Electron transport pathway for a Streptomyces cytochrome P450: cytochrome P450 105D5-catalyzed fatty acid hydroxylation in Streptomyces coelicolor A3(2)

Streptomyces and other bacterial actinomycete species produce many important natural products, including the majority of known antibiotics, and cytochrome P450 (P450) enzymes catalyze important biosynthetic steps. Relatively few electron transport pathways to P450s have been characterized in bacteria, particularly streptomycete species. One of the 18 P450s in Streptomyces coelicolor A3(2), P450 105D5, was found to bind fatty acids tightly and form hydroxylated products when electrons were delivered from heterologous systems. The six ferredoxin (Fdx) and four flavoprotein Fdx reductase (FDR) proteins coded by genes in S. coelicolor were expressed in Escherichia coli, purified, and used to characterize the electron transfer pathway. Of the many possibilities, the primary pathway was NADH --> FDR1 --> Fdx4 --> P450 105D5. The genes coding for FDR1, Fdx4, and P450 105D5 are located close together in the S. coelicolor genome. Several fatty acids examined were substrates, including those found in S. coelicolor extracts, and all yielded several products. Mass spectra of the products of lauric acid imply the 8-, 9-, 10-, and 11-hydroxy derivatives. Hydroxylated fatty acids were also detected in vivo in S. coelicolor. Rates of electron transfer between the proteins were measured; all steps were faster than overall hydroxylation and consistent with rates of NADH oxidation. Substrate binding, product release, and oxygen binding were relatively fast in the catalytic cycle; high kinetic deuterium isotope effects for all four lauric acid hydroxylations indicated that the rate of C-H bond breaking is rate-limiting in every case. Thus, an electron transfer pathway to a functional Streptomyces P450 has been established.     

Inactivation of Mitochondrial Permeability Transition Pore by Octylguanidine and Octylamine

Mitochondrial permeability transition occurs through a Ca²⁺-dependent opening of atransmembrane pore, whose identity has been attributed to that of the adenine nucleotide translocase(ANT). In this work, we induced permeability transition by adding 0.5 M carboxyatractyloside.The process was evaluated analyzing Ca²⁺ efflux, a drop in transmembrane electric gradient,and swelling. We found that the amphiphyllic cations octylguanidine and octylamine, at theconcentration of 100 M, inhibited, almost completely, nonspecific membrane permeability.Hexylguanidine, hexylamine, as well as guanidine chloride and hydroxylamine failed to doso. The inhibition was reversed after the addition of 40 mM Li⁺, Na⁺ K⁺,Rb⁺, or Cs⁺; K⁺ wasthe most effective. We propose that the positive charge of the amines interact with negativecharges of membrane proteins, more likely the ADP/ATP carrier, while the alkyl chain penetratesinto the hydrophobic milieu of the inner membrane, fixing the reagent.     

Biophysical Studies on BEX3, the p75NTR-Associated Cell Death Executor,Reveal a High-Order Oligomer with Partially Folded Regions

BEX3 (Brain Expressed X–linked protein 3) is a member of a mammal-specific placental protein family. Several studies have found the BEX proteins to be associated with neurodegeneration, the cell cycle and cancer. BEX3 has been predicted to be intrinsically disordered and also to represent an intracellular hub for cell signaling. The pro-apoptotic activity of BEX3 in association with a number of additional proteins has been widely supported; however, to the best of our knowledge, very limited data are available on the conformation of any of the members of the BEX family. In this study, we structurally characterized BEX3 using biophysical experimental data. Small angle X-ray scattering and atomic force microscopy revealed that BEX3 forms a specific higher-order oligomer that is consistent with a globular molecule. Solution nuclear magnetic resonance, partial proteinase K digestion, circular dichroism spectroscopy, and fluorescence techniques that were performed on the recombinant protein indicated that the structure of BEX3 is composed of approximately 31% α-helix and 20% β-strand, contains partially folded regions near the N- and C-termini, and a core which is proteolysis-resistant around residues 55–120. The self-oligomerization of BEX3 has been previously reported in cell culture and is consistent with our in vitro data.     

