Phrixothrix Research Articles

BIOLUMINESCENCE IN INSECTS

Lightning in organisms is a wonderful creation of nature. Many insects such as firefly, Railroad worm, Click Beetle, Glow-worm produces light known as bioluminescence. Insects use it as a Mating signal, Predation, and defence. Fluorescence is also used in space research, pest management, medical research, and fluorescent marker gene manipulation.

Genome analysis of Phrixothrix hirtus (Phengodidae) railroad worm shows the expansion of odorant-binding gene families and positive selection on morphogenesis and sex determination genes

Among bioluminescent beetles of the Elateroidea superfamily, Phengodidae is the third largest family, with 244 bioluminescent species distributed only in the Americas, but is still the least studied from the phylogenetic and evolutionary points of view. The railroad worm Phrixothrix hirtus is an essential biological model and symbolic species due to its bicolor bioluminescence, being the only organism that produces true red light among bioluminescent terrestrial species. Here, we performed partial genome assembly of P. hirtus, combining short and long reads generated with Illumina sequencing, providing the first source of genomic information and a framework for comparative analyses of the bioluminescent system in Elateroidea. This is the largest genome described in the Elateroidea superfamily, with an estimated size of ∼3.4 Gb, displaying 32 % GC content, and 67 % transposable elements. Comparative genomic analyses showed a positive selection of genes and gene family expansion events of growth and morphogenesis gene products, which could be associated with the atypical anatomical development and morphogenesis found in paedomorphic females and underdeveloped males. We also observed gene family expansion among distinct odorant-binding receptors, which could be associated with the pheromone communication system typical of these beetles, and retrotransposable elements. Common genes putatively regulating bioluminescence production and control, including two luciferase genes corresponding to lateral lanterns green-emitting and head lanterns red-emitting luciferases with 7 exons and 6 introns, and genes potentially involved in luciferin biosynthesis were found, indicating that there are no clear differences about the presence or absence of gene families associated with bioluminescence in Elateroidea.

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.Graphic abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s43630-021-00111-0.

Same Luciferin in Different Luciferases Emitting Different-Color Light. A Theoretical Study on Beetle Bioluminescence.

Bioluminescent beetles, firefly, click beetle, and railroad worm, naturally emit different-color light via the identical luciferin and bioluminescence (BL) mechanisms. Railroad worm especially emits two colors of light in its dorsal-lateral and cephalic lanterns. Four computational models of bioluminophore (oLu) in luciferases of red-emitting, yellow-green-emitting, red-emitting with additional loop, and red-emitting without C-terminal were built in this paper. To unveil the details of this luciferase effect at the molecular and electronic-state levels, second-order multiconfigurational perturbation calculations were performed following molecular dynamic simulations and time-dependent density functional calculations for the above four oLu-luciferase systems. Via a systematic analysis on properties of oLu at the first singlet state (S1-oLu) in different luciferases, one clearly see the details of the microenvironment and secondary structure of luciferase affecting the excited-state property of S1-oLu, which ultimately result in the variant color of light emission. Typically, the increase in charge transfer of S1-oLu leads to the longer wavelength BL.

RNA-Seq analysis of the bioluminescent and non-bioluminescent species of Elateridae (Coleoptera): Comparison to others photogenic and non-photogenic tissues of Elateroidea species

