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Sample records for chytridiomycete blastocladiella emersonii

  1. Phosphate limitation induces sporulation in the chytridiomycete Blastocladiella emersonii.

    PubMed

    Bongiorno, Vagner Alexandre; Ferreira da Cruz, Angela; Nunis da Silva, Antonio; Corrêa, Luiz Carlos

    2012-09-01

    The cell cycle is controlled by numerous mechanisms that ensure correct cell division. If growth is not possible, cells may eventually promote autophagy, differentiation, or apoptosis. Microorganisms interrupt their growth and differentiate under general nutrient limitation. We analyzed the effects of phosphate limitation on growth and sporulation in the chytridiomycete Blastocladiella emersonii using kinetic data, phase-contrast, and laser confocal microscopy. Under phosphate limitation, zoospores germinated and subsequently formed 2-4 spores, regardless of the nutritional content of the medium. The removal of phosphate at any time during growth induced sporulation of vegetative cells. If phosphate was later added to the same cultures, growth was restored if the cells were not yet committed to sporulation. The cycles of addition and withdrawal of phosphate from growth medium resulted in cycles of germination-growth, germination-sporulation, or germination-growth-sporulation. These results show that phosphate limitation is sufficient to interrupt cell growth and to induce complete sporulation in B. emersonii. We concluded that the determination of growth or sporulation in this microorganism is linked to phosphate availability when other nutrients are not limiting. This result provides a new tool for the dissection of nutrient-energy and signal pathways in cell growth and differentiation. PMID:22913304

  2. Global gene expression analysis during germination in the chytridiomycete Blastocladiella emersonii.

    PubMed

    Salem-Izacc, Silvia M; Koide, Tie; Vêncio, Ricardo Z N; Gomes, Suely L

    2009-02-01

    Blastocladiella emersonii is an aquatic fungus of the Chytridiomycete class. During germination, the zoospore, a motile nongrowing cell, goes through a cascade of morphological changes that culminates with its differentiation into the germling cell, capable of coenocytic vegetative growth. Transcriptome analyses of B. emersonii cells were carried out during germination induced under various environmental conditions. Microarray data analyzing 3,563 distinct B. emersonii genes revealed that 26% of them are differentially expressed during germination in nutrient medium at at least one of the time points investigated. Over 500 genes are upregulated during the time course of germination under those conditions, most being related to cell growth, including genes involved in protein biosynthesis, DNA transcription, energetic metabolism, carbohydrate and oligopeptide transport, and cell cycle control. On the other hand, several transcripts stored in the zoospores are downregulated during germination in nutrient medium, such as genes involved in signal transduction, amino acid transport, and chromosome organization. In addition, germination induced in the presence of nutrients was compared with that triggered either by adenine or potassium ions in inorganic salt solution. Several genes involved in cell growth, induced during germination in nutrient medium, do not show increased expression when B. emersonii zoospores germinate in inorganic solution, suggesting that nutrients exert a positive effect on gene transcription. The transcriptome data also revealed that most genes involved in cell signaling show the same expression pattern irrespective of the initial germination stimulus. PMID:19098129

  3. Structure, expression, and functional analysis of the gene coding for calmodulin in the chytridiomycete Blastocladiella emersonii.

    PubMed

    Simão, R C; Gomes, S L

    2001-04-01

    The single calmodulin (CaM) gene and the corresponding cDNA of the chytridiomycete Blastocladiella emersonii were isolated and characterized. The CaM gene is interrupted by three introns and transcribed in a single 0.7-kb mRNA species encoding a predicted protein 91% identical to human CaM. B. emersonii CaM has been expressed in Escherichia coli as a fusion protein with gluthatione S-transferase (GST) and purified by affinity chromatography and cleavage from the GST portion using a site-specific protease. In the presence of Ca(2+), B. emersonii CaM exhibited a shift in apparent molecular mass similar to that observed with bovine CaM and was able to activate the autophosphorylation of CaM-dependent protein kinase II (CaMKII) from rat brain. CaM expression is developmentally regulated in B. emersonii, with CaM mRNA and protein concentrations increasing during sporulation to maximum levels observed just prior to the release of the zoospores into the medium. Both CaM protein and mRNA levels decrease drastically at the zoospore stage, increasing again during germination. The CaM antagonists compound 48/80, calmidazolium, and W7 were shown to completely inhibit B. emersonii sporulation when added to the cultures at least 120, 150, and 180 min after induction, respectively. All these drugs also inhibited growth and zoospore production in this fungus. The Ca(2+) channel blocker TMB-8 and the CaMKII inhibitor KN93 completely inhibited sporulation if added up to 60 min after induction of this stage, but only KN93 affected fungal growth. The data presented suggest that the Ca(2+)-CaM complex and CaMKII play an important role during growth and sporulation in B. emersonii. PMID:11244068

  4. Large-subunit rRNA sequence of the chytridiomycete Blastocladiella emersonii, and implications for the evolution of zoosporic fungi.