Experimental Evidence for a Revision in the Annotation of Putative Pyridoxamine 5'-Phosphate Oxidases P(N/M)P from Fungi

Pyridoxinamine 5''-phosphate oxidases (P(N/M)P oxidases) that bind flavin mononucleotide (FMN) and oxidize pyridoxine 5''-phosphate or pyridoxamine 5''-phosphate to form pyridoxal 5''-phosphate (PLP) are an important class of enzymes that play a central role in cell metabolism. Failure to generate an adequate supply of PLP is very detrimental to most organisms and is often clinically manifested as a neurological disorder in mammals. In this study, we analyzed the function of YLR456W and YPR172W, two homologous genes of unknown function from S. cerevisiae that have been annotated as putative P(N/M)P oxidases based on sequence homology. Different experimental approaches indicated that neither protein catalyzes PLP formation nor binds FMN. On the other hand, our analysis confirmed the enzymatic activity of Pdx3, the S. cerevisiae protein previously implicated in PLP biosynthesis by genetic and structural characterization. After a careful sequence analysis comparing the putative and confirmed P(N/M)P oxidases, we found that the protein domain (PF01243) that led to the YLR456W and YPR172W annotation is a poor indicator of P(N/M)P oxidase activity. We suggest that a combination of two Pfam domains (PF01243 and PF10590) present in Pdx3 and other confirmed P(N/M)P oxidases would be a stronger predictor of this molecular function. This work exemplifies the importance of experimental validation to rectify genome annotation and proposes a revision in the annotation of at least 400 sequences from a wide variety of fungal species that are homologous to YLR456W and are currently misrepresented as putative P(N/M)P oxidases.     

Global coral disease prevalence associated with sea temperature anomalies and local factors

Coral diseases are taking an increasing toll on coral reef structure and biodiversity and are important indicators of declining health in the oceans. We implemented standardized coral disease surveys to pinpoint hotspots of coral disease, reveal vulnerable coral families and test hypotheses about climate drivers from 39 locations worldwide. We analyzed a 3 yr study of coral disease prevalence to identify links between disease and a range of covariates, including thermal anomalies (from satellite data), location and coral cover, using a Generalized Linear Mixed Model. Prevalence of unhealthy corals, i.e. those with signs of known diseases or with other signs of compromised health, exceeded 10% on many reefs and ranged to over 50% on some. Disease prevalence exceeded 10% on 20% of Caribbean reefs and 2.7% of Pacific reefs surveyed. Within the same coral families across oceans, prevalence of unhealthy colonies was higher and some diseases were more common at sites in the Caribbean than those in the Pacific. The effects of high disease prevalence are potentially extensive given that the most affected coral families, the acroporids, faviids and siderastreids, are among the major reef-builders at these sites. The poritids and agaricids stood out in the Caribbean as being the most resistant to disease, even though these families were abundant in our surveys. Regional warm temperature anomalies were strongly correlated with high disease prevalence. The levels of disease reported here will provide a much-needed local reference point against which to compare future change.     

Efficiency of Opuntia ficus in the phytoremediation of a soil contaminated with used motor oil and lead,compared to that of Lolium perenne and Aloe barbadensis

Industrial pollutants such as heavy metals and hydrocarbons in soils represent a serious concern due to their persistence and negative effects on the environment, affecting cellular processes in living organisms and even causing mutations and cancer. The main objectives of this work were to evaluate the efficiency of Opuntia ficus in the phytoremediation of a soil polluted with used motor oil. Two other species, one with different and one with similar characteristics, relatively, were used for comparison purposes: Lolium perenne and Aloe barbadensis. The effect of the plants on lead solubility and bioaccumulation, the biomass production of each specie and the microbial counts and bacterial identification for each experiment was studied. Total petroleum hydrocarbons (TPH) were measured every 5 weeks throughout the 20-week phytoremediation experiment. At the end of the experiment soluble Pb, Pb extracted by the plant species, microbiological counts, total biomass and bacterial species in soil were analyzed. Even though Lolium perenne showed the highest TPH removal (47%), Opuntia ficus produced the highest biomass and similar removal (46%). Since Opuntia ficus requires low amounts of water and grows fast, it would be a suitable option in the remediation of soils polluted with hydrocarbons and/or heavy metals.     

Dispensing synthetic green leaf volatiles in maize fields increases the release of sesquiterpenes by the plants, but has little effect on the attraction of pest and beneficial insects

Maize plants respond to feeding by arthropod herbivores by producing a number of secondary plant compounds, including volatile organic compounds (VOCs). These herbivore-induced VOCs are not only known to attract natural enemies of the herbivores, but they may also prime inducible defences in neighbouring plants, resulting in stronger and faster defence responses in these VOC-exposed plants. Among the compounds that cause this priming effect, green leaf volatiles (GLVs) have received particular attention, as they are ubiquitous and rapidly emitted upon damage. In this study, we investigated their effects under realistic conditions by applying specially devised dispensers to release four synthetic GLVs at physiologically relevant concentrations in a series of experiments in maize fields. We compared the VOC emission of GLV-exposed maize plants to non-exposed plants and monitored the attraction of herbivores and predators, as well as parasitism of the caterpillar Spodoptera frugiperda, the most common herbivore in the experimental maize fields. We found that maize plants that were exposed to GLVs emitted increased quantities of sesquiterpenes compared to non-exposed plants. In several replicates, herbivorous insects, such as adult Diabrotica beetles and S. frugiperda larvae, were observed more frequently in GLV-treated plots and caused more damage to GLV-exposed plants than to non-exposed plants. Parasitism of S. frugiperda was only weakly affected by GLVs and overall parasitism rates of S. frugiperda were similar in GLV-exposed and non-exposed plots. The effects on insect presence depended on the distance from the GLV-dispensers at which the plants were located. The results are discussed in the context of strategies to improve biological control by enhancing plant-mediated attraction of natural enemies.     

