Green Bioluminescence Research Articles

Spectral tuning of bioluminescence and visual sensitivity in males of Brazilian firefly species inhabiting dim light environments (Coleoptera: Elateroidea: Lampyridae).

Bioluminescence in fireflies is essential for sexual communication, and each species has evolved a specific bioluminescence emission capable of being detected by its visual system. This spectral "tuning"between visual sensitivity and bioluminescent emission has been established in 14species of North American fireflies inhabiting diverse photoecological niches. Here we extend that research to three Brazilian species. Macrolampis omissa inhabits the Cerrado (savannas), while Photinus sp1 and Pyrogaster moestus are often sympatric species inhabiting borders of mesophyll rain forests and secondary growth. P. moestus particularly favors humid areas of the forest. M. omissa and Photinus sp1 are twilight-active fireflies emitting yellow bioluminescence. P. moestus is a "twi-night" species emitting green bioluminescence. It initiates flashing at the end of twilight and continues activity into the night. The visual spectral sensitivity of dark-adapted compound eyes in these three species is similar, showing a maximum in the yellow-green wavelengths and a secondary peak in the near-UV, suggesting the presence of two receptors. The bioluminescence emission spectrum in each species is tuned to its yellow-green visual sensitivity peak. Green chromatic adaptation experiments on Photinus sp1 and P. moestus suggest the presence of a blue receptor. The presence of near-UV, blue, and long-wavelength receptors in the compound eyes would enable a trichromatic color vision in Brazilian firefly species active in dim illumination.

The proton and metal binding sites responsible for the pH-dependent green-red bioluminescence color tuning in firefly luciferases

Firefly luciferases produce yellow-green light under physiological and alkaline conditions, however at acidic pH, higher temperatures or in the presence of heavy metals the color changes to red, a property called pH-sensitivity. Despite many decades of studies, the proton and metal binding sites responsible for pH-sensitivity remain enigmatic. Previously we suggested that the salt bridge E311/R337 keeps a closed conformation of the luciferin phenolate binding site. Here we further investigated the effect of this salt bridge and mutations of the neighbor residues H310 and E/N354, on metal and pH-sensitivity of firefly luciferases emitting distinct bioluminescence colors (Cratomorphus distinctus: 548 nm; Macrolampis sp2: 569 nm). The substitutions of H310 and E/N354 modulate metal sensitivity, whereas the carboxylate of E311 may work as the catalytic base essential for green bioluminescence and pH-sensitivity. Modeling studies showed that H310, E311 and E354 side-chains coordinate Zinc, constituting the metal binding site and the pH-sensor. Electrostatic potential and pKa calculations suggest that the external couple H310/E354 is affected by pH, whereas E311/R337 make a stabilized internal pair which retains excited oxyluciferin ejected proton near its phenolate group into a high energy state, promoting yellow-green bioluminescence. Protonation or metal binding weaken these electrostatic gates and their ability to retain the excited oxyluciferin released proton near its phenolate, promoting red light emission.

Theoretical Insight into the Emission Properties of the Luciferin and Oxyluciferin of Latia.

Latia neritoides is a small limpet-like snail that produces a bright green bioluminescence (BL) via a unique light-emitting system. The process, mechanism, and even light emitter of its light emission remain unknown, although this BL has been known for decades. Unlike the other BL systems, neither the luciferin (Luc) nor the oxyluciferin (OxyLuc) of Latia is fluorescent according to the previous experiments. To help to identify its bioluminophore, we studied the geometrical and electronic structures and absorption and fluorescence spectra of Latia Luc and its six analogs as well as its OxyLuc in the gas phase and in water. The calculated results provide clear evidence of the lack of fluorescence in the Luc and OxyLuc of Latia. For the analogs of Latia Luc, the electron-withdrawing or electron-donating ability of the substituted group affects the fluorescence. The results shed new light on the BL mechanism and will likely aid the understanding of Latia BL.

Glu311 and Arg337 Stabilize a Closed Active-site Conformation and Provide a Critical Catalytic Base and Countercation for Green Bioluminescence in Beetle Luciferases

