Environmental shaping of sponge associated archaeal communities

Background

Archaea are ubiquitous symbionts of marine sponges but their ecological roles and the influence of environmental factors on these associations are still poorly understood.

Methodology/Principal Findings

We compared the diversity and composition of archaea associated with seawater and with the sponges Hymeniacidon heliophila, Paraleucilla magna and Petromica citrina in two distinct environments: Guanabara Bay, a highly impacted estuary in Rio de Janeiro, Brazil, and the nearby Cagarras Archipelago. For this we used metagenomic analyses of 16S rRNA and ammonia monooxygenase (amoA) gene libraries. Hymeniacidon heliophila was more abundant inside the bay, while P. magna was more abundant outside and P. citrina was only recorded at the Cagarras Archipelago. Principal Component Analysis plots (PCA) generated using pairwise unweighted UniFrac distances showed that the archaeal community structure of inner bay seawater and sponges was different from that of coastal Cagarras Archipelago. Rarefaction analyses showed that inner bay archaeaoplankton were more diverse than those from the Cagarras Archipelago. Only members of Crenarchaeota were found in sponge libraries, while in seawater both Crenarchaeota and Euryarchaeota were observed. Although most amoA archaeal genes detected in this study seem to be novel, some clones were affiliated to known ammonia oxidizers such as Nitrosopumilus maritimus and Cenarchaeum symbiosum.

Conclusion/Significance

The composition and diversity of archaeal communities associated with pollution-tolerant sponge species can change in a range of few kilometers, probably influenced by eutrophication. The presence of archaeal amoA genes in Porifera suggests that Archaea are involved in the nitrogen cycle within the sponge holobiont, possibly increasing its resistance to anthropogenic impacts. The higher diversity of Crenarchaeota in the polluted area suggests that some marine sponges are able to change the composition of their associated archaeal communities, thereby improving their fitness in impacted environments.     

Virome analyses of <Emphasis Type="Italic">Hevea brasiliensis</Emphasis> using small RNA deep sequencing and PCR techniques reveal the presence of a potential new virus

Background

Hevea brasiliensis is an important commercial crop due to the high quality of the latex it produces; however, little is known about viral infections in this plant. The only virus described to infect H. brasiliensis until now is a Carlavirus, which was described more than 30?years ago. Virus-derived small interfering RNA (vsiRNAs) are the product of the plant’s antiviral defense triggered by dsRNA viral intermediates generated, during the replication cycle. These vsiRNAs are complementar to viral genomes and have been widely used to identify and characterize viruses in plants.

Methods

In the present study, we investigated the virome of leaf and sapwood samples from native H. brasiliensis trees collected in two geographic areas in the Brazilian Amazon. Small RNA (sRNA) deep sequencing and bioinformatic tools were used to assembly, identify and characterize viral contigs. Subsequently, PCR amplification techniques were performed to experimentally verify the presence of the viral sequences. Finally, the phylogenetic relationship of the putative new virus with related viral genomes was analyzed.

Results

Our strategy allowed the identification of 32 contigs with high similarity to viral reference genomes, from which 23 exhibited homology to viruses of the Tymoviridae family. The reads showed a predominant size distribution at 21?nt derived from both strands, which was consistent with the vsiRNAs profile. The presence and genome position of the viral contigs were experimentally confirmed using droplet digital PCR amplifications. A 1913 aa long fragment was obtained and used to infer the phylogenetic relationship of the putative new virus, which indicated that it is taxonomically related to the Grapevine fleck virus, genus Maculavirus. The putative new virus was named Hevea brasiliensis virus (HBrV) in reference to its host.

Conclusion

The methodological strategy applied here proved to be efficient in detecting and confirming the presence of new viral sequences on a ‘very difficult to manage’ sample. This is the second time that viral sequences, that could be ascribed as a putative novel virus, associated to the rubber tree has been identified.

