Identification of a Novel Partitivirus of Trichoderma harzianum NFCF319 and Evidence for the Related Antifungal Activity

We have reported 15 agarose gel band patterns of double-stranded RNA (dsRNA) from Trichoderma spp. We describe herein that band pattern IX in Trichoderma harzianum NFCF319, which appeared to be a single band but consisted of two dsRNAs of similar size, was identified as a novel mycovirus, designated Trichoderma harzianum partitivirus 1 (ThPV1). The larger segment (dsRNA1) of the ThPV1 genome comprised 2,289 bp and contained a single open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp). The smaller segment (dsRNA2) consisted of 2,245 bp with a single ORF encoding a capsid protein (CP). Evaluation of the deduced amino acid sequence and phylogenetic analysis indicated that ThPV1 is a new member of the genus Betapartitivirus in the family Partitiviridae. Curing of virus infection by single-sporing generated 31 virus-free single-spore clones. No significant differences in growth rate, conidia production, or pigmentation were observed between ThPV1-infected and -cured isogenic strains. In addition, comparison of the newly ThPV1-transmitted isolates with their ThPV1-cured parental strain showed no significant difference in colony morphology or pigmentation. However, inhibition of growth in co-cultured Pleurotus ostreatus and Rhizoctonia solani by T. harzianum was increased in ThPV1-containing strains compared with ThPV1-cured isogenic strains. Moreover, β-1,3-glucanase activity was significantly increased in the ThPV1-containing strains. However, no difference in chitinase activity was observed, suggesting that ThPV1 regulates the activity of a specific fungal enzyme

Heterologous expression of a tannic acid-inducible laccase3 of Cryphonectria parasitica in Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>A tannic acid-inducible and mycoviral-regulated laccase3 (<it>lac</it>3) from the chestnut blight fungus <it>Cryphonectria parasitica </it>has recently been identified, but further characterization was hampered because of the precipitation of protein products by tannic acid supplementation. The present study investigated the heterologous expression of the functional laccase3 using a yeast <it>Saccharomyces cerevisiae</it>.</p> <p>Results</p> <p>Laccase activity in the culture broth of transformants measured using a laccase-specific substrate suggested that the <it>lac</it>3 gene was successfully expressed and the corresponding protein product secreted into the culture media. In addition, activity staining and Western blot analysis of a native gel revealed that the enzyme activity co-existed with the protein product specific to anti-laccase3 antibody, confirming that the cloned <it>lac</it>3 gene is responsible for the laccase activity. When transformants were grown on plates containing tannic acid-supplemented media, brown coloration was observed around transformed cells, indicating the oxidation of tannic acid. However, the enzymatic activity was measurable only in the selective ura^-media and was negligible in nonselective nutrient-rich culture conditions. This was in part because of the increased plasmid instability in the nonselective media. Moreover, the protein product of <it>lac</it>3 appears to be sensitive to the cultured nonselective nutrient-rich broth, because a rapid decline in enzymatic activity was observed when the cultured broth of ura^-media was mixed with that of nonselective nutrient-rich broth. In addition, constitutive expression of the <it>lac</it>3 gene resulted in a reduced cell number of the <it>lac</it>3 transformants compared to that of vector-only transformed control. However, the presence of recombinant vector without <it>lac</it>3 induction did not affect the growth of transformants.</p> <p>Conclusions</p> <p>The results suggest that expression of the <it>lac</it>3 gene has an inhibitory effect on the growth of transformed <it>S. cerevisiae </it>and that the controlled expression of <it>lac</it>3 is appropriate for the possible application of recombinant yeast to the treatment of phenolic compounds.</p

Folding machineries displayed on a cation-exchanger for the concerted refolding of cysteine- or proline-rich proteins

<p>Abstract</p> <p>Background</p> <p><it>Escherichia coli </it>has been most widely used for the production of valuable recombinant proteins. However, over-production of heterologous proteins in <it>E. coli </it>frequently leads to their misfolding and aggregation yielding inclusion bodies. Previous attempts to refold the inclusion bodies into bioactive forms usually result in poor recovery and account for the major cost in industrial production of desired proteins from recombinant <it>E. coli</it>. Here, we describe the successful use of the immobilized folding machineries for <it>in vitro </it>refolding with the examples of high yield refolding of a ribonuclease A (RNase A) and cyclohexanone monooxygenase (CHMO).</p> <p>Results</p> <p>We have generated refolding-facilitating media immobilized with three folding machineries, mini-chaperone (a monomeric apical domain consisting of residues 191–345 of GroEL) and two foldases (DsbA and human peptidyl-prolyl <it>cis-trans </it>isomerase) by mimicking oxidative refolding chromatography. For efficient and simple purification and immobilization simultaneously, folding machineries were fused with the positively-charged consecutive 10-arginine tag at their C-terminal. The immobilized folding machineries were fully functional when assayed in a batch mode. When the refolding-facilitating matrices were applied to the refolding of denatured and reduced RNase A and CHMO, both of which contain many cysteine and proline residues, RNase A and CHMO were recovered in 73% and 53% yield of soluble protein with full enzyme activity, respectively.</p> <p>Conclusion</p> <p>The refolding-facilitating media presented here could be a cost-efficient platform and should be applicable to refold a wide range of <it>E. coli </it>inclusion bodies in high yield with biological function.</p

