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

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The influence of the region between residues 220 and 344 and beyond in Phrixotrix railroad worm luciferases green and red bioluminescence

To find the regions having a major influence on the bioluminescence spectra of railroad worm luciferases, we constructed new chimeric luciferases switching the fragments from residues 1-219 and from 220-545 between Phrixotrix viviani (PxvGR; λmax = 548 nm) green light-emitting luciferase and Phrixothrix hirtus (PxhRE; λmax = 623 nm) red light-emitting luciferases. The emission spectrum (λmax = 571 nm) and KM for luciferin in the chimera PxRE220GR (1-219, PxhRE; 220-545, PxvGR) suggested that the region above residue 220 of PxvGR had a major effect on the active site. However, switching the sequence between the residues 226-344 from PxvGR luciferase into PxhRE (PxREGRRE) luciferase resulted in red light emission (λmax = 603 nm), indicating that the region 220-344 by itself does not determine the emission spectrum. Furthermore, the sequence before residue 220 of the green-emitting luciferase is incompatible for light emission with the sequence above residue 220 of PxhRE. These results suggest that the fragments before and after residue 220, which correspond to distinct subdomains, may fold differently in the green- and red-emitting luciferases, affecting the active site conformation.

Rubiaceae em um remanescente de floresta atlântica no Rio Grande do Norte, Brasil

Empresa de Pesquisa Agropecuária do Rio Grande do Norte

Active-site properties of Phrixotrix railroad worm green and red bioluminescence-eliciting luciferases

The luciferases of the railroad worm Phrixotrix (Coleoptera: Phengodidae) are the only beetle luciferases that naturally produce true red bioluminescence. Previously, we cloned the green- (PxGR) and red-emitting (PxRE) luciferases of railroad worms Phrixotrix viviani and P. hirtus[OLE1]. These luciferases were expressed and purified, and their active-site properties were determined. The red-emitting PxRE luciferase displays flash-like kinetics, whereas PxGR luciferase displays slow-type kinetics. The substrate affinities and catalytic efficiency of PxRE luciferase are also higher than those of PxGR luciferase. Fluorescence studies with 8-anilino-1-naphthalene sulfonic acid and 6-p-toluidino-2-naphthalene sulfonic acid showed that the PxRE luciferase luciferin-binding site is more polar than that of PxGR luciferase, and it is sensitive to guanidine. Alutagenesis and modelling studies suggest that several invariant residues in the putative luciferin-binding site of PxRE luciferase cannot interact with excited oxyluciferin. These results suggest that one portion of the luciferin-binding site of the red-emitting luciferase is tighter than that of PxGR luciferase, whereas the other portion could be more open and polar.

The influence of the loop between residues 223-235 in beetle luciferase bioluminescence spectra: A solvent gate for the active site of pH-sensitive luciferases

Beetle luciferases emit a wide range of bioluminescence colors, ranging from green to red. Firefly luciferases can shift the spectrum to red in response to pH and temperature changes, whereas click beetle and railroadworm luciferases do not. Despite many studies on firefly luciferases, the origin of pH-sensitivity is far from being understood. Through comparative site-directed mutagenesis and modeling studies, using the pH-sensitive luciferases (Macrolampis and Cratomorphus distinctus fireflies) and the pH-insensitive luciferases (Pyrearinus termitilluminans, Phrixotrix viviani and Phrixotrix hirtus) cloned by our group, here we show that substitutions dramatically affecting bioluminescence colors in both groups of luciferases are clustered in the loop between residues 223-235 (Photinus pyralis sequence). The substitutions at positions 227, 228 and 229 (P. pyralis sequence) cause dramatic redshift and temporal shift in both groups of luciferases, indicating their involvement in labile interactions. Modeling studies showed that the residues Y227 and N229 are buried in the protein core, fixing the loop to other structural elements participating at the bottom of the luciferin binding site. Changes in pH and temperature (in firefly luciferases), as well as point mutations in this loop, may disrupt the interactions of these structural elements exposing the active site and modulating bioluminescence colors. © 2007 The Authors.