     

The 5-hydroxytryptamine(1A) receptor is stably palmitoylated, and acylation is critical for communication of receptor with Gi protein

In the present study, we verified that the mouse 5-hydroxytryptamine(1A) (5-HT(1A)) receptor is modified by palmitic acid, which is covalently attached to the protein through a thioester-type bond. Palmitoylation efficiency was not modulated by receptor stimulation with agonists. Block of protein synthesis by cycloheximide resulted in a significant reduction of receptor acylation, suggesting that palmitoylation occurs early after synthesis of the 5-HT(1A) receptor. Furthermore, pulse-chase experiments demonstrated that fatty acids are stably attached to the receptor. Two conserved cysteine residues 417 and 420 located in the proximal C-terminal domain were identified as acylation sites by site-directed mutagenesis. To address the functional role of 5-HT(1A) receptor acylation, we have analyzed the ability of acylation-deficient mutants to interact with heterotrimeric G(i) protein and to modulate downstream effectors. Replacement of individual cysteine residues (417 or 420) resulted in a significantly reduced coupling of receptor with G(i) protein and impaired inhibition of adenylyl cyclase activity. When both palmitoylated cysteines were replaced, the communication of receptors with G alpha(i) subunits was completely abolished. Moreover, non-palmitoylated mutants were no longer able to inhibit forskolin-stimulated cAMP formation, indicating that palmitoylation of the 5-HT(1A) receptor is critical for the enabling of G(i) protein coupling/effector signaling. The receptor-dependent activation of extracellular signal-regulated kinase was also affected by acylation-deficient mutants, suggesting the importance of receptor palmitoylation for the signaling through the G beta gamma-mediated pathway, in addition to the G alpha(i)-mediated signaling.     

Swimming training induces liver adaptations to oxidative stress and insulin sensitivity in rats submitted to high-fat diet

Oxidative stress, physical inactivity and high-fat (FAT) diets are associated with hepatic disorders such as metabolic syndrome (MS). The therapeutic effects of physical training (PT) were evaluated in rats with MS induced by FAT diet for 13 weeks, on oxidative stress and insulin signaling in the liver, during the last 6 weeks. FAT-sedentary (SED) rats increased body mass, retroperitoneal fat, mean arterial pressure (MAP) and heart rate (HR), and total cholesterol, serum alanine aminotransferase, glucose and insulin. Livers of FAT-SED rats increased superoxide dismutase activity, thiobarbituric acid-reactive substances, protein carbonyl and oxidized glutathione (GSSG); and decreased catalase activity, reduced glutathione/GSSG ratio, and the mRNA expression of insulin receptor substrate 1 (IRS-1) and serine/threonine kinase 2. FAT-PT rats improved in fitness and reduced their body mass, retroperitoneal fat, and glucose, insulin, total cholesterol, MAP and HR; and their livers increased superoxide dismutase and catalase activities, the reduced glutathione/GSSG ratio and the expression of peroxisome proliferator-activated receptor gamma and insulin receptor compared to FAT-SED rats. These findings indicated adaptive responses to PT by restoring the oxidative balance and insulin signaling in the liver and certain biometric and biochemical parameters as well as MAP in MS rats.     

Recent Findings in the Chemistry of Odorants from Four Baccharis Species and Their Impact as Chemical Markers

Baccharis is a widespread genus belonging to the Asteraceae family that includes almost 400 species exclusively from the Americas. Even when studied in detail, the taxonomic classification among species from this genus is not yet fully defined. Within the framework of our study of the volatile composition of the Baccharis genus, four species (B. trimera, B. milleflora, B. tridentata, and B. uncinella) were collected from the ‘Campos de Cima da Serra’ highlands of the Brazilian state of Rio Grande do Sul. The aerial parts were dried and extracted by the simultaneous distillation extraction (SDE) procedure. This is the first time that SDE has been applied to obtain and compare the volatile‐extract composition in the Baccharis genus. Characterization of the volatile extracts allowed the identification of 180 peaks with many coeluting components; these latter being detailed for the first time for this genus. The multivariate statistical analyses allowed separating the volatile extracts of the four populations of Baccharis into two separate groups. The first one included the B. milleflora, B. trimera, and B. uncinella volatile extracts. The three species showed a high degree of similarity in their volatile composition, which was characterized by the presence of high contents of sesquiterpene compounds, in particular of spathulenol. The second group comprised the extract of B. tridentata, which contained α‐pinene, β‐pinene, limonene, and (E)‐β‐ocimene in high amounts.     

Solution Structure of the SGTA Dimerisation Domain and Investigation of Its Interactions with the Ubiquitin-Like Domains of BAG6 and UBL4A

Background

The BAG6 complex resides in the cytosol and acts as a sorting point to target diverse hydrophobic protein substrates along their appropriate paths, including proteasomal degradation and ER membrane insertion. Composed of a trimeric complex of BAG6, TRC35 and UBL4A, the BAG6 complex is closely associated with SGTA, a co-chaperone from which it can obtain hydrophobic substrates.