Bioluminescence in the superfamily Elateroidea is produced by specific light organs distributed in different parts of the body depending on the life stage and family. Whereas the luciferin-luciferase system is well known, the molecular control of bioluminescence, the biosynthetic route of luciferin, and the molecular differentiation of photogenic tissue in Elateridae remain enigmatic. With the aim to improve the knowledge about these questions, we performed the RNA-Seq analysis of lanterns and fat body of the Brazilian click beetle larvae: Pyrearinus fragilis (luminescent) and Conoderus sp. (non-luminescent) and compared the gene products contents and their abundance among these tissues. For the first time, we showed that the fat body of luminescent click beetle displays luminescence. We also analyzed some gene products expression patterns in lantern and fat body of Elateridae. Several of them, which are potentially involved with bioluminescence and its control are also found in the transcriptional profile of the firefly Aspisoma lineatum (Lampyridae) and the railroad worm Phrixothrix hirtus (Phengodidae). These gene products were observed in both lanterns and fat body of elaterid species, however with low abundance in the fat body. The results indicate that in Elateridae, the bioluminescent tissue also originated from the fat body. In P. fragilis, we observed two luciferase/luciferase-like enzymes with high abundance, one in the lantern and a different one in the fat body, indicating the possibility of two distinct isoenzymes involved in the bioluminescence in the lantern and fat body of Elateridae species, such as observed in fireflies.

Use of a special Brazilian red-light emitting railroad worm Luciferase in bioassays of NEK7 protein Kinase and Creatine Kinase

BackgroundLuciferases, enzymes that catalyze bioluminescent reactions in different organisms, have been extensively used for bioanalytical purposes. The most well studied bioluminescent system is that of firefly and other beetles, which depends on a luciferase, a benzothiazolic luciferin and ATP, and it is being widely used as a bioanalytical reagent to quantify ATP. Protein kinases are proteins that modify other proteins by transferring phosphate groups from a nucleoside triphosphate, usually ATP.MethodsHere, we used a red-light emitting luciferase from Phrixotrix hirtus railroad worm to determine the activity of kinases in a coupled assay, based on luminescence that is generated when luciferase is in the presence of its substrate, the luciferin, and ATP.ResultsIn this work we used, after several optimization reactions, creatine kinase isoforms as well as NEK7 protein kinase in the absence or presence of ATP analogous inhibitors to validate this new luminescence method.ConclusionWith this new approach we validated a luminescence method to quantify kinase activity, with different substrates and inhibition screening tests, using a novel red-light emitting luciferase as a reporter enzyme.

Transcriptional comparison of the photogenic and non-photogenic tissues of Phrixothrix hirtus (Coleoptera: Phengodidae) and non-luminescent Chauliognathus flavipes (Coleoptera: Cantharidae) give insights on the origin of lanterns in railroad worms

Bioluminescence in beetles is found in the Elateroidea superfamily, which includes Elateridae, Lampyridae, Rhagophthalmidae, and Phengodidae families. Despite sharing the same biochemical system, it is unclear whether the lanterns and the bioluminescent system share a common origin or not. To better understand the molecular differentiation of photogenic from non-photogenic tissues and the molecular physiology of these tissues, we compared the transcriptional profiles of the lateral lanterns and fat body in Phrixothrix hirtus railroad worm (Phengodidae) and the abdomen of the non-luminescent Chauliognathus flavipes (Cantharidae). We observed gene products involved in pheromone biosynthesis in all tissues and four distinct opsin gene products in C. flavipes, more than expected when compared to the transcriptional analysis of lampyrid eyes. We also observed several gene products potentially associated with bioluminescence in P. hirtus lantern and in C. flavipes body, whereas in the fat body of P. hirtus we found only a single copy of luciferase homolog enzyme. Altogether these results suggest that the photogenic tissue of phengodid lanterns has distinct origin from fireflies lanterns, and also the existence of a latent bioluminescent potential in the fat body tissue of some Elateroidea families.

