Enhanced Beetle Luciferase for High-Resolution Bioluminescence Imaging

We developed an enhanced green-emitting luciferase (ELuc) to be used as a bioluminescence imaging (BLI) probe. ELuc exhibits a light signal in mammalian cells that is over 10-fold stronger than that of the firefly luciferase (FLuc), which is the most widely used luciferase reporter gene. We showed that ELuc produces a strong light signal in primary cells and tissues and that it enables the visualization of gene expression with high temporal resolution at the single-cell level. Moreover, we successfully imaged the nucleocytoplasmic shuttling of importin α by fusing ELuc at the intracellular level. These results demonstrate that the use of ELuc allows a BLI spatiotemporal resolution far greater than that provided by FLuc.     

Effects of aldehyde and internal ions on bioluminescence expression of Photobacterium phosphoreum

In a complex medium, cells of Photobacterium phosphoreum (strain 496) grow equally well with 1% and 3% NaCl, but luminescence occurs only with 3% NaCl in the medium. However, the suppression of luminescence is not attributable to the lack of luciferase; log phase cells growing in 1% NaCl will develop luminescence following a shift to 3% NaCl, which is accompanied by an increase of intracellular potassium. Tetradecanal stimulates bioluminescence in a 1% NaCl culture, and also in the presence of nalidixic acid, an inhibitor or gyrase. It is thus suggested that the suppression of luminescence in 1% NaCl or in 3% NaCl with nalidixic acid is due to a deficiency in the synthesis of intracellular aldehyde. The increase in intracellular potassium that occurs upon shifting from 1% to 3% NaCl may also relate to aldehyde synthesis gene expression via activation of gyrase, or via an increase in negative supercoiling of the chromosome. However, since an initial decrease of light intensity is still observed during culture even with the addition of tetradecanal, an additional factor related to cell density must also be involved in bioluminescence expression.Abbreviations nal nalidixic acid - nal-r nalidixic acid resistant strain     

Gaussia princeps luciferase: a bioluminescent substrate for oxidative protein folding

Gaussia princeps luciferase (GLuc) generates an intense burst of blue light when exposed to coelenterazine in the absence of ATP. Here we show that this 5‐disulfide containing enzyme can be used as a facile and convenient substrate for studies of oxidative protein folding. Reduced GLuc (rGLuc), with 10 free cysteine residues, is completely inactive as a luciferase but >60% bioluminescence activity, compared to controls, can be recovered using a range of oxidizing regimens in the absence of the exogenous shuffling activity of protein disulfide isomerase (PDI). The sulfhydryl oxidase QSOX1 can be assayed using rGLuc in a simple bioluminescence plate reader format. Similarly, low concentrations of rGLuc can be oxidized by millimolar levels of dehydroascorbate, hydrogen peroxide or much lower concentrations of sodium tetrathionate. The oxidative refolding of rGLuc in the presence of a range of glutathione redox buffers is only marginally accelerated by micromolar levels of PDI. This modest rate enhancement probably results from a relatively simple disulfide connectivity in native GLuc; reflecting two homologous domains each carrying two disulfide bonds with a single interdomain disulfide. When GLuc is reoxidized under denaturing conditions the resulting scrambled protein (sGLuc) can be used in a sensitive bioluminescence assay for reduced PDI in the absence of added exogenous thiols. Finally, the general facility by which rGLuc can recover bioluminescent activity in vitro provides a sensitive method for the assessment of inhibitors of oxidative protein folding.     

Tracing location by applying Emerald luciferase in an early phase of murine endometriotic lesion formation

The pathogenesis of endometriosis has not been fully elucidated. We focused on the behavior of the ectopic endometrium, that is, the origin of the endometriotic lesion, before adhering to the peritoneal cavity. To observe lesion formation in the very early phase, we developed a novel endometriosis animal model using bioluminescence technology. We established a new transgenic mouse that expressed Emerald luciferase (ELuc) under the control of the CAG promoter. This transgenic mouse, called the CAG-ELuc mouse, showed strong bioluminescence emission; we succeeded in tracing the lesion location by the emission of ELuc. The accuracy of tracing by ELuc was high (57.7–100% of correspondence) and depended on the dosage of E2 administration. In the very early phase after transplantation, the process of lesion formation can be observed non-invasively and chronologically. We have verified that the preferred location of the uterus (transplanted grafts) was fixed immediately after the transplantation of the grafts.     

