Biosensing firefly luciferin synthesis in bacteria reveals a cysteine-dependent quinone detoxification route in Coleoptera.

Luciferin biosynthetic origin and alternative biological functions during the evolution of beetles remain unknown. We have set up a bioluminescent sensing method for luciferin synthesis from cysteine and benzoquinone using E. coli and Pichia pastoris expressing the bright Amydetes vivianii firefly and P. termitilluminans click beetle luciferases. In the presence of D-cysteine and benzoquinone, intense bioluminescence is quickly produced, indicating the expected formation of D-luciferin. Starting with L-cysteine and benzoquinone, the bioluminescence is weaker and delayed, indicating that bacteria produce L-luciferin, and then racemize it to D-luciferin in the presence of endogenous esterases, CoA and luciferase. In bacteria the p-benzoquinone toxicity (IC50 ~ 25 µM) is considerably reduced in the presence of cysteine, maintaining cell viability at 3.6 mM p-benzoquinone concomitantly with the formation of luciferin. Transcriptional analysis showed the presence of gene products involved with the sclerotization/tanning in the photogenic tissues, suggesting a possible link between these pathways and bioluminescence. The lack of two enzymes involved with the last steps of these pathways, indicate the possible accumulation of toxic quinone intermediates in the lanterns. These results and the abundance of cysteine producing enzymes suggest that luciferin first appeared as a detoxification byproduct of cysteine reaction with accumulated toxic quinone intermediates during the evolution of sclerotization/tanning in Coleoptera.

Synthesis of bioluminescent gold nanoparticle-luciferase hybrid systems for technological applications.

Bioluminescent gold nanoparticles (AuNPs) were synthesized in situ using dithiol-terminated polyethylene glycol (PEG(SH)2) as reducer and stabilizing agents. Hybrid Au/F3O4 nanoparticles were also produced in a variation of synthesis, and both types of nanostructures had the polymer capping replaced by L-cysteine (Cys). The four types of nanoparticles, PEG(SH)2AuNPs, PEG(SH)2Au/F3O4NPs, CysAuNPs, and CysAu/F3O4NPs were associated with purified recombinant Pyrearinus termitilluminans green emitting click beetle luciferase (PyLuc) and Phrixotrix hirtus (RELuc) red-emitting railroad worm luciferase. Enzyme association with PEG(SH)2 was also investigated as a control. Luciferases were chosen because they catalyze bioluminescent reactions used in a wide range of bioanalytical applications, including ATP assays, gene reporting, high-throughput screening, bioluminescence imaging, biosensors and other bioluminescence-based assays. The immobilization of PyLuc and RELuc promoted partial suppression of the enzyme luminescence activity in a functionalization-dependent way. Association of PyLuc and RELuc with AuNPs increased the enzyme operational stability in relation to the free enzyme, as evidenced by the luminescence intensity from 0 to 7 h after substrate addition. The stability of the immobilized enzymes was also functionalization-dependent and the association with CysAuNPs was the condition that combined more sustained luminescent activity with a low degree of luminescence quenching. The higher enzymatic stability and sustained luminescence of luciferases associated with nanoparticles may improve the applicability of bioluminescence for bioimaging and biosensing purposes.

A Very Bright Far-Red Bioluminescence Emitting Combination Based on Engineered Railroad Worm Luciferase and 6'-Amino-Analogs for Bioimaging Purposes.