    PubMed

    Van der Auwera, G; De Wachter, R

    1996-11-01

    The 5.8S and 28S ribosomal RNA sequences of the chytridiomycete Blastocladiella emersonii were determined. These data were combined with 18S rRNA sequences in order to carry out a phylogenetic analysis based on distance matrix, parsimony, and maximum likelihood methods. The new data confirmed that chytridiomycetes are true fungi and not protists, as was already suggested on the basis of biochemical, ultrastructural, and 18S rRNA data. Within the fungal clade, B. emersonii formed the first line of divergence. The position of the fungi within the eukaryotic "crown" taxa was also reassessed, and the alveolate-stramenopile cluster appeared as their sister group. The stramenopiles also comprise a number of zoosporic fungi, which resemble chytridiomycetes in so many respects, e.g., production of motile spores, thallus morphology, and absorptive nutrition, that they have been classified together with them in the past. This suggests that the possible common ancestor of the fungi, stramenopiles, and alveolates may have been a zoosporic fungus, which would mean that zoosporic fungi are paraphyletic instead of polyphyletic as previously suggested. PMID:8875862

  5. Comparative expression analysis of members of the Hsp70 family in the chytridiomycete Blastocladiella emersonii.

    PubMed

    Georg, Raphaela de Castro; Gomes, Suely Lopes

    2007-01-15

    Sequencing of a large number of expressed sequence tags from Blastocladiella emersonii revealed the presence of ten distinct putative members of the 70 kDa-heat shock protein (Hsp70) family in this fungus. The amino acid sequence deduced from eight of these cDNAs showed significant similarity to members of the Saccharomyces cerevisiae Hsp70 family, and the remaining displayed high sequence homology with hsp70 gene products from other organisms. The hsp70-3 gene was the most highly expressed at normal temperatures and was poorly induced during heat shock. Except for hsp70-4 and hsp70-6, all other hsp70 genes were induced to different degrees upon exposure of B. emersonii cells to heat shock, with hsp70-1 gene presenting the highest transcript levels. Phylogenetic analysis of complete B. emersonii putative Hsp70 protein sequences indicated that Hsp70-1 and Hsp70-3 corresponded to cytosolic proteins, whereas Hsp70-7 and Hsp70-9 are probably localized in the endoplasmic reticulum and mitochondria, respectively. PMID:17185163

  6. Characterization and expression of two genes encoding isoforms of a putative Na, K-ATPase in the chytridiomycete Blastocladiella emersonii.

    PubMed

    Fietto, Luciano Gomes; Pugliese, Luciana; Gomes, Suely Lopes

    2002-06-01

    A P-type ATPase gene (BePAT1) from the aquatic fungus Blastocladiella emersonii, which surprisingly showed high similarity with the alpha-subunit of Na, K-ATPases from animal cells, has been reported recently [Biochim. Biophys. Acta 1383 (1998) 183]. In the present study, we describe the characterization of a second gene, denominated BePAT2, and show that these two genes have a different intron-exon structure but encode putative proteins with greater than 90% amino acid identity. Northern blot and multiplex reverse transcription and polymerase chain reaction (RT-PCR) assays have revealed that BePAT1 and BePAT2 genes have a non-coordinate, developmentally regulated expression during B. emersonii life cycle. Phosphoenzyme formation experiments using the immunopurified enzymes have indicated the presence of a Na, K-ATPase-like activity. Furthermore, immunofluorescence studies using B. emersonii zoospores localized the ATPases on the plasma membrane of these cells. PMID:12031485

  7. Gene discovery and expression profile analysis through sequencing of expressed sequence tags from different developmental stages of the chytridiomycete Blastocladiella emersonii.

    PubMed

    Ribichich, Karina F; Salem-Izacc, Silvia M; Georg, Raphaela C; Vêncio, Ricardo Z N; Navarro, Luci D; Gomes, Suely L

    2005-02-01

    Blastocladiella emersonii is an aquatic fungus of the chytridiomycete class which diverged early from the fungal lineage and is notable for the morphogenetic processes which occur during its life cycle. Its particular taxonomic position makes this fungus an interesting system to be considered when investigating phylogenetic relationships and studying the biology of lower fungi. To contribute to the understanding of the complexity of the B. emersonii genome, we present here a survey of expressed sequence tags (ESTs) from various stages of the fungal development. Nearly 20,000 cDNA clones from 10 different libraries were partially sequenced from their 5' end, yielding 16,984 high-quality ESTs. These ESTs were assembled into 4,873 putative transcripts, of which 48% presented no matches with existing sequences in public databases. As a result of Gene Ontology (GO) project annotation, 1,680 ESTs (35%) were classified into biological processes of the GO structure, with transcription and RNA processing, protein biosynthesis, and transport as prevalent processes. We also report full-length sequences, useful for construction of molecular phylogenies, and several ESTs that showed high similarity with known proteins, some of which were not previously described in fungi. Furthermore, we analyzed the expression profile (digital Northern analysis) of each transcript throughout the life cycle of the fungus using Bayesian statistics. The in silico approach was validated by Northern blot analysis with good agreement between the two methodologies. PMID:15701807