Do wood-based panels made with agro-industrial residues provide environmentally benign alternatives? An LCA case study of sugarcane bagasse addition to particle board manufacturing

Purpose

Sugarcane bagasse is one of the main agro-industrial residues which can be used to produce wood-based panels. However, more investigations related to its environmental performance assessment are needed, focusing on questions such as: Does it provide environmental benefits? What are its main environmental impacts? Could it substitute wood as raw material? Accordingly, this paper presents a life cycle assessment (LCA) study of particle board manufactured with sugarcane bagasse residues.

Methods

The cradle-to-gate assessment of 1 m³ of particle board made with sugarcane bagasse (PSB) considered three main subsystems: bagasse generation, bagasse distribution, and PSB production. For the inventory of PSB, dataset from two previous LCA studies related to the conventional particle board production and the ethanol life cycle for the Brazilian context were used. The allocation criterion for the bagasse generation subsystem was 9.08 % (economic base). The potential environmental impact phase was assessed by applying the CML and USEtox methods. PSB was compared with the conventional particle board manufactured in Brazil by the categories of the CML and USETox, and including land use indicators. Finally, two scenarios were analyzed to evaluate the influence of the allocation criteria and the consumption of sugarcane bagasse.

Results and discussion

All hotspots identified by CML and USETox methods are mainly related to the PSB production subsystem (24–100 % of impacts) due to heavy fuel oil, electricity, and urea-formaldehyde resin supply chain. The bagasse generation subsystem was more relevant to the eutrophication category (75 % of impacts). The bagasse distribution subsystem was not relevant because the impacts on all categories were lower than 1 %. PSB can substitute the conventional particle board mainly because of its lower contribution to abiotic depletion and ecotoxicity. Regarding land use impacts, PSB showed lower values according to all indicators (38–40 % of all impacts), which is explained by the lower demand for land occupation in comparison to that of the traditional particle board.

Conclusions

PSB can replace the traditional particle board due to its better environmental performance. The analysis of the economic allocation criterion was relevant only for the EP category, being important to reduce diesel and N-based fertilizers use during sugarcane cultivation. Regarding the influence of the sugarcane bagasse consumption, it is suggested that the sugarcane bagasse be mixed up to 75 % during particle board manufacturing so that good quality properties and environmental performance of panels can be provided.     

Implementation of a novel in vitro model of infection of reconstituted human epithelium for expression of virulence genes in methicillin-resistant Staphylococcus aureus strains isolated from catheter-related infections in Mexico

Background

Methicillin-resistant Staphylococcus aureus (MRSA) are clinically relevant pathogens that cause severe catheter-related nosocomial infections driven by several virulence factors.

Methods

We implemented a novel model of infection in vitro of reconstituted human epithelium (RHE) to analyze the expression patterns of virulence genes in 21 MRSA strains isolated from catheter-related infections in Mexican patients undergoing haemodialysis. We also determined the phenotypic and genotypic co-occurrence of antibiotic- and disinfectant-resistance traits in the S. aureus strains, which were also analysed by pulsed-field-gel electrophoresis (PFGE).

Results

In this study, MRSA strains isolated from haemodialysis catheter-related infections expressed virulence markers that mediate adhesion to, and invasion of, RHE. The most frequent pattern of expression (present in 47.6% of the strains) was as follows: fnbA, fnbB, spa, clfA, clfB, cna, bbp, ebps, eap, sdrC, sdrD, sdrE, efb, icaA, and agr. Seventy-one percent of the strains harboured the antibiotic- and disinfectant-resistance genes ermA, ermB, tet(M), tet(K), blaZ, qacA, qacB, and qacC. PFGE of the isolated MRSA revealed three identical strains and two pairs of identical strains. The strains with identical PFGE patterns showed the same phenotypes and genotypes, including the same spa type (t895), suggesting hospital personnel manipulating the haemodialysis equipment could be the source of catheter contamination.

Conclusion

These findings help define the prevalence of MRSA virulence factors in catheter-related infections. Some of the products of the expressed genes that we detected in this work may serve as potential antigens for inclusion in a vaccine for the prevention of MRSA-catheter-related infections.