Beetle luciferases elicit the emission of different bioluminescence colors from green to red. Whereas firefly luciferases emit yellow-green light and are pH-sensitive, undergoing a typical red-shift at acidic pH and higher temperatures and in the presence of divalent heavy metals, click beetle and railroadworm luciferases emit a wider range of colors from green to red but are pH-independent. Despite many decades of study, the structural determinants and mechanisms of bioluminescence colors and pH sensitivity remain enigmatic. Here, through modeling studies, site-directed mutagenesis, and spectral and kinetic studies using recombinant luciferases from the three main families of bioluminescent beetles that emit different colors of light (Macrolampis sp2 firefly, Phrixotrix hirtus railroadworm, and Pyrearinus termitilluminans click beetle), we investigated the role of E311 and R337 in bioluminescence color determination. All mutations of these residues in firefly luciferase produced red mutants, indicating that the preservation of opposite charges and the lengths of the side chains of E311 and R337 are essential for keeping a salt bridge that stabilizes a closed hydrophobic conformation favorable for green light emission. Kinetic studies indicate that residue R337 is important for binding luciferin and creating a positively charged environment around excited oxyluciferin phenolate. In Pyrearinus green-emitting luciferase, the R334A mutation causes a 27 nm red-shift, whereas in Phrixotrix red-emitting luciferase, the L334R mutation causes a blue-shift that is no longer affected by guanidine. These results provide compelling evidence that the presence of arginine at position 334 is essential for blue-shifting the emission spectra of most beetle luciferases. Therefore, residues E311 and R337 play both structural and catalytic roles in bioluminescence color determination, by stabilizing a closed hydrophobic conformation favorable for green light emission, and also providing a base to accept excited oxyluciferin phenol proton, and a countercation to shield the negative charge of E311 and to stabilize excited oxyluciferin phenolate, blue-shifting emission spectra in most beetle luciferases.

Dual-Color Monitoring Overcomes the Limitations of Single Bioluminescent Reporters in Fast-Growing Microbes and Reveals Phase-Dependent Protein Productivity during the Metabolic Rhythms of Saccharomyces cerevisiae

Luciferase is a useful, noninvasive reporter of gene regulation that can be continuously monitored over long periods of time; however, its use is problematic in fast-growing microbes like bacteria and yeast because rapidly changing cell numbers and metabolic states also influence bioluminescence, thereby confounding the reporter's signal. Here we show that these problems can be overcome in the budding yeast Saccharomyces cerevisiae by simultaneously monitoring bioluminescence from two different colors of beetle luciferase, where one color (green) reports activity of a gene of interest, while a second color (red) is stably expressed and used to continuously normalize green bioluminescence for fluctuations in signal intensity that are unrelated to gene regulation. We use this dual-luciferase strategy in conjunction with a light-inducible promoter system to test whether different phases of yeast respiratory oscillations are more suitable for heterologous protein production than others. By using pulses of light to activate production of a green luciferase while normalizing signal variation to a red luciferase, we show that the early reductive phase of the yeast metabolic cycle produces more luciferase than other phases.

Luciferase from Fulgeochlizus bruchi (Coleoptera:Elateridae), a Brazilian click-beetle with a single abdominal lantern: molecular evolution, biological function and comparison with other click-beetle luciferases

Bioluminescent click-beetles emit a wide range of bioluminescence colors (λ(Max) = 534-594 nm) from thoracic and abdominal lanterns, which are used for courtship. Only the luciferases from Pyrophorus and Pyrearinus species were cloned and sequenced. The Brazilian Fulgeochlizus bruchi click-beetle, which inhabits the Central-west Cerrado (Savannas), is noteworthy because, differently from other click-beetles, the adult stage displays only a functional abdominal lantern, which produces a bright green bioluminescence for sexual attraction purposes, and lacks functional thoracic lanterns. We cloned the cDNA for the abdominal lantern luciferase of this species. Notably, the primary sequence of this luciferase showed slightly higher identity with the green emitting dorsal lantern luciferases of the Pyrophorus genus instead of the abdominal lanterns luciferases. This luciferase displays a blue-shifted spectrum (λ(Max) = 540 nm), which is pH-insensitive from pH 7.5 to 9.5 and undergoes a slight red shift and broadening above this pH; the lowest K(M) for luciferin among studied click-beetle luciferases, and the highest optimum pH (9.0) ever reported for a beetle luciferase. At pH 9.0, the K(M) for luciferin increases, showing a decrease of affinity for this substrate, despite the higher activity. The slow luminescence decay rate of F. bruchi luciferase in vitro reaction could be an adaptation of this luciferase for the long and sustained in vivo luminescence display of the click-beetle during the courtship, and could be useful for in vivo intracellular imaging.

Abstracts of the 17th International Symposium on Bioluminescence and Chemiluminescence ‐ (ISBC 2012)

WARNING : The light-emitting molecular structures responsible for the chemiluminescence and fluorescence phenomena are not necessarily the same!