  8. The mitochondrial view of Blastocladiella emersonii.

    PubMed

    Tambor, José Humberto M; Ribichich, Karina F; Gomes, Suely L

    2008-11-15

    The mitochondrial genome of the chytrid Blastocladiella emersonii was sequenced and annotated, revealing the complete set of oxidative phosphorylation genes and tRNAs/rRNAs necessary for the translation process. Phylogenetic reconstructions reinforce the proposal of the new phylum Blastocladiomycota. Evidences of gene duplication due to inserted elements suggest shared susceptibility to gene invasion/exchange between chytrids and zygomycetes. The gene content of B. emersonii is very similar to Allomyces macrogynus but the content of intronic and changeable elements is much lower suggesting a stronger resistance to this kind of exchange. In addition, a total of 401 potential nuclear transcripts encoding mitochondrial proteins were obtained after B. emersonii EST database scanning using Saccharomyces cerevisiae, Homo sapiens and Arabidopsis thaliana data as probes and TargetP tool to find N-terminal mitochondrial signal in translated sequences. PMID:18721866

  9. Transcriptome analysis in response to heat shock and cadmium in the aquatic fungus Blastocladiella emersonii.

    PubMed

    Georg, Raphaela C; Gomes, Suely L

    2007-06-01

    The global transcriptional response of the chytridiomycete Blastocladiella emersonii to environmental stress conditions was explored by sequencing a large number of expressed sequence tags (ESTs) from three distinct cDNA libraries, constructed with mRNA extracted from cells exposed to heat shock and different concentrations of cadmium chloride. A total of 6,350 high-quality EST sequences were obtained and assembled into 2,326 putative unigenes, 51% of them not previously described in B. emersonii. To approximately 59% of the unigenes it was possible to assign an orthologue in another organism, whereas 41% of them remained without a putative identification, with transcripts related to protein folding and antioxidant activity being highly enriched in the stress libraries. A microarray chip was constructed encompassing 3,773 distinct ESTs from the B. emersonii transcriptome presently available, which correspond to a wide range of biological processes. Global gene expression analysis of B. emersonii cells exposed to stress conditions revealed a large number of differentially expressed genes: 122 up- and 60 downregulated genes during heat shock and 189 up- and 110 downregulated genes during exposure to cadmium. The main functional categories represented among the upregulated genes were protein folding and proteolysis, proteins with antioxidant properties, and cellular transport. Interestingly, in response to cadmium stress, B. emersonii cells induced genes encoding six different glutathione S-transferases and six distinct metacaspases, as well as genes coding for several proteins of sulfur amino acid metabolism, indicating that cadmium causes oxidative stress and apoptosis in this fungus. All sequences described in this study have been submitted to the GenBank EST section with the accession numbers EE 730389 to EE 736848. PMID:17449658

  10. Regulation of adenylyl cyclase from Blastocladiella emersonii by guanine nucleotides.

    PubMed

    Terenzi, H; Maia, J C

    1993-11-01

    GTP gamma S stimulates adenylyl cyclase in particulate fractions of Blastocladiella emersonii zoospores. Cholera toxin catalyses the ADP-ribosylation of a membrane protein of a molecular weight (46,000) similar to that of the alpha subunit of Gs found in vertebrate cells. A membrane protein of 46 kDa can also be recognized in Western blots by an antipeptide antiserum (RM/1) raised against the C-terminus of G alpha 2-subunits. These results suggest that a G-protein mediates the regulation of Blastocladiella adenylyl cyclase by guanine nucleotides. PMID:8224237

  11. Expression of genes encoding cytosolic and endoplasmic reticulum HSP90 proteins in the aquatic fungus Blastocladiella emersonii.

    PubMed

    Pugliese, Luciana; Georg, Raphaela C; Fietto, Luciano G; Gomes, Suely L

    2008-03-31

    HSP90 proteins are important molecular chaperones involved in multiple cellular processes. This work reports the characterization of cDNAs encoding two distinct HSP90 proteins (named HSP90A and HSP90B) from the chytridiomycete Blastocladiella emersonii. Deduced amino acid sequences of HSP90A and HSP90B exhibit signatures of the cytosolic and endoplasmic reticulum (ER) HSP90 proteins, respectively. A genomic clone encoding HSP90A was also characterized indicating the presence of a single intron of 184 bp interrupting the coding region, located near the amino-terminus of the protein. Expression of both HSP90A and HSP90B genes increases significantly during heat shock at 38 degrees C, with highest induction ratios observed in cells stressed during germination of the fungus. Changes in the amount of HSP90A transcript were also evaluated during B. emersonii life cycle at physiological temperature (27 degrees C), and its levels were found to increase both during germination and sporulation of the fungus. HSP90A protein levels were analyzed during B. emersonii life cycle and significant changes were observed only during sporulation. Furthermore, during heat stress a large increase in the amount of HSP90A protein was observed. Induction of HSP90A and HSP90B genes during heat stress indicates the importance of both genes in the response to high temperature in B. emersonii. PMID:18281163

  12. Acquisition of thermotolerance during development of Blastocladiella emersonii.

    PubMed

    Silva, A M; Juliani, M H; da Costa, J J; Bonato, M C

    1987-04-14

    In the fungus Blastocladiella emersonii the synthesis of heat-shock proteins is developmentally regulated; particular subsets of heat-shock proteins are induced by heat shock during sporulation, germination and growth and some heat shock-related proteins are spontaneously expressed during sporulation (Bonato et al., 1987, Eur. J. Biochem., in press). Nevertheless, acquisition of thermotolerance can be induced at any stage of the life cycle. The development of thermotolerance is correlated with the enhanced synthesis of some heat-shock proteins: hsp 82a, hsp 82b, hsp 76, hsp 70, hsp 60, hsp 25, hsp 17b. Other hsps are not specifically involved in thermotolerance. PMID:3579921

  13. Blastocladiella emersonii expresses a centrin similar to Chlamydomonas reinhardtii isoform not found in late-diverging fungi.