Beetle luciferases produce bioluminescence (BL) colors ranging from green to red, having been extensively used for many bioanalytical purposes, including bioimaging of pathogen infections and metastasis proliferation in living animal models and cell culture. For bioimaging purposes in mammalian tissues, red bioluminescence is preferred, due to the lower self-absorption of light at longer wavelengths by hemoglobin, myoglobin and melanin. Red bioluminescence is naturally produced only by Phrixothrix hirtus railroad worm luciferase (PxRE), and by some engineered beetle luciferases. However, Far-Red (FR) and Near-Infrared (NIR) bioluminescence is best suited for bioimaging in mammalian tissues due to its higher penetrability. Although some FR and NIR emitting luciferin analogs have been already developed, they usually emit much lower bioluminescence activity when compared to the original luciferin-luciferases. Using site-directed mutagenesis of PxRE luciferase in combination with 6'-modified amino-luciferin analogs, we finally selected novel FR combinations displaying BL ranging from 636-655 nm. Among them, the combination of PxRE-R215K mutant with 6'-(1-pyrrolidinyl)luciferin proved to be the best combination, displaying the highest BL activity with a catalytic efficiency ~2.5 times higher than the combination with native firefly luciferin, producing the second most FR-shifted bioluminescence (650 nm), being several orders of magnitude brighter than commercial AkaLumine with firefly luciferase. Such combination also showed higher thermostability, slower BL decay time and better penetrability across bacterial cell membranes, resulting in ~3 times higher in vivo BL activity in bacterial cells than with firefly luciferin. Overall, this is the brightest FR emitting combination ever reported, and is very promising for bioimaging purposes in mammalian tissues.

Revisiting Coleoptera a + T-rich region: structural conservation, phylogenetic and phylogeographic approaches in mitochondrial control region of bioluminescent Elateridae species (Coleoptera).

The control region (CR) or A + T-rich region in Coleoptera mt genome is poorly characterized, including the Elateroidea bioluminescent species. Here, we provided the first attempt to characterize and compare the structure and organization of the CR of different species within Elateridae. We also revisited some sequenced Coleoptera CR and observed consensus T-stretches, non-conserved sequences near the stem-loop and unusual inner tRNAs-like sequences. All these features are probably involved in the replication start of the mt genome. The phylogenetic relationships in Elateridae bioluminescent groups using partial sequence of CR showed the monophyly of Pyrearinus pumilus group and Pyrearinus as a polyphyletic genus, corroborating our previous results. The wider genetic variation obtained by CR analysis could separate two different lineages that occur within P. termitilluminans populations. In Elateridae, the CR exhibited high polymorphism within and between populations, which was also observed in other Coleoptera species, suggesting that the CR could be described as a suitable molecular marker to be applied in phylogenetic and phylogeographic studies.

Organization and comparative analysis of the mitochondrial genomes of bioluminescent Elateroidea (Coleoptera: Polyphaga).

Mitochondrial genome organization in the Elateroidea superfamily (Coleoptera), which include the main families of bioluminescent beetles, has been poorly studied and lacking information about Phengodidae family. We sequenced the mitochondrial genomes of Neotropical Lampyridae (Bicellonycha lividipennis), Phengodidae (Brasilocerus sp.2 and Phrixothrix hirtus) and Elateridae (Pyrearinus termitilluminans, Hapsodrilus ignifer and Teslasena femoralis). All species had a typical insect mitochondrial genome except for the following: in the elaterid T. femoralis genome there is a non-coding region between NADH2 and tRNA-Trp; in the phengodids Brasilocerus sp.2 and P. hirtus genomes we did not find the tRNA-Ile and tRNA-Gln. The P. hirtus genome showed a ~1.6kb non-coding region, the rearrangement of tRNA-Tyr, a new tRNA-Leu copy, and several regions with higher AT contents. Phylogenetics analysis using Bayesian and ML models indicated that the Phengodidae+Rhagophthalmidae are closely related to Lampyridae family, and included Drilus flavescens (Drilidae) as an internal clade within Elateridae. This is the first report that compares the mitochondrial genomes organization of the three main families of bioluminescent Elateroidea, including the first Neotropical Lampyridae and Phengodidae. The losses of tRNAs, and translocation and duplication events found in Phengodidae mt genomes, mainly in P. hirtus, may indicate different evolutionary rates in these mitochondrial genomes. The mitophylogenomics analysis indicates the monophyly of the three bioluminescent families and a closer relationship between Lampyridae and Phengodidae/Rhagophthalmidae, in contrast with previous molecular analysis.