Beetles (Coleoptera) of Peru: A Survey of the Families. Phengodidae LeConte, 1861

Diversity in Peru: 2 subfamilies, 7 genera, 12 species. Recognition: Phengodidae are commonly called glow-worms because the larviform females and larvae have bioluminescent organs in transverse bands along the body. They glow yellow-green using eleven paired photic organs of 2 thoracic through 9th abdominal segments; some genera have head organs that glow red. Some adult males are also bioluminescent, and at least one species has bioluminescent eggs. The adult male body length is 3.3–25 mm; larviform females are generally larger than males. The antenna has 10–12 antennomeres in adult males, and may be filiform, serrate, bipectinate or plumose. Adult males have truncate or dehiscent elytra. Phengodid larvae are slender, mostly well-sclerotized, and rounded in crosssection (compared to the flattened larvae of Lampyridae and Lycidae), resembling those of click beetles. Habitat: These may be nocturnal, active at nights. Winged males may be attracted at light traps. Females may be collected from loose bark while larvae are usually found under leaf litter or rotting logs. The larviform females and larvae are specialist predators of millipedes and other litter arthropods. Notes: The American family Phengodidae is now considered as separate from Asiatic Rhagophthalmidae, although sister group relationships of the families were supported by both morphological evidence (Lawrence et al., 1995), and comprehensive molecular phylogenies (Bocakova et al., 2007; Kundrata et al., 2014). Previous studies suggested rhagophthalmids as a clade of fireflies (Crowson, 1955; Suzuki, 1997). The following checklist is derived from Pic in Titschack (1954; as Drilidae), Zaragoza-Caballero (1984), and specimens housed in SEMC. The family was monographed by Wittmer (1976), but a number of new genera have been added since. Lawrence et al. (2000) reported the minute Penicillophorus (2–4 mm) as occurring in Colombia and Peru. The “railroad worm”, Phrixothrix, occurs in Peru but it is unclear if it is a pest (see Costa et al., 1999). An interactive key to the world fauna is available online (Lawrence et al., 2000); see Zaragoza-Caballero (2010) for an updated key to the genera of the large subfamily Mastinocerinae. Taximastinocerus is reported as new genus record for Peru based on 19 specimens (probably multiple species) collected by Chaboo in 2010–2011 with flight intercept traps and Malaise traps at the CICRA Biological Field Station, Madre de Dios, Peru.

The Luciferin Binding Site Residues C/T311 (S314) Influence the Bioluminescence Color of Beetle Luciferases through Main-Chain Interaction with Oxyluciferin Phenolate

Beetle luciferases emit different bioluminescence colors from green to red; however, no clear relationship between the identity of the luciferin binding site residues and bioluminescence colors was found in different luciferases, and it is unclear whether critical interactions affecting emission spectra occur on the thiazolyl or on the benzothiazolyl sides of the luciferin binding site. Through homology modeling and site-directed mutagenesis using our multicolor set of beetle luciferases (Pyrearinus termitilluminans larval click beetle, Pte, λ(max) = 534 nm; Phrixothrix hirtus railroad worm red emitting, PxRE, λ(max) = 623 nm; and Macrolampis sp2 firefly, Mac, λ(max) = 564 nm), we show that the residues C/T311 (S314) play an important role in bioluminescence color determination. Modeling studies indicate that the main-chain carbonyls of these residues are close to both oxyluciferin phenolate and AMP, whereas the side chains pack against second-shell residues. The C311(S314)A mutation considerably red shifts the spectra of the green-yellow-emitting luciferases (Pte λ(max) = 534 to 590 nm; Mac λ(max) = 564 to 583/613 nm) and affects the K(M) values for luciferin and ATP, but not the spectrum of the red-emitting luciferase. On the other hand, whereas the exchange between C/T311 (S314) caused smaller effects on the emission spectra of green-yellow-emitting luciferases, the C311T substitution (naturally found in green-emitting railroad worm luciferases) resulted in the largest reported blue shift in P. hirtus red-emitting luciferase (λ(max) = 623 to 606 nm). Altogether, these results indicate that the stability of residues C/T311 (S314) and the size of the cavity around oxyluciferin phenolate affect bioluminescence colors and suggest, for the first time, the occurrence of a critical interaction between main-chain carbonyls of position 311 (314) residues and oxyluciferin phenolate.