    PubMed

    Ribichich, Karina Fabiana; Gomes, Suely Lopes

    2005-08-15

    Centrins are members of the calcium-binding EF-hand protein superfamily which can be divided into two subfamilies, probably associated with different functions: one related to Chlamydomonas reinhardtii centrin, CrCenp, and the other, represented by Saccharomyces cerevisiae isoform, ScCdc31p. ESTs encoding the two isoforms (BeCen1 and BeCen3) from the chytridiomycete Blastocladiella emersonii were isolated, and expression of the CrCenp-type centrin, BeCen1, was analyzed throughout the fungus life cycle. Becen1 mRNA levels increase transiently during sporulation and protein levels present a similar pattern. Immunolocalization studies seem to localize BeCen1 at the basal body zone and in the cytoplasm surrounding the nuclear cap, a zoospore organelle. PMID:16051227

  14. Transcriptional response to hypoxia in the aquatic fungus Blastocladiella emersonii.

    PubMed

    Camilo, César M; Gomes, Suely L

    2010-06-01

    Global gene expression analysis was carried out with Blastocladiella emersonii cells subjected to oxygen deprivation (hypoxia) using cDNA microarrays. In experiments of gradual hypoxia (gradual decrease in dissolved oxygen) and direct hypoxia (direct decrease in dissolved oxygen), about 650 differentially expressed genes were observed. A total of 534 genes were affected directly or indirectly by oxygen availability, as they showed recovery to normal expression levels or a tendency to recover when cells were reoxygenated. In addition to modulating many genes with no putative assigned function, B. emersonii cells respond to hypoxia by readjusting the expression levels of genes responsible for energy production and consumption. At least transcriptionally, this fungus seems to favor anaerobic metabolism through the upregulation of genes encoding glycolytic enzymes and lactate dehydrogenase and the downregulation of most genes coding for tricarboxylic acid (TCA) cycle enzymes. Furthermore, genes involved in energy-costly processes, like protein synthesis, amino acid biosynthesis, protein folding, and transport, had their expression profiles predominantly downregulated during oxygen deprivation, indicating an energy-saving effort. Data also revealed similarities between the transcriptional profiles of cells under hypoxia and under iron(II) deprivation, suggesting that Fe(2+) ion could have a role in oxygen sensing and/or response to hypoxia in B. emersonii. Additionally, treatment of fungal cells prior to hypoxia with the antibiotic geldanamycin, which negatively affects the stability of mammalian hypoxia transcription factor HIF-1alpha, caused a significant decrease in the levels of certain upregulated hypoxic genes. PMID:20418381

  15. Agrobacterium tumefasciens-mediated transformation of the aquatic fungus Blastocladiella emersonii.

    PubMed

    Vieira, André L G; Camilo, César M

    2011-08-01

    Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting model of study of cellular function and differentiation. We constructed binary T-DNA vectors containing hygromycin phosphotransferase (hph) or enhanced green fluorescent protein (egfp) genes, under the control of Aspergillus nidulans trpC promoter and terminator sequences. 24 h of co-cultivation in induction medium (IM) agar plates, followed by transfer to PYG-agar plates containing cefotaxim to kill Agrobacterium tumefsciens and hygromycin to select transformants, resulted in growth and sporulation of resistant transformants. Genomic DNA from the pool o resistant zoospores were shown to contain T-DNA insertion as evidenced by PCR amplification of hph gene. Using a similar protocol we could also evidence the expression of enhanced green fluorescent protein (EGFP) in zoospores derived from transformed cells. This protocol can also open new perspectives for other non-transformable closely related fungi, like the Chytridiomycete class. PMID:21396477

  16. An analysis of developmental timing in Blastocladiella emersonii sporulation.

    PubMed

    Peralta, R M; Lodi, W R

    1988-07-01

    We propose a model of time regulation for the expression of the Blastocladiella emersonii sporulation phenotypes based on new methods (Soll, 1986) which analyse the effect of temperature on the rate limiting processes, i.e., "timers" of certain events during development. By using reciprocal shift experiments (transferring sporulating cells from 22 to 27 degrees C and vice versa) we characterized the timers of the phenotypes: septate zoosporangium, papillate zoosporangium, cleavage zoosporangium, and empty zoosporangium, considering the number of the components, sensitivity, duration, and the mutual dependency of each limiting factor. The timers for the first three phenotypes started at zero time of sporulation induction and acted in parallel. The fourth phenotype, empty zoosporangium, has a timer which appears to act sequentially to that of the papillate zoosporangium. We also studied the effects of polyoxin D, calcofluor white, and congo red on sporulation. The first drug prevents the appearance of the septate zoosporangium and the other two prevent the expression of the papillate zoosporangium. In spite of the morphological blockage, the zoosporogenesis proceeds, resulting in the formation of normal zoospores. These results are interpreted as additional evidence for the parallel model of control proposed here. PMID:2454856