Survey of Bioluminescent Coleoptera in the Atlantic Rain Forest of Serra da Paranapiacaba in São Paulo State (Brazil) / Levantamento de Coleópteros bioluminescentes na Mata Atlântica da Serra da Paranapiacaba

Brazil is the country with the largest number of bioluminescent beetle species in the world. However, estimates suggest that this number could much be higher, since many species remain to be discovered. In this work we made a survey of the species of bioluminescent beetles in Serra de Paranapiacaba - the largest remnant of Atlantic Forest in São Paulo State. The survey was done at Intervales State Park, Carlos Botelho State Park and municipality of Tapiraí and the following species were collected: Aspisoma lineatum, Aspisoma physonotum, Aspisoma fenestrata, Cratomorphus besckey, Cratomorphus distinctus, Photinus penai, Photinus sp1, Photinus sp9, Ethra aff. malledicta or axilaris, Ethra aff. adicta , Lucidotini incertae sedis, Cladodes flabellicornis, Cladodes demoulini, Amydetes lucernuta, Bicellonycha sp8, Bicellonycha ornaticollis, Pyrogaster lunifer, Pyrogaster moestus, Pyrogaster sp2, Pyrogaster sp5, Pyrogaster sp6, Photuris lugubris, Photuris sp1, Photuris sp7, Stenophrixotrix sp1, Brasilocerus sp1, Pseudophengodes sp1, Hapsodrilus pyrotis, Hypsiophtalmus sp1, Ptesimopsia luculenta, Pyroptesis cincticollis, Pyrearinus brevicolis, Pyrearinus candelarius, Pyrearinus micatus, Pyrophorus divergens. Our data show that Serra de Paranapiacaba is the second richest area in São Paulo state, especially in elaterids, with unique species typical of this area and species common to other investigated sites such as the Biological Station of Boracéia (in Salesópolis county) and the urbanized areas in the between Campinas - Sorocaba- São Paulo, originally covered to the Atlantic Rainforest.

O Brasil é o país que possui o maior número de espécies de coleópteros bioluminescentes no mundo. Entretanto, estimativas sugerem que este número possa ser bem maior, dado que muitas espécies ainda não foram descritas. Neste trabalho foi realizado um levantamento das espécies de coleópteros bioluminescentes em três localidades na Serra da Paranapiacaba - a maior área remanescente contígua de Mata Atlântica no país, com associação ao respectivo habitat. No Parque Estadual Intervales, Parque Estadual Carlos Botelho e Tapiraí foram coletadas as seguintes espécies: (Lampyidae) Aspisoma lineatum, Aspisoma physonotum, Aspisoma fenestrata, Cratomorphus besckey, Cratomorphus distinctus, Photinus penai, Photinus sp1, Photinus sp9, Ethra aff. malledicta ou axilaris, Ethra aff. adicta, Lucidotini incertae sedis, Cladodes flabellicornis, Cladodes demoulini, Amydetes sp1, Bicellonycha sp8, Bicellonycha ornaticollis, Pyrogaster lunifer, Pyrogaster moestus, Pyrogaster sp2, Pyrogaster sp5, Pyrogaster sp6, Photuris lugubris, Photuris sp1, Photuris sp7; (Phengodidae) Stenophrixotrix sp1, Brasilocerus sp2, Pseudophengodes sp1; (Elateridae) Hapsodrilus pyrotis, Hypsiophtalmus sp1, Ptesimopsia luculenta, Pyroptesis cincticollis, Pyrearinus brevicolis, Pyrearinus candelarius, Pyrearinus micatus, Pyrophorus divergens. Estes dados mostram que esta constitui a segunda área mais rica em espécies luminescentes do Estado de São Paulo, depois da Est. Biológica de Boracéia, especialmente em elaterídeos, com espécies únicas características destas localidades e espécies comuns è outras áreas investigadas, como as da Estação Biológica de Boracéia (Salesópolis, SP) e áreas urbanizadas no triângulo Campinas - Sorocaba - São Paulo, originalmente cobertas por Mata Atlântica.

Novel application of pH-sensitive firefly luciferases as dual reporter genes for simultaneous ratiometric analysis of intracellular pH and gene expression/location.