Aspectos evolutivos da bioluminescência de elateroidea (coleoptera) do Brasil

Pós-graduação em Ciências Biológicas (Biologia Celular e Molecular) - IBRC

Aspectos comparativos das luciferases pH-insensitivas de Pyrearinus termitilluminans (Coleoptera: Elateridae) e Phrixotrix spp (Coleoptera: Phengodidae): expressão, purificação e propriedades do sítio ativo

Pós-graduação em Ciências Biológicas (Biologia Celular e Molecular) - IBRC

Revision of Trogulidae Sundevall, 1833 (Arachnida: Opiliones)

The relationship and phylogeny of the western Palearctic harvestmen family Trogulidae is investigated. The traditional system of seven genera and approximately 40 species appeared to be artificially composed but a phylogenetic approach and a comprehensive revision has long been sought after. Species are poorly characterised due to their uniform morphology and species evaluation is furthermore complicated by the variability of the few characters used for species delineation. To meet these demands a molecular genetic analysis is accomplished using the nuclear 28S rRNA gene and the mitochondrial cytochrome b gene. This analysis incorporates most genera and species of Trogulidae as well as a comprehensive set of Nemastomatidae and Dicranolasmatidae as outgroup taxa. Phylogenetic results of Bayesian analysis, Maximum Parsimony, Maximum Likelihood and Neighbor Joining are compared with distributional data, morphological characters and results of canonical discriminant analysis of morphometric characters and general congruence of these data sets is shown. To demonstrate the applicability of this method the revision of two species-groups within Trogulus is set out in detail. The Trogulus hirtus species-group and the Trogulus coriziformis species-group are revised. The former is in the central and north-western Balkan Peninsula. T. tricarinatus ssp. hirtus is raised to species level and four new species are described (T. karamanorum [man.n.], T. melitensis [man.n.], T. pharensis [man.n]; T. thaleri [man.n.]). The Trogulus coriziformis species-group is confined to the western Mediterranean area. T. coriziformis, T. aquaticus are re-described, T. cristatus and T. lusitanicus are re-established and four species are described as new (T. balearicus, T. huberi, T. prietoi, T. pyrenaicus). In both species-groups two further cryptic species probably exist but were not described. The species groups are shown to represent different phylogenetic levels and this information is used for the revisional work on the genus Trogulus as well as for the generic system of Trogulidae. Family status of Dicranolasmatidae is rejected and Dicranolasma is shown to be best incorporated within Trogulidae. Calathocratus, Platybessobius and Trogulocratus appear to be polyphyletic and are best to be united within Calathocratus, the oldest name of this set. The cryptic diversity within Trogulidae, especially in Trogulus and the composed genus Calathocratus rates to 150-235% and is thereby remarkably high for a group of the generally well researched European fauna. Genetic features of the group such as heteroplasmy, the possibility of major gene rearrangements and usability of the cytochrome b gene for phylogenetic studies in Opiliones are outlined.

Marine diatoms in deep sea sediments

Long-term evolution is thought to take opportunities that arise as a consequence of mass extinction (as argued, for example, by Gould, 2002) and the following biotic recovery, but there is absolutely no evidence for this being the case. However, our study shows that eutrophication by oceanic mixing also played a part in the enhancement of several evolutionary events amongst marine organisms, and these results could indicate that the rates of oceanic biodiversification may be slowed if upwelling becomes weakened by future global warming. This paper defines three distinct evolutionary events of resting spores of the marine diatom genus Chaetoceros, to reconstruct past upwelling through the analysis of several DSDP, ODP and land-based successions from the North, South and equatorial Pacific as well as the Atlantic Ocean during the past 40 million years. The Atlantic Chaetoceros Explosion (ACE) event occurred across the E/O boundary in the North Atlantic, and is characterized by resting spore diversification that occurred as a consequence of the onset of upwelling following changes in thermohaline circulation through global cooling in the early Oligocene. Pacific Chaetoceros Explosion events-1 and -2 (PACE-1 and PACE-2) are characterized by relatively higher occurrences of iron input following the Himalayan uplift and aridification at 8.5 Ma and ca. 2.5 Ma in the North Pacific region. These events not only enhanced the diversification and increased abundance of primary producers, including that of Chaetoceros, other diatoms and seaweeds, but also stimulated the evolution of zooplankton and larger predators, such as copepods and marine mammals, which ate these phytoplankton and plants. Current thinking suggests new evolutionary niches open up after a mass extinction, but our study finds that eutrophication can also stimulate evolutionary diversification. Moreover, in the opposite fashion, our results show that as thermohaline circulation abates, global warming progresses and the ocean surface becomes warmer, many marine organisms will be affected by the environmental degradation.