Spectral Tuning between Vision and Bioluminescence in Beetles, An Example of von Uexkull's Functional Cycle

Spectral Tuning between Vision and Bioluminescence in Beetles, An Example of von Uexkull's Functional Cycle

Swima (Annelida, Acrocirridae), holopelagic worms from the deep Pacific

Two new species of Swima, a recently established genus of annelid worms, are introduced, one from deep water off the North American West Coast and the other from the Philippines. The acrocirrid genus now contains three named species, Swima bombiviridis, Swima fulgida sp. nov., and Swima tawitawiensis sp. nov.Swima are holopelagic, occurring only in the water column, and thus far have only been observed below 2700 m. The worms are relatively large, sometimes reaching over 30 mm in length and 5 mm in width. They have gelatinous bodies and fans of long swimming chaetae, which are flattened into paddles in S. tawitawiensis sp. nov. Members of Swima are distinguished from other swimming acrocirrids by their transparent bodies, single medial subulate branchiae, and simple nuchal organs that do not skirt the bases of lateral subulate branchiae. Swima fulgida sp. nov. is distinguished from other members of the genus by its darkly pigmented anterior gut, whereas S. tawitawiensis sp. nov. is distinguished by possessing three subulate head appendages instead of just one and by the shape of its noto- and neurochaetae. Swima species possess four pairs of elliptical, transformed segmental branchiae that produce green bioluminescence when autotomized. These ‘bombs’ were observed in various states of regeneration on a single individual. Swima are neutrally buoyant, often observed hanging immobile in the water column, and are active, agile swimmers. Although not previously documented in the literature, these worms are not rare in the deep water column. Since the worms were first noticed in 2001, they have been observed on more than half of the Monterey Bay Aquarium Research Institute's midwater remotely operated vehicle dives that went to sufficient depth. The discovery of Swima underscores our lack of knowledge of deep pelagic fauna. © 2011 The Linnean Society of London, Zoological Journal of the Linnean Society, 2011, 163, 663–678.

Green-Fluorescent Protein from the Bioluminescent Jellyfish Clytia gregaria Is an Obligate Dimer and Does Not Form a Stable Complex with the Ca2+-Discharged Photoprotein Clytin

Green-fluorescent protein (GFP) is the origin of the green bioluminescence color exhibited by several marine hydrozoans and anthozoans. The mechanism is believed to be Förster resonance energy transfer (FRET) within a luciferase-GFP or photoprotein-GFP complex. As the effect is found in vitro at micromolar concentrations, for FRET to occur this complex must have an affinity in the micromolar range. We present here a fluorescence dynamics investigation of the recombinant bioluminescence proteins from the jellyfish Clytia gregaria, the photoprotein clytin in its Ca(2+)-discharged form that is highly fluorescent (λ(max) = 506 nm) and its GFP (cgreGFP; λ(max) = 500 nm). Ca(2+)-discharged clytin shows a predominant fluorescence lifetime of 5.7 ns, which is assigned to the final emitting state of the bioluminescence reaction product, coelenteramide anion, and a fluorescence anisotropy decay or rotational correlation time of 12 ns (20 °C), consistent with tight binding and rotation with the whole protein. A 34 ns correlation time combined with a translational diffusion constant and molecular brightness from fluorescence fluctuation spectroscopy all confirm that cgreGFP is an obligate dimer down to nanomolar concentrations. Within the dimer, the two chromophores have a coupled excited-state transition yielding fluorescence depolarization via FRET with a transfer correlation time of 0.5 ns. The 34 ns time of cgreGFP showed no change upon addition of a 1000-fold excess of Ca(2+)-discharged clytin, indicating no stable complexation below 0.2 mM. It is proposed that any bioluminescence FRET complex with micromolar affinity must be one formed transiently by the cgreGFP dimer with a short-lived (millisecond) intermediate in the clytin reaction pathway.

Molecular insights on the evolution of the lateral and head lantern luciferases and bioluminescence colors in Mastinocerini railroad-worms (Coleoptera: Phengodidae)

Among bioluminescent beetles of Elateroidea superfamily, railroad-worms (Phengodidae) produce the widest range of colors, from green to red, using the same luciferin-luciferase system. Members of the Mastinocerini tribe display additional unique cephalic organs that emit red-shifted light, with Phrixothrix railroad-worms being the most dramatic cases with head lanterns emitting red light. Although the luciferases from the head lanterns of Phrixothrix hirtus and from the lateral lanterns of P. vivianii were previously cloned, the luciferases from both lanterns of the same species were not cloned yet. Therefore the origin and evolution of head and lateral lanterns luciferases in Phengodidae remains unknown. In the present work, we cloned by PCR the cDNA for lateral lantern luciferases of three Mastinocerini species: Phrixothrix hirtus, Brasilocerus sp(3). and Taximastioncerus sp. The results suggest that the head and lateral lanterns luciferases in Mastinocerini are coded by paralogous genes, and that the ancestral luciferase in the Phengodinae subfamily produced green bioluminescence. The evolutionary history of bioluminescence colors within Phengodinae is discussed.