Multiply mutated Gaussia luciferases provide prolonged and intense bioluminescence

Gaussia luciferase (GLuc) from the copepod Gaussia princeps is both the smallest and brightest known luciferase. GLuc catalyzes the oxidation of coelenterazine to produce an intense blue light but with a very short emission half-life. We report mutated GLucs with much longer luminescence half-lives that retain the same initial intensity as the wild-type enzyme. The GLuc variants were produced using cell-free protein synthesis to provide high yields and rapid production of fully active product as well as simple non-natural amino acid substitution. By incorporating homopropargylglycine and attaching PEG using azide-alkyne click reactions, we also show that the four methionines in GLuc are surface accessible. The mutants provide a significantly improved reporter protein for both in vivo and in vitro studies, and the successful non-natural amino acid incorporation and PEG attachment indicate the feasibility of producing useful bioconjugates using click attachment reactions.     

Soluble electron acceptors affect bioluminescence from Shewanella woodyi

Shewanella woodyi cultures were used to correlate bioluminescence intensity with changes in the electrochemical potential of a saltwater medium using soluble electron acceptors. A relationship between the concentration of NaNO₃ or CoCl₂ to bioluminescence intensity was confirmed using aerobic cultures of S. woodyi at 20°C with glucose as the sole carbon source. In general, increasing the concentration of nitrate or Co(II) reduced the bioluminescence per cell, with complete luminescence being repressed at ≥5 mM nitrate and ≥0.5 mM Co(II). Results from cell viability fluorescent staining concluded that increasing the concentration of Co(II) or nitrate did not affect the overall viability of the cells when compared with cultures lacking Co(II) or nitrate. These data show that potentials of <0.2 V vs Normal Hydrogen Electrode (NHE) repress the luminescence from the cells, but the exact mechanism is unclear. Our results indicated that the luminescence intensity from S. woodyi could be systematically reduced using these two soluble electron acceptors, making S. woodyi a potential model bacterium for whole‐cell luminescence bioelectrochemical sensor applications.     

Preparation and characterization of fusion protein truncated Pseudomonas Exotoxin A (PE38KDEL) in Escherichia coli

Pseudomonas Exotoxin A (PE) and truncated PE have been used to prepare immunotoxin with monoclonal antibodies. Truncated Pseudomonas Exotoxin A (PE38KDEL) was expressed with the pET-32a(+) vector in Escherichia coli under control of a T7 promoter. The recombinant protein was purified by His-Ni(2+) metal affinity chromatography and gel filtration. The biological activity of PE38KDEL was evaluated by the inhibition assay of protein synthesis in rabbit reticulocyte lysate system, and the cytotoxicity was tested in Hut 102 and hepatocellular cell lines by the MTS assay. PE38KDEL can significantly inhibit luciferase synthesis in cell-free protein synthesis assay and was slightly cytotoxic in the Hut 102 and hepatocellular cell lines. The results suggest that PE38KDEL would be useful for the preparation of more potent immunotoxins.     

Alternative luciferase for monitoring bacterial cells under adverse conditions

The availability of cloned luciferase genes from fireflies (luc) and from bacteria (luxAB) has led to the widespread use of bioluminescence as a reporter to measure cell viability and gene expression. The most commonly occurring bioluminescence system in nature is the deep-sea imidazolopyrazine bioluminescence system. Coelenterazine is an imidazolopyrazine derivative which, when oxidized by an appropriate luciferase enzyme, produces carbon dioxide, coelenteramide, and light. The luciferase from the marine copepod Gaussia princeps (Gluc) has recently been cloned. We expressed the Gluc gene in Mycobacterium smegmatis using a shuttle vector and compared its performance with that of an existing luxAB reporter. In contrast to luxAB, the Gluc luciferase retained its luminescence output in the stationary phase of growth and exhibited enhanced stability during exposure to low pH, hydrogen peroxide, and high temperature. The work presented here demonstrated the utility of the copepod luciferase bioluminescent reporter as an alternative to bacterial luciferase, particularly for monitoring responses to environmental stress stimuli.     