Firefly luciferases are widely used as bioluminescent reporter genes for bioimaging and biosensors. Aiming at simultaneous analyses of different gene expression and cellular events, luciferases and GFPs that exhibit distinct bioluminescence and fluorescence colors have been coupled with each promoter, making dual and multicolor reporter systems. Despite their wide use, firefly luciferase bioluminescence spectra are pH-sensitive, resulting in a typical large red shift at acidic pH, a side-effect that may affect some bioanalytical purposes. Although some intracellular pH-indicators employ dual color and fluorescent dyes, none has been considered to benefit from the characteristic spectral pH-sensitivity of firefly luciferases to monitor intracellular pH-associated stress, an important indicator of cell homeostasis. Here we demonstrate a linear relationship between the ratio of intensities in the green and red regions of the bioluminescence spectra and pH using firefly luciferases cloned in our laboratory (Macrolampis sp2 and Cratomorphus distinctus), allowing estimation of E. coli intracellular pH, thus providing a new analytical method for ratiometric intracellular pH-sensing. This is the first dual reporter system that employs a single luciferase gene to simultaneously monitor intracellular pH using spectral changes, and gene expression and/or ATP concentration using the bioluminescence intensity, showing great potential for real time bioanalysis of intracellular processes associated with metabolic changes such as apoptosis, cell death, inflammation and tissue acidification, among the other physiological changes.

A transcriptional survey of the cDNA library of Macrolampis sp2 firefly lanterns (Coleoptera: Lampyridae).

The biochemistry of firefly bioluminescence is well understood; however, the molecular physiology of the lanterns is still poorly studied, especially the biosynthesis and origin of beetle luciferin which are almost unknown. Using a cDNA library previously constructed from Macrolampis sp2 lanterns, we randomly selected and sequenced 572 cDNAs in order to have a first transcriptional profile of the most represented messages found in the lanterns and therefore to better understand their molecular physiology. As expected, high percentage of the gene products (~22%) displayed high similarity with Coleoptera genome products. About 7% represented mitochondrial genes, including several copies of cytochrome oxidase, which are also expected for this tissue. Luciferase genes were especially abundant, representing ca 2% of the products. Gene products involved with cysteine and sulfur metabolism such as the cystathionine ß-lyase and the S-adenosylmethionine synthetase were abundant. Noteworthy, an abundance of proteins involved with hormone metabolism was found, suggesting a possible link between bioluminescence and hormone metabolism.

A new blue-shifted luciferase from the Brazilian Amydetes fanestratus (Coleoptera: Lampyridae) firefly: molecular evolution and structural/functional properties.

Firefly luciferases usually produce bioluminescence in the yellow-green region, with colors in the green and yellow-orange extremes of the spectrum being less common. Several firefly luciferases have already been cloned and sequenced, and site-directed mutagenesis studies have already identified important regions and residues for bioluminescence colors. However the structural determinants and mechanisms of bioluminescence colors turned out to be elusive, mainly when comparing luciferases with a high degree of divergence. Thus comparison of more similar luciferases producing colors in the two extremes of the spectrum could be revealing. The South-American fauna of fireflies remains largely unstudied, with some unique taxa that are not found anywhere else in the world and that produce a wide range of bioluminescence colors. Among them, fireflies of the genus Amydetes are especially interesting because its taxonomical status as an independent subfamily or as a tribe is not yet solved, and because they usually produce a continuous bright blue-shifted bioluminescence. In this work we cloned the cDNA for the luciferase of the Atlantic rain forest Amydetes fanestratus firefly, which is found near Sorocaba municipality (São Paulo, Brazil). Despite showing a higher degree of identity with the South-American Cratomorphus, the European Lampyris and the Asiatic Pyrocoelia, phylogenetical analysis of the luciferase sequence support the inclusion of Amydetes as an independent subfamily. Amydetes luciferase displays one of the most blue-shifted emission spectra (λ(max) = 538 nm) among beetle luciferases, with lower pH-sensitivity and higher affinity for ATP when compared to other luciferases, making this luciferase attractive for sensitive ATP and reporter assays.

The origin of luciferase activity in Zophobas mealworm AMP/CoA-ligase (protoluciferase): luciferin stereoselectivity as a switch for the oxygenase activity.