  17. Regulation of tubulin and actin synthesis and accumulation during Blastocladiella emersonii development.

    PubMed

    da Silva, A M; Juliani, M H

    1988-06-01

    Actin and alpha and beta-tubulin have been identified in Blastocladiella emersonii by two-dimensional gel electrophoresis and Western blotting. The kinetics of synthesis of these proteins were compared by pulse-labeling experiments with [35S]methionine and with the accumulation of their corresponding mRNAs, translated in a cell-free system. Large increases occur in the rates of actin and alpha- and beta-tubulin biosynthesis during sporulation and there is an accumulation of the corresponding mRNAs. In parallel to the increased synthesis, these cytoskeletal proteins accumulate during the late stage of sporulation. PMID:3409324

  18. Effect of heat shock on S6 phosphorylation during the development of Blastocladiella emersonii.

    PubMed

    da Silva, A M; Juliani, M H; Bonato, M C

    1987-11-01

    Changes in phosphorylation of ribosomal protein S6 during heat shock, induction of thermotolerance and recovery from heat shock at different stages of Blastocladiella emersonii development were investigated. Independently of the initial state of S6 phosphorylation (maximal or intermediate), a rapid and complete dephosphorylation of S6 is induced by heat shock and S6 remains unphosphorylated during the acquired thermotolerance. During recovery from heat shock rephosphorylation of S6 occurs always to the levels characteristic of that particular stage, coincidently with the turn off of heat shock protein synthesis. PMID:3454866

  19. A Cyclic GMP-Dependent K+ Channel in the Blastocladiomycete Fungus Blastocladiella emersonii

    PubMed Central

    Avelar, Gabriela Mól; Glaser, Talita; Leonard, Guy; Richards, Thomas A.; Ulrich, Henning

    2015-01-01

    Phototaxis in flagellated zoospores of the aquatic fungus Blastocladiella emersonii depends on a novel photosensor, Blastocladiella emersonii GC1 (BeGC1), comprising a type I (microbial) rhodopsin fused to a guanylyl cyclase catalytic domain, that produces the conserved second messenger cyclic GMP (cGMP). The rapid and transient increase in cGMP levels during the exposure of zoospores to green light was shown to be necessary for phototaxis and dependent on both rhodopsin function and guanylyl cyclase activity. It is noteworthy that BeGC1 was localized to the zoospore eyespot apparatus, in agreement with its role in the phototactic response. A putative cyclic nucleotide-gated channel (BeCNG1) was also identified in the genome of the fungus and was implicated in flagellar beating via the action of a specific inhibitor (l-cis-diltiazem) that compromised zoospore motility. Here we show that B. emersonii expresses a K+ channel that is activated by cGMP. The use of specific channel inhibitors confirmed the activation of the channel by cGMP and its K+ selectivity. These characteristics are consistent with the function of an ion channel encoded by the BeCNG1 gene. Other blastocladiomycete fungi, such as Allomyces macrogynus and Catenaria anguillulae, possess genes encoding a similar K+ channel and the rhodopsin–guanylyl cyclase fusion protein, while the genes encoding both these proteins are absent in nonflagellated fungi. The presence of these genes as a pair seems to be an exclusive feature of blastocladiomycete fungi. Taken together, these data demonstrate that the B. emersonii cGMP-activated K+ channel is involved in the control of zoospore motility, most probably participating in the cGMP-signaling pathway for the phototactic response of the fungus. PMID:26150416

  20. A Cyclic GMP-Dependent K+ Channel in the Blastocladiomycete Fungus Blastocladiella emersonii.

    PubMed

    Avelar, Gabriela Mól; Glaser, Talita; Leonard, Guy; Richards, Thomas A; Ulrich, Henning; Gomes, Suely L

    2015-09-01

    Phototaxis in flagellated zoospores of the aquatic fungus Blastocladiella emersonii depends on a novel photosensor, Blastocladiella emersonii GC1 (BeGC1), comprising a type I (microbial) rhodopsin fused to a guanylyl cyclase catalytic domain, that produces the conserved second messenger cyclic GMP (cGMP). The rapid and transient increase in cGMP levels during the exposure of zoospores to green light was shown to be necessary for phototaxis and dependent on both rhodopsin function and guanylyl cyclase activity. It is noteworthy that BeGC1 was localized to the zoospore eyespot apparatus, in agreement with its role in the phototactic response. A putative cyclic nucleotide-gated channel (BeCNG1) was also identified in the genome of the fungus and was implicated in flagellar beating via the action of a specific inhibitor (l-cis-diltiazem) that compromised zoospore motility. Here we show that B. emersonii expresses a K(+) channel that is activated by cGMP. The use of specific channel inhibitors confirmed the activation of the channel by cGMP and its K(+) selectivity. These characteristics are consistent with the function of an ion channel encoded by the BeCNG1 gene. Other blastocladiomycete fungi, such as Allomyces macrogynus and Catenaria anguillulae, possess genes encoding a similar K(+) channel and the rhodopsin-guanylyl cyclase fusion protein, while the genes encoding both these proteins are absent in nonflagellated fungi. The presence of these genes as a pair seems to be an exclusive feature of blastocladiomycete fungi. Taken together, these data demonstrate that the B. emersonii cGMP-activated K(+) channel is involved in the control of zoospore motility, most probably participating in the cGMP-signaling pathway for the phototactic response of the fungus. PMID:26150416