Apple Maggot, Apple Maggot Fly, Rhagoletis pomonella (Walsh) (Insecta: Diptera: Tephritidae)

The apple maggot, Rhagoletis pomonella (Walsh), also known as the apple maggot fly and “railroad worm,” is native to North America. Originally, it fed on the fruit of wild hawthorn (Crataegus spp.), but then became a primary pest of cultivated apples, especially in the northeastern United States and southeastern Canada. Summer- and early fall-maturing varieties are particularly vulnerable, but hard winter apples are sometimes infested. Thin skinned sweet and subacid varieties are most susceptible, but acid varieties may be attacked. This revised 4-page fact sheet was written by H. V. Weems, Jr. and T. R. Fasulo, and published by the UF Department of Entomology and Nematology, March 2012. EENY-261/IN505: Apple Maggot, Apple Maggot Fly, Rhagoletis pomonella (Walsh) (Insecta: Diptera: Tephritidae) (ufl.edu)

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!

Dual-Color Bioluminescence Analysis for Quantitatively Monitoring G-Protein-Coupled Receptor and β-Arrestin Interactions

G protein-coupled receptors (GPCRs) are crucial elements in mammalian signal transduction, and are considered to represent potent drug targets. We have previously developed a GPCR assay system in cultured cells based on complementation of split fragments of click beetle (Pyrearinus termitilluminans) luciferase. The interaction of GPCRs with its target, β-arrestin, resulted in strong emission of bioluminescence upon stimulation with its specific ligand. In this study, we improved precision of the GPCR assay system by using railroad worm (Phrixothrix hirtus) luciferase as an internal control. We generated stable cell lines harboring the railroad worm luciferase and quantitatively evaluate the extent of GPCR-β-arrestin interactions. We showed concentration-dependent bioluminescence responses for four GPCRs: β2-adrenoceptor, endothelin receptor type A, α2-adrenoceptor and human μ-opioid receptor. We also demonstrated that the variation of responses was reduced significantly by normalizing the data with bioluminescence from railroad worm luciferase. This assay system represents a simple and reliable approach for screening drug candidates in a high throughput manner.

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.

Relationship between stability and bioluminescence color of firefly luciferase

Firefly luciferase catalyzes the oxidation of luciferin in the presence of ATP, Mg(2+) and molecular oxygen. The bioluminescence color of firefly luciferases is identified by the luciferase structure and assay conditions. Amongst different types of beetles, luciferase from Phrixotrix railroad worm (PhRE) with a unique additional residue (Arg353) naturally emits red bioluminescence color. By insertion of Arg356 in luciferase of Lampyris turkestanicus, corresponding to Arg353 in Phrixotrix hirtus, the color of the emitted light was changed to red. To understand the effect of this position on the bioluminescence color shift, four residues with similar sizes but different charges (Arg, Lys, Glu, and Gln) were inserted into Photinus pyralis luciferase. Comparison of mutants with native luciferase shows that mutation brought an increase in the content of secondary structure and globular compactness of (P. pylalis) luciferase. Comparative study of chemical denaturation of native and mutant luciferases by activity measurement, intrinsic and extrinsic fluorescence, circular dichroism, and DSC techniques revealed that insertion of positively charged residues (Arg, Lys) in the flexible loop (352-358) plays a significant role on the stability of (P. pyralis) luciferase and changes the light color to red.

