Metagenomic Analysis of the Microbiota from the Crop of an Invasive Snail Reveals a Rich Reservoir of Novel Genes

The shortage of petroleum reserves and the increase in CO₂ emissions have raised global concerns and highlighted the importance of adopting sustainable energy sources. Second-generation ethanol made from lignocellulosic materials is considered to be one of the most promising fuels for vehicles. The giant snail Achatina fulica is an agricultural pest whose biotechnological potential has been largely untested. Here, the composition of the microbial population within the crop of this invasive land snail, as well as key genes involved in various biochemical pathways, have been explored for the first time. In a high-throughput approach, 318 Mbp of 454-Titanium shotgun metagenomic sequencing data were obtained. The predominant bacterial phylum found was Proteobacteria, followed by Bacteroidetes and Firmicutes. Viruses, Fungi, and Archaea were present to lesser extents. The functional analysis reveals a variety of microbial genes that could assist the host in the degradation of recalcitrant lignocellulose, detoxification of xenobiotics, and synthesis of essential amino acids and vitamins, contributing to the adaptability and wide-ranging diet of this snail. More than 2,700 genes encoding glycoside hydrolase (GH) domains and carbohydrate-binding modules were detected. When we compared GH profiles, we found an abundance of sequences coding for oligosaccharide-degrading enzymes (36%), very similar to those from wallabies and giant pandas, as well as many novel cellulase and hemicellulase coding sequences, which points to this model as a remarkable potential source of enzymes for the biofuel industry. Furthermore, this work is a major step toward the understanding of the unique genetic profile of the land snail holobiont.     

DNA damage, RAD9 and fertility/infertility of Echinococcus granulosus hydatid cysts

Hydatidosis, caused by the larval stage of the platyhelminth parasite Echinococcus granulosus, affects human and animal health. Hydatid fertile cysts are formed in intermediate hosts (human and herbivores) producing protoscoleces, the infective form to canines, at their germinal layers. Infertile cysts are also formed, but they are unable to produce protoscoleces. The molecular mechanisms involved in hydatid cysts fertility/infertility are unknown. Nevertheless, previous work from our laboratory has suggested that apoptosis is involved in hydatid cyst infertility and death. On the other hand, fertile hydatid cysts can resist oxidative damage due to reactive oxygen and nitrogen species. On these foundations, we have postulated that when oxidative damage of DNA in the germinal layers exceeds the capability of DNA repair mechanisms, apoptosis is triggered and hydatid cysts infertility occurs. We describe a much higher percentage of nuclei with oxidative DNA damage in dead protoscoleces and in the germinal layer of infertile cysts than in fertile cysts, suggesting that DNA repair mechanisms are active in fertile cysts. rad9, a conserved gene, plays a key role in cell cycle checkpoint modulation and DNA repair. We found that RAD9 of E. granulosus (EgRAD9) is expressed at the mRNA and protein levels. As it was found in other eukaryotes, EgRAD9 is hyperphosphorylated in response to DNA damage. Our results suggest that molecules involved in DNA repair in the germinal layer of fertile hydatid cysts and in protoscoleces, such as EgRAD9, may allow preserving the fertility of hydatid cysts in the presence of ROS and RNS.     

Recombinant subunits as tools for the structural and functional characterization of Echinococcus granulosus antigen B

Antigen B (AgB) is a major protein component of the Echinococcus granulosus metacestode. It is oligomeric and this raises several questions regarding the subunit structure and composition of AgB. Several genes that encode different AgB subunits have been identified, and some of these have been cloned and expressed to produce recombinant subunits. The study of these recombinant subunits may provide new insights into the structure, physical-chemical properties, and functional aspects of AgB. Like native AgB, the AgB8/1, AgB8/2, and AgB8/3 recombinant subunits produced in our laboratory form 120-160 kDa oligomers that have stable secondary structures, are strongly antigenic and immunogenic, and selectively bind hydrophobic compounds. Here, we review these results and discuss their implications for the elucidation of the structure and function of AgB. This includes a possible role for AgB in host-parasite interactions.     