A New pH-ISFET Based Dissolved Oxygen Sensor

A new dissolved oxygen sensor based on pH-ISFET has been discussed. A platinum working electrode surrounding a pH-sensing gate of the pH-ISFET electrolyzes dissolved oxygen, resulting in a corresponding pH change. The pH-ISFET can determine dissolved oxygen concentration through detecting this pH change. --Summar

Algae–bacteria interactions: Evolution, ecology and emerging applications

AbstractAlgae and bacteria have coexisted ever since the early stages of evolution. This coevolution has revolutionized life on earth in many aspects. Algae and bacteria together influence ecosystems as varied as deep seas to lichens and represent all conceivable modes of interactions — from mutualism to parasitism. Several studies have shown that algae and bacteria synergistically affect each other's physiology and metabolism, a classic case being algae–roseobacter interaction. These interactions are ubiquitous and define the primary productivity in most ecosystems. In recent years, algae have received much attention for industrial exploitation but their interaction with bacteria is often considered a contamination during commercialization. A few recent studies have shown that bacteria not only enhance algal growth but also help in flocculation, both essential processes in algal biotechnology. Hence, there is a need to understand these interactions from an evolutionary and ecological standpoint, and integrate this understanding for industrial use. Here we reflect on the diversity of such relationships and their associated mechanisms, as well as the habitats that they mutually influence. This review also outlines the role of these interactions in key evolutionary events such as endosymbiosis, besides their ecological role in biogeochemical cycles. Finally, we focus on extending such studies on algal–bacterial interactions to various environmental and bio-technological applications

Enhanced overall efficiency of GaInN-based light-emitting diodes with reduced efficiency droop by Al-composition-graded AlGaN/GaN superlattice electron blocking layer

AlxGa1-xN/GaN superlattice electron blocking layers (EBLs) with gradually decreasing Al composition toward the p-type GaN layer are introduced to GaInN-based high-power light-emitting diodes (LEDs). GaInN/GaN multiple quantum well LEDs with 5- and 9-period Al-composition-graded AlxGa1-xN/GaN EBL show comparable operating voltage, higher efficiency as well as less efficiency droop than LEDs having conventional bulk AlGaN EBL, which is attributed to the superlattice doping effect, enhanced hole injection into the active region, and reduced potential drop in the EBL by grading Al compositions. Simulation results reveal a reduction in electron leakage for the superlattice EBL, in agreement with experimental results. (C) 2013 AIP Publishing LLC.open1133sciescopu

The ancient phosphatidylinositol 3-kinase signaling system is a master regulator of energy and carbon metabolism in algae

Algae undergo a complete metabolic transformation under stress by arresting cell growth, inducing autophagy and hyperaccumulating biofuel precursors such as triacylglycerols and starch. However, the regulatory mechanisms behind this stress-induced transformation are still unclear. Here, we use biochemical, mutational, and “omics” approaches to demonstrate that PI3K signaling mediates the homeostasis of energy molecules and influences carbon metabolism in algae. In Chlamydomonas reinhardtii, the inhibition and knockdown (KD) of algal class III PI3K led to significantly decreased cell growth, altered cell morphology, and higher lipid and starch contents. Lipid profiling of wild-type and PI3K KD lines showed significantly reduced membrane lipid breakdown under nitrogen starvation (-N) in the KD. RNA-seq and network analyses showed that under -N conditions, the KD line carried out lipogenesis rather than lipid hydrolysis by initiating de novo fatty acid biosynthesis, which was supported by tricarboxylic acid cycle down-regulation and via acetyl-CoA synthesis from glycolysis. Remarkably, autophagic responses did not have primacy over inositide signaling in algae, unlike in mammals and vascular plants. The mutant displayed a fundamental shift in intracellular energy flux, analogous to that in tumor cells. The high free fatty acid levels and reduced mitochondrial ATP generation led to decreased cell viability. These results indicate that the PI3K signal transduction pathway is the metabolic gatekeeper restraining biofuel yields, thus maintaining fitness and viability under stress in algae. This study demonstrates the existence of homeostasis between starch and lipid synthesis controlled by lipid signaling in algae and expands our understanding of such processes, with biotechnological and evolutionary implications.Ministry of Science, ICT and Future Planning 2015M3A6A2065697Ministry of Oceans and Fisheries 2015018