Beetle luciferases evolved from AMP/CoA-ligases. However, it is unclear how the new luciferase activity evolved. In order to clarify this question, we compared the luminescence and catalytic properties of a recently cloned luciferase-like enzyme from Zophobas mealworm, an AMP/CoA-ligase displaying weak luminescence activity, with those of cloned luciferases from the three main families of luminescent beetles: Phrixthrix hirtus railroad worm; Pyrearinus termitilluminans click beetle and Photinus pyralis firefly. The catalytic constant of the mealworm enzyme was 2-4 orders of magnitude lower than that of beetle luciferases, but 3 orders of magnitude above the non-catalyzed chemiluminescence of luciferyl-adenylate in buffer. Studies with D- and L-luciferin and their adenylates show that the luminescence reaction of the luciferase-like enzyme and beetle luciferases are stereoselective for D-luciferin and its adenylate, and that the selectivity is determined mainly at the adenylation step. Modelling studies showed that the luciferin binding site cavity of this enzyme is smaller and more hydrophobic than that of beetle luciferases. Therefore Zophobas mealworm enzyme displays true luciferase activity, keeping the attributes of an ancient protoluciferase. These results suggest that stereoselectivity for D-luciferin may have been a key event for the origin of oxygenase/luciferase activity in AMP/CoA-ligases, and that efficient luciferase activity may have further evolved mainly by increasing the catalytic constant of the oxidative reaction and the quantum yield of bioluminescence.

Bovine serum albumin displays luciferase-like activity in presence of luciferyl adenylate: insights on the origin of protoluciferase activity and bioluminescence colours.

Luciferyl adenylate, the key intermediate in beetle bioluminescence, is produced through adenylation of d-luciferin by beetle luciferases and also by mealworm luciferase-like enzymes which produce a weak red chemiluminescence. However, luciferyl adenylate is only weakly chemiluminescent in water at physiological pH and it is unclear how efficient bioluminescence evolved from its weak chemiluminescent properties. We found that bovine serum albumin (BSA) and neutral detergents enhance luciferyl adenylate chemiluminescence by three orders of magnitude, simulating the mealworm luciferase-like enzyme chemiluminescence properties. These results suggest that the beetle protoluciferase activity arose as an enhanced luciferyl adenylate chemiluminescence in the protein environment of the ancestral AMP-ligase. The predominance of luciferyl adenylate chemiluminescence in the red region under most conditions suggests that red luminescence is a more primitive condition that characterized the original stages of protobioluminescence, whereas yellow-green bioluminescence may have evolved later through the development of a more structured and hydrophobic active site.

Two bioluminescent diptera: the North American Orfelia fultoni and the Australian Arachnocampa flava. Similar niche, different bioluminescence systems.

Orfelia fultoni is the only bioluminescent dipteran (Mycetophilidae) found in North America. Its larvae live on stream banks in the Appalachian Mountains. Like their Australasian relative Arachnocampa spp., they build sticky webs to which their bioluminescence attracts flying prey. They bear two translucent lanterns at the extremities of the body, histologically distinct from the single caudal lantern of Arachnocampa spp., and emit the bluest bioluminescence recorded for luminescent insects (lambda(max) = 460 nm versus 484 nm from Arachnocampa). A preliminary characterization of these two bioluminescent systems indicates that they are markedly different. In Orfelia a luciferin-luciferase reaction was demonstrated by mixing a hot extract prepared with dithiothreitol (DTT) under argon with a crude cold extract. Bioluminescence is not activated by adenosine triphosphate (ATP) but is strongly stimulated by DTT and ascorbic acid. Using gel filtration, we isolated a luciferase fraction of approximately 140 kDa and an additional high molecular weight fraction (possibly a luciferin-binding protein) that activated bioluminescence in the presence of luciferase and DTT. The Arachnocampa luciferin-luciferase system involves a 36 kDa luciferase and a luciferin soluble in ethyl acetate under acidic conditions; the bioluminescence is activated by ATP but not by DTT. The present findings indicate that the bioluminescence of O. fultoni constitutes a novel bioluminescent system unrelated to that of Arachnocampa.