Internal and secreted bioluminescence of the marine polychaete Odontosyllis phosphorea (Syllidae)

Abstract. The syllid polychaete Odontosyllis phosphorea produces brilliant displays of green bioluminescence during mating swarms. We studied freshly collected individuals of O. phosphorea in the laboratory to understand the characteristics of its luminescent system. Light emission appeared as an intense glow after stimulation with potassium chloride, and was associated with secreted mucus. The mucus was viscous, blue in color, and exhibited a long‐lasting glow that was greatly intensified by addition of peroxidase or ammonium persulfate. The emission spectrum of mucus‐associated bioluminescence was unimodal, with a maximum emission in the green spectrum between 494 and 504 nm. The fluorescence emission spectrum was similar, but the fluorescence intensity was low unless it originated from mucus that had already produced light, suggesting that the oxidized product of the light production is the source of fluorescence. Individuals as small as 0.5–1.0 mm produced bioluminescence that was mainly internal and not secreted as mucus. The early occurrence of bioluminescence in the life cycle of members of O. phosphorea suggests that bioluminescence may be used for purposes other than attracting mates. The luminous system was functional at temperatures as low as −20°C and was degraded above 40°C. Mixing hot and cold extracts of the mucus did not result in reconstituting original levels of light emission. Additionally, mucus samples exposed to oxygen depletion by bubbling with argon or nitrogen were still able to produce intense bioluminescence. These results suggest that bioluminescence from the mucus may involve a photoprotein rather than a luciferin–luciferase reaction.

An Integrated-Molecule-Format Multicolor Probe for Monitoring Multiple Activities of a Bioactive Small Molecule

Bioactive small molecules, including steroids, activate multiple signaling pathways in mammalian cells. However, current technologies cannot illuminate such multiple effects of a ligand in mammalian cells. Here, we demonstrate integrated-molecule-format multicolor systems simultaneously visualizing bifacial activities of a ligand, where estrogen receptor alpha (ERalpha) was exemplified to demonstrate the present technology. First, we developed a single-molecule-format probe emitting red bioluminescence for imaging interaction between the phosphorylated ligand binding domain of ERalpha (ER LBD) and the Src homology-2 (SH2) domain of Src. The SH2 domain-linked ER LBD was sandwiched between dissected N- and C-terminal fragments of Pyrophorus plagiophthalamus (click beetle) luciferase emitting red bioluminescence. Second, another single-molecule-format bio-luminescent probe emitting green bioluminescence was constructed to visualize intramolecular interaction between ER LBD and LXXLL motifs. Mammalian cells carrying the two probes emit red and/or green light in response to agonistic and antagonistic activities of a ligand, which correspond to its genomic and nongenomic activities, respectively. Third, the two probes were assembled to make an single-molecule-format multicolor indicator, in which all of the components for ligand sensing and multiple-light emission were integrated. The probe emitted characteristic light spectra in response to various agonists and antagonists. This is the first example where (i) protein phosphorylation was recognized with a single bioluminescent probe and (ii) bifacial activities of a ligand, either agonistic or antagonistic, were simultaneously visualized with multiple colors.

Functional complementation of high-efficiency resonance energy transfer: a new tool for the study of protein binding interactions in living cells

Green bioluminescence in Renilla species is generated by a approximately 100% efficient RET (resonance energy transfer) process that is caused by the direct association of a blue-emitting luciferase [Rluc (Renilla luciferase)] and an RGFP (Renilla green fluorescent protein). Despite the high efficiency, such a system has never been evaluated as a potential reporter of protein-protein interactions. To address the question, we compared and analysed in mammalian cells the bioluminescence of Rluc and RGFP co-expressed as free native proteins, or as fused single-chain polypeptides and tethered partners of self-assembling coiled coils. Here, we show that: (i) no spontaneous interactions generating detectable BRET (bioluminescence RET) signals occur between the free native proteins; (ii) high-efficiency BRET similar to that observed in Renilla occurs in both fusion proteins and self-interacting chimaeras, but only if the N-terminal of RGFP is free; (iii) the high-efficiency BRET interaction is associated with a dramatic increase in light output when the luminescent reaction is triggered by low-quantum yield coelenterazine analogues. Here, we propose a new functional complementation assay based on the detection of the high-efficiency BRET signal that is generated when the reporters Rluc and RGFP are brought into close proximity by a pair of interacting proteins to which they are linked. To demonstrate its performance, we implemented the assay to measure the interaction between GPCRs (G-protein-coupled receptors) and beta-arrestins. We show that complementation-induced BRET allows detection of the GPCR-beta-arrestin interaction in a simple luminometric assay with high signal-to-noise ratio, good dynamic range and rapid response.