  1. Endogenous electrical currents in the water mold Blastocladiella emersonii during growth and sporulation.

    PubMed

    Stump, R F; Robinson, K R; Harold, R L; Harold, F M

    1980-11-01

    We have explored the pattern of electrical currents generated by single cells of the water mold Blastocladiella emersonii at several stages of its life cycle. Extracellular currents were measured with a vibrating probe constructed after the design of Jaffe and Nuccitelli [Jaffe, L. F. & Nuccitelli, R. (1974) J. Cell Biol. 63, 614-628]. In growing cells positive current, of the order of 1 microA/cm2, enters the rhizoid and leaves from the thallus; circumstantial evidence suggests that protons carry much of the current. Sporulation is associated with reversal of the current pattern, such that positive current enters the thallus and leaves from the rhizoidal region; the ions that carry the current have not been identified. These current patterns appear to play a role in the spatial localization of fungal growth and development. PMID:6256753

  2. Analysis of Blastocladiella emersonii ribosomal proteins in four two-dimensional gel electrophoresis systems.

    PubMed

    Bonato, M C; Maia, J C; Juliani, M H

    1985-01-01

    Ribosomal proteins of the aquatic fungus Blastocladiella emersonii were isolated and characterized on four different two-dimensional polyacrylamide gel electrophoresis systems. 40S and 60S ribosomal subunit proteins from zoospores were identified. The position of every protein was determined in each electrophoretic system using the "four-corners" method (Madjar et al., Molecular and General Genetics, 171: 121-134, 1979). Thirty-two and 39 proteins were identified in the 40S and 60S ribosomal subunits, respectively. The molecular weights of individual proteins in the 40S subunit ranged from 10 000 to 37 000, with a number-average molecular weight of 20 000. The molecular weight range for the 60S subunit was 13 000-51 000 with a number-average molecular weight of 21 000. Proteins from ribosomes of different cell types were compared and found to be qualitatively indistinguishable. The only consistent difference in the patterns of proteins was in the S6 protein of the 40S subunit, which is the major phosphoprotein of Blastocladiella ribosomes. PMID:3830281

  3. Coordinate pretranslational control of cAMP-dependent protein kinase subunit expression during development in the water mold Blastocladiella emersonii.

    PubMed

    Marques, M do V; Borges, A C; de Oliveira, J C; Gomes, S L

    1992-02-01

    The aquatic fungus Blastocladiella emersonii provides a system for studying the regulation of expression of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA). Blastocladiella cells contain a single PKA with properties very similar to type II kinases of mammalian tissues. During development cAMP-dependent protein kinase activity and its associated cAMP-binding activity change drastically. We have previously shown that the increase in cAMP-binding activity during sporulation is due to de novo synthesis of R subunit and to an increase in the translatable mRNA coding for R (Marques et al., Eur. J. Biochem. 178, 803, 1989). In the present work we have continued these studies to investigate the mechanism by which the changes in the level of kinase activity take place. The C subunit of Blastocladiella has been purified; antiserum has been raised against it and used to determine amounts of C subunit throughout the fungus' life cycle. A sharp increase in C subunit content occurs during sporulation and peaks at the zoospore stage. Northern blot analyses, using Blastocladiella C and R cDNA probes, have shown that the levels of C and R mRNAs parallel their intracellular protein concentrations. These results indicate a coordinate pretranslational control for C and R subunit expression during differentiation in Blastocladiella. PMID:1309711

  4. Changes in the pattern of protein synthesis during zoospore germination in Blastocladiella emersonii.

    PubMed

    Silva, A M; Maia, J C; Juliani, M H

    1987-05-01

    Using two-dimensional gel electrophoresis, we analyzed the pattern of proteins synthesized during Blastocladiella emersonii zoospore germination in an inorganic solution, in both the presence and absence of actinomycin D. During the transition from zoospore to round cells (the first 25 min), essentially no qualitative differences were noticeable, indicating that the earliest stages of germination are entirely preprogrammed with stored RNA. Later in germination (after 25 min), however, changes in the pattern of protein synthesis were found. Some of these proteins (a total of 6 polypeptides) correspond possibly to a selective translation of stored messages, whereas the majority of the changed proteins (22 polypeptides) corresponds to newly synthesized mRNA. Thus, multiple levels of protein synthesis regulation seem to occur during zoospore germination, involving both transcriptional and translational controls. We also analyzed the pattern of protein synthesis during germination in a nutrient medium; synthesis of specific polypeptides occurred during late germination. During early germination posttranslational control was also observed, several labeled proteins from zoospores being specifically degraded or charge modified. PMID:3571161