Autoinduction of bacterial bioluminescence in a carbon limited chemostat

Several strains of four species of luminous marine bacteria were maintained in a chemostat at a constant dilution rate and a variety of steady state densities by carbon (glycerol) limitation in order to study the relationship between culture density and bioluminescence activity. In general, luminescence per cell was constant at high culture density, and decreased dramatically at low culture density. For Vibrio fischeri, luminescence decreased to nondectable levels when the culture was maintained at low density; such dark cells were stimulated to synthesize luciferase and became luminous within minutes when purified autoinducer was added to the chemostat. Two strains, Photobacterium phosphoreum NZ11D and Photobacterium leiognathi S1, did not show the decrease in light intensity at low culture density that was characteristic of all other strains tested; they appeared to be constitutive for bioluminescence.Abbreviations BCM basal salts glycerol medium - BM basal salts medium - BSA bovine serum albumin - D dilution rate - DTT dilitiothreitol - LU light unit=2×10¹⁰ quanta s^-1 - OD optical density - SWC sea water complete medium - specific growth rate     

Inhibition of bioluminescence in Photobacterium phosphoreum by sulfamethizole and its stimulation by thymine

In bioluminescent bacteria very few agents have been reported that can selectively inhibit the luminescence. In sensitivity tests with Photobacterium phosphoreum, using 55 different antibiotics, it was found that sulfamethizole, an inhibitor of dihydropteroate synthetase and the formation of folic acid, inhibited bioluminescence more than growth. Likewise, in mutants requiring thymine for growth, the luminescence per cell was much less in a medium low in thymine. In neither case could the decreased specific luminescence be attributed to a decrease in the cellular level of luciferase or aldehyde factor; the involvement of additional but unidentified factors in the regulation of in vivo bioluminescence is postulated.     

The effect of Hg2+ on the bioluminescence of Photobacterium leiognathi

Photobacteria were used as test objects for rapid monitoring of ecotoxicants. Specific inhibitory effects of Hg²⁺ on bioluminescence and cell growth as well as the lux gene expression of Photobacterium leiognathi were studied. The 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide reduction assay was used to evaluate cellular proliferation and mortality. The luminescent inhibition effect on P. leiognathi cells was found to increase as cellular mortality increased; y = 0.744x ‐ 4.8916, R² = 0.9794. However, this trend was not observed in cell growth processes. Quantitation of lux mRNAs by semi‐quantitative RT‐qPCR indicated that increases and decreases in luciferase mRNA integral level coincided with changes in luminescence intensity (R² = 0.93). Addition of Hg²⁺ changed luminescence but without concomitant changes in extractable luciferase activity. Nevertheless, the presence of Hg²⁺ changed lux gene expression. This phenomenon requires further research. Copyright © 2012 John Wiley & Sons, Ltd.     

Rapid and highly sensitive detection of cadmium chloride induced cytotoxicity using the HSP70B' promoter in live cells

One unique to detect cytotoxicity is to utilize reporter gene assays for promoters that respond to stress-induced effects. In the present study, we discovered that the DNA sequence from nt -287 to +110 of the heat shock protein 70B' (HSP70B') gene could be used as a functional promoter to detect cytotoxicity of cadmium chloride. We thus detected cytotoxicity induced by cadmium chloride with the luciferase assay using this functional HSP70B' promoter, as well as the cell viability test based on the quantification of intracellular ATP. The luciferase assay using the functional HSP70B' promoter resulted in nearly maximal luciferase activity after only 12 h of exposure to cadmium chloride, however, with intracellular ATP quantification, the decrease in cell viability only reached a plateau after 24 h of exposure. Cytotoxicity detection limits for cadmium chloride with the functional HSP70B' promoter assay or cell viability based on ATP quantification were 130 ng/mL and 530 ng/mL, respectively. Our results therefore suggest that the novel reporter gene assay using a functional region of the HSP70B' promoter has significant advantages for the detection of cytotoxicity in terms of both speed and sensitivity, when compared to the cell viability test based on ATP quantification.     