Methodology and Principal Findings

SGTA consists of an N-terminal dimerisation domain (SGTA_NT), a central tetratricopeptide repeat (TPR) domain, and a glutamine rich region towards the C-terminus. Here we solve a solution structure of the SGTA dimerisation domain and use biophysical techniques to investigate its interaction with two different UBL domains from the BAG6 complex. The SGTA_NT structure is a dimer with a tight hydrophobic interface connecting two sets of four alpha helices. Using a combination of NMR chemical shift perturbation, isothermal titration calorimetry (ITC) and microscale thermophoresis (MST) experiments we have biochemically characterised the interactions of SGTA with components of the BAG6 complex, the ubiquitin-like domain (UBL) containing proteins UBL4A and BAG6. We demonstrate that the UBL domains from UBL4A and BAG6 directly compete for binding to SGTA at the same site. Using a combination of structural and interaction data we have implemented the HADDOCK protein-protein interaction docking tool to generate models of the SGTA-UBL complexes.

Significance

This atomic level information contributes to our understanding of the way in which hydrophobic proteins have their fate decided by the collaboration between SGTA and the BAG6 complex.     

Visitation rate of pollinators and nectar robbers to the flowers and inflorescences of Tabebuia aurea (Bignoniaceae): effects of floral display size and habitat fragmentation

Large floral displays favour pollinator attraction and the import and export of pollen. However, large floral displays also have negative effects, such as increased geitonogamy, pollen discounting and nectar/pollen robber attraction. The size of the floral display can be measured at different scales (e.g. the flower, inflorescence or entire plant) and variations in one of these scales may affect the behaviour of flower visitors in different ways. Moreover, the fragmentation of natural forests may affect flower visitation rates and flower visitor behaviour. In the present study, video recordings of the inflorescences of a tree species (Tabebuia aurea) from the tropical savannah of central Brazil were used to examine the effect of floral display size at the inflorescence and tree scales on the visitation rate of pollinators and nectar robbers to the inflorescence, the number of flowers approached per visit, the number of visits per flower of potential pollinators and nectar robbers, and the interaction of these variables with the degree of landscape disturbance. Nectar production was quantified with respect to flower age. Although large bees are responsible for most of the pollination, a great diversity of flower insects visit the inflorescences of T. aurea. Other bee and hummingbird species are highly active nectar robbers. Increases in inflorescence size increase the visitation rate of pollinators to inflorescences, whereas increases in the number of inflorescences on the tree decrease visitation rates to inflorescences and flowers. This effect has been strongly correlated with urban environments in which trees with the largest floral displays are observed. Pollinating bees (and nectar robbers) visit few flowers per inflorescence and concentrate visits to a fraction of available flowers, generating an overdispersed distribution of the number of visits per inflorescence and per flower. This behaviour reflects preferential visits to young flowers (including flower buds) with a greater nectar supply.     

A rapid method for the determination of bacterial fatty acid composition

Heat treatment of spores of non-proteolytic strains of Clostridium botulinum at 75–90°C, and enumeration of survivors on a nutrient medium containing lysozyme gave biphasic survival curves. A majority of spores were inactivated rapidly by heating, and the apparent heat-resistance of these spores was similar to that observed by enumeration on medium without lysozyme. A minority of spores showed much greater heat-resistance, due to the fact that the spore coat was permeable to lysozyme, which diffused into the spore from the medium and replaced the heat-inactivated germination system. The proportion of heated spores permeable to lysozyme was between 0.2 and 1.4% for spores of strains 17B (type B) and Beluga (type E), but was about 20% for spores of strain Foster B96 (type E). After treatment of heated spores with alkaline thioglycolate, all were permeable to lysozyme. D-values for heated spores that were permeable to lysozyme (naturally and after treatment with thioglycolate) were: for strain 17B, D₈₅°C, 100 min; D₉₀°C, 18.7 min; D₉₅°C, 4.4 min; for strain Beluga, D₈₅°C, 46 min; D₉₀°C, 11.8 min; D₉₅°C, 2.8 min. The z-values for these spores of strains 17B and Beluga were 7.6°C and 8.3°C.