Mixed-valence porphyrin π-cation radical derivatives: Electrochemical investigations

The electrochemistry of [Cu(OEP)] and [Ni(OEP)] are compared with the mixed-valence π-cations and . These electrochemical studies, carried out with cyclic voltammetry and hydrodynamic voltammetry, show that the mixed valence π-cations have distinct electrochemical properties, although the differences between the [M(OEP)]^+/0 and processes are subtle.     

New findings of Neurospora in Europe and comparisons of diversity in temperate climates on continental scales

The life cycles of the conidiating species of Neurospora are adapted to respond to fire, which is reflected in their natural history. Neurospora is found commonly on burned vegetation from the tropic and subtropical regions around the world and through the temperate regions of western North America. In temperate Europe it was unknown whether Neurospora would be as common as it is in North America because it has been reported only occasionally. In 2003 and 2004 a multinational effort surveyed wildfire sites in southern Europe. Neurospora was found commonly from southern Portugal and Spain (37 degrees N) to Switzerland (46 degrees N). Species collected included N. crassa, N. discreta, N. sitophila and N. tetrasperma. The species distribution and spatial dynamics of Neurospora populations showed both similarities and differences when compared between temperate Europe and western North America, both regions of similar latitude, climate and vegetation. For example the predominant species in western North America, N. discreta phylogenetic species 4B, is common but not predominant in Europe, whereas species rare in western North America, N. crassa NcB and N. sitophila, are much more common in Europe. The meiotic drive element Spore killer was also common in European populations of N. sitophila and at a higher proportion than anywhere else in the world. The methods by which organisms spread and adapt to new environments are fundamental ecosystem properties, yet they are little understood. The differences in regional diversity, reported here, can form the basis of testable hypotheses. Questions of phylogeography and adaptations can be addressed specifically by studying Neurospora in nature.     

Arabidopsis Synaptotagmin 1 Is Required for the Maintenance of Plasma Membrane Integrity and Cell Viability

Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca²⁺-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca²⁺-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness.     

eIF5A dimerizes not only in vitro but also in vivo and its molecular envelope is similar to the EF-P monomer

The protein eukaryotic initiation factor 5A (eIF5A) is highly conserved among archaea and eukaryotes, but not in bacteria. Bacteria have the elongation factor P (EF-P), which is structurally and functionally related to eIF5A. eIF5A is essential for cell viability and the only protein known to contain the amino acid residue hypusine, formed by post-translational modification of a specific lysine residue. Although eIF5A was initially identified as a translation initiation factor, recent studies strongly support a function for eIF5A in the elongation step of translation. However, the mode of action of eIF5A is still unknown. Here, we analyzed the oligomeric state of yeast eIF5A. First, by using size-exclusion chromatography, we showed that this protein exists as a dimer in vitro, independent of the hypusine residue or electrostatic interactions. Protein–protein interaction assays demonstrated that eIF5A can form oligomers in vitro and in vivo, in an RNA-dependent manner, but independent of the hypusine residue or the ribosome. Finally, small-angle X-ray scattering (SAXS) experiments confirmed that eIF5A behaves as a stable dimer in solution. Moreover, the molecular envelope determined from the SAXS data shows that the eIF5A dimer is L-shaped and superimposable on the tRNA^Phe tertiary structure, analogously to the EF-P monomer.     

Cloning and characterization of the ribosomal RNA genes of Rhynchosciara americana

The ribosomal RNA genes (rDNA) of Rhynchosciara americana were analysed using Southern transfers of DNA cleaved with EcoRI, HindIII, BamHI and PstI. The results show that the rDNA is heterogeneous in structure. Following digestion with EcoRI and hybridization to rRNA three bands corresponding to fragments of 9.5, 7.5 and 5.5 kilobases (kb) were detected. Recombinants containing EcoRI fragments of R. americana DNA were prepared using the vector gtB. Three different recombinants (gtRa1, gtRa23 and gtRa5) were isolated containing the rDNA fragments of 9.5, 7.5 and 5.5 kb, respectively. These fragments were transferred to pBR325 and analysed with restriction enzymes and Southern hybridization with 28 S and 18 S rRNA. The gt recombinants were further analysed by R-loop mapping. The data show that the rDNA occurs in two different repeating gene units. A shorter repeat of 9.5 kb and a longer repeat of 13 kb, in which the 28 S rRNA coding sequence contains an insertion of 3.5 kb.