A Gaussia Luciferase Cell-Based System to Assess the Infection of Cell Culture- and Serum-Derived Hepatitis C Virus

Robust replication of hepatitis C virus (HCV) in cell culture occurs only with the JFH-1 (genotype 2a) recombinant genome. The aim of this study was to develop a system for HCV infection quantification analysis and apply it for the selection of patient sera that may contain cell culture infectious viruses, particularly of the most clinically important genotype 1. Initially, a hepatoma cell line (designated Huh-7.5/EG(4A/4B)GLuc) was generated that stably expressed the enhanced green fluorescent protein (EGFP) fused in-frame to the secreted Gaussia luciferase via a recognition sequence of the viral NS3/4A protease. Upon HCV infection, NS3/4A cleaved at its signal and the Gaussia was secreted to the culture medium, thus facilitating the infection quantification. The Huh-7.5/EG(4A/4B)GLuc cell line provided a rapid and highly sensitive quantification of HCV infection in cell culture using JFH-1-derived viruses. Furthermore, the Huh-7.5/EG(4A/4B)GLuc cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target distinct stages of the HCV life cycle; the Ź-factor of this assay ranged from 0.72 to 0.75. Additionally, eighty-six sera derived from HCV genotype 1b infected liver transplant recipients were screened for their in vitro infection and replication potential. Approximately 12% of the sera contained in vitro replication-competent viruses, as deduced by the Gaussia signal, real time quantitative PCR, immunofluorescence and capsid protein secretion. We conclude that the Huh-7.5/EG(4A/4B)GLuc cell line is an excellent system not only for the screening of in vitro replication-competent serum-derived viruses, but also for the subsequent cloning of recombinant isolates. Additionally, it can be utilized for high-throughput screening of antiviral compounds.     

Alternative Luciferase for Monitoring Bacterial Cells under Adverse Conditions

The availability of cloned luciferase genes from fireflies (luc) and from bacteria (luxAB) has led to the widespread use of bioluminescence as a reporter to measure cell viability and gene expression. The most commonly occurring bioluminescence system in nature is the deep-sea imidazolopyrazine bioluminescence system. Coelenterazine is an imidazolopyrazine derivative which, when oxidized by an appropriate luciferase enzyme, produces carbon dioxide, coelenteramide, and light. The luciferase from the marine copepod Gaussia princeps (Gluc) has recently been cloned. We expressed the Gluc gene in Mycobacterium smegmatis using a shuttle vector and compared its performance with that of an existing luxAB reporter. In contrast to luxAB, the Gluc luciferase retained its luminescence output in the stationary phase of growth and exhibited enhanced stability during exposure to low pH, hydrogen peroxide, and high temperature. The work presented here demonstrated the utility of the copepod luciferase bioluminescent reporter as an alternative to bacterial luciferase, particularly for monitoring responses to environmental stress stimuli.     

Juglone, a naphthoquinone from walnut, exerts cytotoxic and genotoxic effects against cultured melanoma tumor cells

This study demonstrates cytotoxic and genotoxic potential of juglone, a chief constituent of walnut, and its underlying mechanisms against melanoma cells. MTT assay and clonogenic assay were used to study cytotoxicity, micronucleus assay to assess genotoxicity, glutathione (GSH) assay and 2′,7′-dicholorofluorescein diacetate (DCFH-DA) assay to evaluate the oxidative stress induction. Apoptosis/necrosis induction was analysed by flow cytometry. We observed a concentration-dependent decrease in cell survival with a corresponding increase in the lactate dehydrogenase levels. A dose-dependent increase in the frequency of micronucleated binucleate cells indicated the potential of juglone to induce cytogenetic damage in melanoma tumor cells. Moreover, results of the micronuclei study indicated division delay in the proliferating cell population by showing decrease in the cytokinesis blocked proliferation index. Further, juglone-induced apoptosis and necrosis could be demonstrated by oligonucleosomal ladder formation, microscopic analysis, increase in the hypodiploid fraction (sub Go peak in DNA histogram), as well as an increased percentage of AnnexinV(+)/PI(+) cells detected by flow cytometry. A significant concentration-dependent decrease in the glutathione levels and increase in dichlorofluorescein (DCF) fluorescence after juglone treatment confirmed the ability of juglone to generate intracellular reactive oxygen species. The cytotoxic effect of juglone can be attributed to mechanisms including the induction of oxidative stress, cell membrane damage, and a clastogenic action leading to cell death by both apoptosis and necrosis.     