  5. Phosphorylation of ribosomal protein S6 in the aquatic fungus Blastocladiella emersonii.

    PubMed

    Bonato, M C; da Silva, A M; Maia, J C; Juliani, M H

    1984-11-01

    The changes in the degree of phosphorylation of ribosomal protein S6 during the life cycle of the aquatic fungus Blastocladiella emersonii were analyzed by two-dimensional gel electrophoresis. Three phosphorylated derivatives of S6 are present throughout the entire life cycle. However, under certain germination conditions, more highly phosphorylated derivatives of S6 appear. Nonetheless, the resumption of protein synthesis that occurs during germination is not dependent on those highly phosphorylated derivatives of S6. The pattern and sites of phosphorylation of S6 labelled in vivo with [32P]orthophosphate have been compared with those of 40S ribosomal subunit labelled in vitro by partially purified protein kinases. Three major phosphopeptides were found in S6 isolated from the zoospore, while six phosphopeptides were found after zoospore germination (in germling cells). The phosphopeptide patterns of S6 phosphorylated by the cAMP-dependent protein kinase and by casein kinases I and II were completely distinct. Only the cAMP-dependent protein kinase gives rise to a phosphopeptide found in 32P-labelled cells, indicating that one of sites phosphorylated in vivo is also phosphorylated in vitro by the cAMP-dependent protein kinase. PMID:6092077

  6. Developmental regulation of hexosamine biosynthesis by protein phosphatases 2A and 2C in Blastocladiella emersonii.

    PubMed

    Etchebehere, L C; Simon, M N; Campanhã, R B; Zapella, P D; Véron, M; Maia, J C

    1993-08-01

    Extracts of the aquatic fungus Blastocladiella emersonii were found to contain protein phosphatases type 1, type 2A, and type 2C with properties analogous to those found in mammalian tissues. The activities of all three protein phosphatases are developmentally regulated, increasing during sporulation, with maximum level in zoospores. Protein phosphatases 2A and 2C, present in zoospore extracts, catalyze the dephosphorylation of L-glutamine:fructose-6-phosphate amidotransferase (EC 2.6.1.16, amidotransferase), a key regulatory enzyme in hexosamine biosynthesis. The protein phosphatase inhibitor okadaic acid induces encystment and inhibits germ tube formation but does not affect the synthesis of the chitinous cell wall. These results strongly suggest that phosphatase 2C is responsible for the dephosphorylation of amidotransferase in vivo. This dephosphorylation is inhibited by uridine-5'-diphospho-N-acetylglucosamine, the end product of hexosamine synthesis and the substrate for chitin synthesis. This result demonstrates a dual role of uridine-5'-diphospho-N-acetylglucosamine by inhibiting the activity of the phosphorylated form of amidotransferase and by preventing its dephosphorylation by protein phosphatases. PMID:8394312

  7. Evidence that Blastocladiella emersonii zoospore maintenance factor is a sulfhydryl group-containing cyclic ribotide.

    PubMed

    Gottschalk, W K; Sonneborn, D R

    1985-06-10

    Blastocladiella emersonii zoospore maintenance factor (ZMF), released into the medium during zoospore production, mediates a reversible developmental block to zoospore encystment (Gottschalk, W. K., and Sonneborn, D. R. (1981) Exp. Mycol. 5, 1-14 and (1982) Dev. Biol. 93, 165-180). Crude ZMF and purified ZMF display indistinguishable sensitivities/insensitivities to inactivations by several different chemical or enzymatic treatments. Such data have provided additional support for the conclusion (Gottschalk, W. K., and Sonneborn, D. R. (1985) J. Biol. Chem. 260, 6588-6591) that ZMF biological activity resides in a single molecular species. The inactivation analyses have provided substantial evidence that ZMF is a newly discovered SH-containing cyclic ribotide. At least one SH-containing side group and at least one free amino group linked to an imidazole, as well as a ribosyl moiety containing a cyclic 3',5'-phosphate, a 2'-free hydroxyl, and a 1'-linkage to the imidazole, appear to be essential structural requirements for ZMF-mediated encystment blockage. The proposed structure of biologically functional ZMF is similar to that of a key intermediate in the de novo pathway of purine nucleotide biosynthesis (5'-phosphoribosyl-5-aminoimidazole-4-N-succino-carboxamide), except that ZMF, and not 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide, contains a cyclic phosphate and at least one reduced SH group. PMID:3997839