Bioluminescence in the Limpet‐Like Snail, Latia neritoides

AbstractFor Abstract see ChemInform Abstract in Full Text.

Bioluminescence in the Limpet-Like Snail, Latia neritoides

Abstract Latia neritoides is a small limpet-like snail that produces a bright green bioluminescence; it is found only in New Zealand streams. The light-emitting system is unique. Although Latia bioluminescence has been studied since 1880, its mechanism is unclear. Shimomura and Johnson clarified the elements of the mechanism, including the structures of luciferin and luciferase, in 1968. However, neither the emitter nor the mechanism of the excited state of luciferin has been determined. We studied molecular mechanisms to clarify the characteristics of luciferin and luciferase and to produce a new application for this system.

Cloning and Molecular Characterization of the cDNA for the Brazilian Larval Click‐beetle Pyrearinus termitilluminans Luciferase

Abstract. The larval click‐beetle Pyrearinus termitilluminans elicits the phenomenon of luminous termite mounds in the central‐west region of Brazil. The bioluminescence (BL) spectrum of this larva (Λmax= 534 run) is one of the most blue‐shifted reported among known luminescent Cole‐optera. We have isolated mRNA from larval thoracic lanterns and constructed a cDNA library into a δZAP II vector. An expression library was obtained after excision of the pBluescript plasmid. This library was screened by photodetection and one clone that emitted green BL (Δmax= 538 nm) was isolated. The 2.2 kb cDNA insert includes a 543 residue open reading frame showing 82% homology with the luciferase isoenzymes of Pyrophorus pla‐giophthalamus (Coleoptera: Elateridae). As expected, the region between residues 223 and 247 that contains the putative active site for BL color determination showed a higher degree of homology among click‐beetle luciferases that elicit closer BL colors. The in vitro BL spectrum of recombinant P. termitilluminans luciferase also peaks at 538 nm and, as in the case of native enzyme, does not show any bathochromic shift upon decreasing the pH.

Cloning and molecular characterization of the cDNA for the Brazilian larval click-beetle Pyrearinus termitilluminans luciferase.

The larval click-beetle Pyrearinus termitilluminans elicits the phenomenon of luminous termite mounds in the central-west region of Brazil. The bioluminescence (BL) spectrum of this larva (lambda max = 534 nm) is one of the most blue-shifted reported among known luminescent Coleoptera. We have isolated mRNA from larval thoracic lanterns and constructed a cDNA library into a lambda ZAP II vector. An expression library was obtained after excision of the pBluescript plasmid. This library was screened by photodetection and one clone that emitted green BL (lambda max = 538 nm) was isolated. The 2.2 kb cDNA insert includes a 543 residue open reading frame showing 82% homology with the luciferase isoenzymes of Pyrophorus plagiophthalamus (Coleoptera: Elateridae). As expected, the region between residues 223 and 247 that contains the putative active site for BL color determination showed a higher degree of homology among click-beetle luciferases that elicit closer BL colors. The in vitro BL spectrum of recombinant P. termitilluminans luciferase also peaks at 538 nm and, as in the case of native enzyme, does not show any bathochromic shift upon decreasing the pH.

`Green mice' as a source of ubiquitous green cells

The green fluorescent protein (GFP) is responsible for the green bioluminescence of the jellyfish Aequorea victoria. Many classes of GFP mutants exist that display modified fluorescence spectra and an increased extinction coefficient. We produced transgenic mouse lines with an `enhanced' GFP (EGFP) cDNA under the control of a chicken beta-actin promoter and cytomegalovirus enhancer. All of the tissues from these transgenic lines, with the exception of erythrocytes and hair, were green under excitation light. The fluorescent nature of the cells from these transgenic mouse lines would facilitate their use in many kinds of cell transplantation experiments.

Using GFP to see the light

Green fluorescent protein (GFP) is responsible for the green bioluminescence from the jellyfish Aequorea victoria. The intense fluorescence of GFP is due to the nature of a chromophore composed of modified amino acids within the polypeptide. Formation of the fluorescent chromophore is species independent and apparently does not require any additional factors. Hence, because the gene product is easily detectable by its intense fluorescence, the gfp cDNA has become a unique reporter system. The advantages of GFP are being exploited in a variety of experimental systems and this is illustrated in the accompanying articles on Dicyostelium, plants, Drosophila and mammalian cells.