Enhanced red‐emitting railroad worm luciferase for bioassays and bioimaging

A luciferase from the railroad worm (Phrixothrix hirtus) is the only red-emitting bioluminescent enzyme in nature that is advantageous in multicolor luciferase assays and in bioluminescence imaging (BLI). However, it is not used widely in scientific or industrial applications because of its low activity and stability. By using site-directed mutagenesis, we produced red-emitting mutants with higher activity and better stability. Compared with the wild-type (WT), the luminescent activities from extracts of cultured mammalian cells expressing mutant luciferase were 9.8-fold in I212L/N351K, 8.4-fold in I212L, and 7.8-fold in I212L/S463R; and the cell-based activities were 3.6-fold in I212L/N351K and 3.4-fold in N351K. The remaining activities after incubation at 37 degrees C for 10 min were 50.0% for I212L/S463R, 31.8% for I212L, and 23.0% for I212L/N351K, but only 5.2% for WT. To demonstrate an application of I212L/N351K, cell-based BLI was performed, and the luminescence signal was 3.6-fold higher than in WT. These results indicate that the mutants might improve the practicability of this signaling in bioassays and BLI.

The effective role of positive charge saturation in bioluminescence color and thermostability of firefly luciferase

Luciferases are the enzymes that catalyze the reactions that produce light in bioluminescence. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. Amongst different beetle luciferases, those from phrixothrix rail-road worm with a unique additional residue (Arg353) emit red bioluminescence color naturally. Insertion of Arg(356) in Lampyris turkestanicus luciferase changed the emitted light to red with a bimodal bioluminescence spectrum. By insertion and substitution of positively-charged residues, different specific mutation (E354R/Arg(356), E354K/Arg(356), E354R, E354K) lead to changes of the bioluminescence color. Bioluminescence emission spectra indicate that substitution of E354 by R along with insertion of Arg(356) produces a luciferase that emits red light with a single peak bioluminescence spectrum. The comparison of mutants with native luciferase shows that mutations of firefly luciferase resulted in structural and functional thermostability. Comparative study of native and mutant luciferase (E354R/Arg(356)) by intrinsic and extrinsic fluorescence, CD spectropolarimetry, and homology modeling revealed mutation brought about an increase in content of secondary structure and globular compactness of L. turkestanicus luciferase. On the other hand, pK(a) of amino acids in the flexible loop decreased upon introducing of positive charges.

Effect of Charge Distribution in a Flexible Loop on the Bioluminescence Color of Firefly Luciferases

Firefly luciferase is a monooxygenase that catalyzes the ATP-dependent conversion of firefly luciferin into a luciferyl-adenylate, which is oxidized to an electronically excited oxyluciferin in a multistep reaction and produces visible light with a remarkable quantum yield. The bioluminescence color of firefly luciferases is determined by the luciferase structure and assay conditions. Among different beetles, only luciferase from Phrixotrix railroad worm (Ph(RE)) emits red bioluminescence, naturally. The presence of Arg353 in Ph(RE) luciferase, which corresponds to the deleted residue in the other luciferases, is an important distinctive structural feature of it. Insertion of Arg356 into a green-emitter luciferase (Lampyris turkestanicus), corresponding to Arg353 in Phrixotrix hirtus, changed the emitted light from green to red. To further clarify the effect of this position on the light shift mechanism, four residues with similar sizes but different charges (Arg, Lys, Glu, and Gln) were inserted into Photinus pyralis luciferase, using site-specific insertion mutagenesis. Insertion of a residue with a positive side chain (Arg356 and Lys356) changed the light color to red, while insertion of a residue with a negative side chain (Glu356) had little effect on color. Insertion of a neutral residue (Gln356) at this position was performed without any change in bioluminescence spectra. Insertion of positively charged residues in this loop took place with a series of structural changes which were confirmed by fluorescence spectroscopy and homology modeling. Homology modeling reveals the appearance of a bulge in a flexible loop (T352-P359) upon mutation which shifts to the left side with a color change from green to red.