Effect of concentrating and exposing the bioluminescent bacteria to the non‐luminescent allo‐bacterial extracellular products on their luminescence

Bioluminescence is a biochemical process occurring in many organisms. Bacterial bioluminescence has been investigated extensively that lead to many applications of such knowledge. Quorum sensing in the bioluminescent bacteria is a chemical signal process to recognize the strength of its own population to start luminescence in harmony. There is a mechanism in these bacteria to also recognize inter‐species strength. When there is a higher number of these bacteria, the possibility and frequency of cell–cell physical contact will be high. In this study, the physical proximity was artificially enhanced between cells and the effect on luminescence in the concentrated cells in the normal culture medium and in the presence of other non‐bacterial cell‐free supernatants was investigated. The role of such physical contact in the quorum sensing in the bioluminescence is not known. Increase in the luminescence of V. fischeri when concentrated shows that the presence of physical proximity facilitates the quorum sensing for their bioluminescence. Copyright © 2009 John Wiley & Sons, Ltd.     

Suitability of Macrolampis firefly and Pyrearinus click beetle luciferases for bacterial light off toxicity biosensor

Bioluminescence is widely used in biosensors. For water toxicity analysis, the naturally bioluminescent bacteria Vibrio fischeri have been used extensively. We investigated the suitability of two new beetle luciferases for Escherichia coli light off biosensors: Macrolampis firefly and Pyrearinus termitilluminans click beetle luciferases. The bioluminescence detection assay using this system is very sensitive, being comparable or superior to V. fischeri. The luciferase of P. termitilluminans produces a strong and sustained bioluminescence that is useful for less sensitive and inexpensive assays that require integration of the emission, whereas Macrolampis luciferase displays a flash-like luminescence that is useful for fast and more sensitive assays. The effect of heavy metals and sanitizing agents was analyzed. Zinc, copper, 1-propanol, and iodide had inhibitory effects on bioluminescence and growth assays; however, in these cases the bioluminescence was not a very reliable indicator of cell growth and metabolic activity because these agents also inhibited the luciferase. On the other hand, mercury and silver strongly affected cell bioluminescence and growth but not the luciferase activity, indicating that bioluminescence was a reliable indicator of cell growth and metabolic activity in this case. Finally, bioluminescent E. coli immobilized in agarose matrix gave a more stable format for environmental assays.     

Enumeration of bacterial cell numbers by amplified firefly bioluminescence without cultivation

We recently developed a novel bioluminescent enzymatic cycling assay for ATP and AMP with the concomitant use of firefly luciferase and pyruvate orthophosphate dikinase (PPDK), where AMP and pyrophosphate produced from ATP by firefly luciferase were converted back into ATP by PPDK. Background luminescence derived from contaminating ATP and AMP in the reagent was reduced using adenosine phosphate deaminase which degrades ATP, ADP, and AMP, resulting in constant and highly amplified bioluminescence with low background luminescence. To detect bacterial cells without cultivation, we applied the above bioluminescent enzymatic cycling reagent to rapid microbe detection system. ATP spots (0.31-5.0 amol/spot) at the level of a single bacterial cell were detected with 5 min signal integration, signifying that integrated luminescence was amplified 43 times in comparison to traditional ATP bioluminescence. Consequently, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Lactobacillus brevis in beer were detected without cultivation. Significant correlation was observed between the number of signal spots obtained using this novel system and the colony-forming units observed with the conventional colony-counting method (R(2)=0.973).     

Biophysical characterization of highly active recombinant Gaussia luciferase expressed in Escherichia coli

Recently, the smallest bioluminescent protein (MW: 19.9 kDa), Gaussia luciferase (GLuc), has been isolated from the marine copepod Gaussia princeps and has attracted much attention as a reporter protein. However, preparation of large quantities of homogeneous natively folded recombinant GLuc appears to be difficult due to its ten cysteines. Here, we report the biophysical characterization of recombinant GLuc expressed using a novel Escherichia coli expression system based on a cold induced expression vector (pCold). Using this system, a large fraction of the protein was expressed in the soluble fraction. GLuc, purified exclusively from the supernatant using nickel affinity chromatography, yielded a large amount of pure GLuc with a native disulfide bond pattern (Soluble-GLuc). Soluble-GLuc had a strong bioluminescence activity and it retained 65% of its activity after 30 min incubation at 95 °C. Soluble-GLuc remained fully folded until 40 °C, as assessed by circular dichroism; and the thermal denaturation curve was S-shaped, indicating a cooperative transition, with a midpoint temperature of 56 °C. These results indicate that both the structure and bioluminescence activity of GLuc remain stable at high temperatures, and they strongly suggest GLuc's potential as a reporter protein.