal4-Lux demonstrated an EC50 value (7.9 × 10-12 M) comparable to that of the current EPA-approved HeLa-9903 firefly luciferase-based estrogen receptor transcription assay (4.6 × 10-12 M). Screening against an expanded array of common ER agonists likewise produced similar relative effect potencies as compared with existing assays. The self-initiated autobioluminescent signal of the bioreporter permitted facile monitoring of the effects of endocrine disrupting chemicals, which decreased the cost and hands-on time required to perform these assays. These characteristics make the HEK293ERE/Gal4-Lux bioreporter potentially suitable as a high-throughput human cell-based assay for screening estrogenic activity.

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.

The 9H-Fluoren Vinyl Ether Derivative SAM461 Inhibits Bacterial Luciferase Activity and Protects Artemia franciscana From Luminescent Vibriosis.

Vibrio campbellii is a major pathogen in aquaculture. It is a causative agent of the so-called "luminescent vibriosis," a life-threatening condition caused by bioluminescent Vibrio spp. that often involves mass mortality of farmed shrimps. The emergence of multidrug resistant Vibrio strains raises a concern and poses a challenge for the treatment of this infection in the coming years. Inhibition of bacterial cell-to-cell communication or quorum sensing (QS) has been proposed as an alternative to antibiotic therapies. Aiming to identify novel QS disruptors, the 9H-fluroen-9yl vinyl ether derivative SAM461 was found to thwart V. campbellii bioluminescence, a QS-regulated phenotype. Phenotypic and gene expression analyses revealed, however, that the mode of action of SAM461 was unrelated to QS inhibition. Further evaluation with purified Vibrio fischeri and NanoLuc luciferases revealed enzymatic inhibition at micromolar concentrations. In silico analysis by molecular docking suggested binding of SAM461 in the active site cavities of both luciferase enzymes. Subsequent in vivo testing of SAM461 with gnotobiotic Artemia franciscana nauplii demonstrated naupliar protection against V. campbellii infection at low micromolar concentrations. Taken together, these findings suggest that suppression of luciferase activity could constitute a novel paradigm in the development of alternative anti-infective chemotherapies against luminescent vibriosis, and pave the ground for the chemical synthesis and biological characterization of derivatives with promising antimicrobial prospects.

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.