Site-directed mutagenesis of firefly luciferase: implication of conserved residue(s) in bioluminescence emission spectra among firefly luciferases

The bioluminescence colours of firefly luciferases are determined by assay conditions and luciferase structure. Owing to red light having lower energy than green light and being less absorbed by biological tissues, red-emitting luciferases have been considered as useful reporters in imaging technology. A set of red-emitting mutants of Lampyris turkestanicus (Iranian firefly) luciferase has been made by site-directed mutagenesis. Among different beetle luciferases, those from Phrixothrix (railroad worm) emit either green or red bioluminescence colours naturally. By substitution of three specific amino acids using site-specific mutagenesis in a green-emitting luciferase (from L. turkestanicus), the colour of emitted light was changed to red concomitant with decreasing decay rate. Different specific mutations (H245N, S284T and H431Y) led to changes in the bioluminescence colour. Meanwhile, the luciferase reaction took place with relative retention of its basic kinetic properties such as K(m) and relative activity. Structural comparison of the native and mutant luciferases using intrinsic fluorescence, far-UV CD spectra and homology modelling revealed a significant conformational change in mutant forms. A change in the colour of emitted light indicates the critical role of these conserved residues in bioluminescence colour determination among firefly luciferases. Relatively high specific activity and emission of red light might make these mutants suitable as reporters for the study of gene expression and bioluminescence imaging.

The Influence of Ala243 (Gly247), Arg215 and Thr226 (Asn230) on the Bioluminescence Spectra and pH-Sensitivity of Railroad Worm, Click Beetle and Firefly Luciferases¶

Among beetle luciferases, the pH-sensitive firefly luciferases have been studied extensively. Much less is known about pH-insensitive luciferases, which include click beetle and railroad worm luciferases. Previously, we found that the residues R215 and T226 (N230) are important for green light emission. Here we show that the conserved residue A243 in pH-insensitive luciferases and the corresponding G247 in pH-sensitive luciferases affect the emission spectrum and influence pH-sensitivity. In contrast to railroad worm green light–emitting (PxvGR) and firefly luciferases, the substitution of R215 in Pyrearinus termitilluminans click beetle luciferase (Pte) had no effect on the spectrum, showing that R215 is not essential for green light emission in all beetle luciferases. A homology-based model of Pte luciferase shows that R215 and T226 are close enough to interact. To investigate if there was an interaction between these conserved residues, double mutants were constructed. The double substitution R215S/T226N in Pte luciferase abolished the activity. In PxvGR luciferase the same double mutant resulted in a redshift (λmax= 595 nm), whose magnitude was lower than the value expected for an additive effect. These results suggest that the effects of R215S and T226N are partially interdependent. The double substitution T226N/A243G had an additive redshift effect on the spectrum of PxvGR luciferase, whereas it had a smaller effect on the spectrum of Pte luciferase. Altogether, these results suggest that the above substitutions have different effects on the active site of click beetle and railroad worm luciferases.

Few substitutions affect the bioluminescence spectra of Phrixotrix (Coleoptera: Phengodidae) luciferases: a site‐directed mutagenesis survey

Phrixotrix (railroad worm) luciferases produce bioluminescence in the green and red regions of the spectrum, depending on the location of the lanterns, and are the only luciferases naturally producing red bioluminescence. Comparison of the luciferase sequences showed a set of substitutions that could be involved in bioluminescence colour determination: (a) unique substitutions in the red luciferase replacing otherwise invariant residues; (b) conserved basic residues in the green-yellow emitting luciferases; and (c) an additional R353 residue in red-emitting luciferase (Viviani et al., 1999). To investigate whether these sites have a functional role in bioluminescence colour determination, we performed a site-directed mutagenesis. Natural substitutions in the region 220-344 and residues in the putative luciferin-binding site were also investigated. With the exception of the previously identified substitution of R215 and T226 (Viviani et al., 2002), which display dramatic red-shift effects on the spectrum of green-yellow-emitting luciferases, only a few substitutions had a moderate effect on the spectrum of the green-emitting luciferase. In contrast, no single substitution affected the spectrum of the red-emitting luciferase. The results suggest that the identity of the active site residues is not so critical for determining red bioluminescence in PxRE luciferase. Rather, the conformation assumed during the emitting step could be critical to set up proper interactions with excited oxyluciferin.