  8. Phosphorylation-dependent regulation of amidotransferase during the development of Blastocladiella emersonii.

    PubMed

    Etchebehere, L C; Maia, J C

    1989-08-01

    The enzyme amidotransferase [2-amino-2-deoxy-D-glucose-6-phosphate ketol isomerase (amino-transferring); EC 2.6.1.16] catalyzes the first step in the hexosamine biosynthetic pathway. In Blastocladiella emersonii the sensitivity of the enzyme to the inhibitor uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) is developmentally regulated. The inhibitable form of amidotransferase activity present in the zoospore is converted to a noninhibitable form during germination. The latter form is present throughout the growth phase and sensitivity to UDP-GlcNAc gradually returns to the zoospore level during sporulation [C.P. Selitrennikoff, N.E. Dalley, and D.R. Sonneborn (1980) Proc. Natl. Acad. Sci. USA 77, 5998-6002]. The following evidence suggests that a phosphorylation/dephosphorylation mechanism underlies this interconversion: (i) Both the vegetative and zoospore enzymes have the same molecular weight of 140,000, but the vegetative enzyme elutes significantly earlier on a DEAE-cellulose column than does the zoospore enzyme. (ii) The increased sensitivity to UDP-GlcNAc occurring in vivo and in vitro correlates with increased phosphorylation of a polypeptide of apparent Mr 76,000. This component copurifies with amidotransferase activity through ion-exchange chromatography and sucrose density gradient centrifugation. (iii) Desensitization and concurrent dephosphorylation of sensitive amidotransferase can be observed in vitro after treatment with a partially purified magnesium-dependent phosphoprotein phosphatase from zoospores. PMID:2546495

  9. Calcium-dependent anion channel in the water mold, Blastocladiella emersonii.

    PubMed

    Caldwell, J H; Van Brunt, J; Harold, F M

    1986-01-01

    Injection of depolarizing current into vegetative cells of the water mold Blastocladiella emersonii elicits a regenerative response that has the electrical characteristics of an action potential. Once they have been taken past a threshold of about -40 mV, cells abruptly depolarize to +20 mV or above; after an interval ranging from several hundred milliseconds to a few seconds, the cells spontaneously return to their resting potential near -100 mV. When the action potential was analyzed with voltage-clamp recording, it proved to be biphasic. The initial phase reflects an influx of calcium ions through voltage-sensitive channels that also carry Sr2+ ions. The delayed, and more extended, phase of inward current results from the efflux of chloride and other anions. The anion channels are broadly selective, passing chloride, nitrate, phosphate, acetate, succinate and even PIPES. The anion channels open in response to the entry of calcium ions, but do not recognize Sr2+. Calcium channels, anion channels and calcium-specific receptors that link the two channels appear to form an ensemble whose physiological function is not known. Action potentials rarely occur spontaneously but can be elicited by osmotic downshock, suggesting that the ion channels may be involved in the regulation of turgor. PMID:2420994

  10. Global gene expression analysis during sporulation of the aquatic fungus Blastocladiella emersonii.

    PubMed

    Vieira, André L G; Gomes, Suely L

    2010-03-01

    The Blastocladiella emersonii life cycle presents a number of drastic biochemical and morphological changes, mainly during two cell differentiation stages: germination and sporulation. To investigate the transcriptional changes taking place during the sporulation phase, which culminates with the production of the zoospores, motile cells responsible for the dispersal of the fungus, microarray experiments were performed. Among the 3,773 distinct genes investigated, a total of 1,207 were classified as differentially expressed, relative to time zero of sporulation, at at least one of the time points analyzed. These results indicate that accurate transcriptional control takes place during sporulation, as well as indicating the necessity for distinct molecular functions throughout this differentiation process. The main functional categories overrepresented among upregulated genes were those involving the microtubule, the cytoskeleton, signal transduction involving Ca(2+), and chromosome organization. On the other hand, protein biosynthesis, central carbon metabolism, and protein degradation were the most represented functional categories among downregulated genes. Gene expression changes were also analyzed in cells sporulating in the presence of subinhibitory concentrations of glucose or tryptophan. Data obtained revealed overexpression of microtubule and cytoskeleton transcripts in the presence of glucose, probably causing the shape and motility problems observed in the zoospores produced under this condition. In contrast, the presence of tryptophan during sporulation led to upregulation of genes involved in oxidative stress, proteolysis, and protein folding. These results indicate that distinct physiological pathways are involved in the inhibition of sporulation due to these two classes of nutrient sources. PMID:20038607

  11. Purification of a naturally produced, low molecular weight organic factor that reversibly blocks encystment of Blastocladiella emersonii zoospores.

    PubMed

    Gottschalk, W K; Sonneborn, D R

    1985-06-10

    The water mold Blastocladiella emersonii releases zoospore maintenance factor into the medium during zoosporogenesis. Extracellular factor mediates a reversible developmental block that maintains the motile, cell wall-less zoospore phenotype. A method for purifying the factor is reported that results in 75-120% recovery of biological activity. Analyses of purified factor by thin layer chromatography support the conclusion that factor activity resides in a single organic, low molecular weight molecular species. Other data (Gottschalk, W.K. & Sonneborn, D. R. (1985) J. Biol. Chem. 260, 6592-6599) independently support this conclusion and, in addition, support the conclusion that biological activity resides in an SH-containing cyclic ribotide. PMID:3997838

  12. Characterization and submitochondrial localization of the alpha subunit of the mitochondrial processing peptidase from the aquatic fungus Blastocladiella emersonii.

    PubMed

    Rocha, C R; Gomes, S L

    1999-07-01

    In an effort to investigate the molecular mechanisms responsible for the drastic morphological changes the mitochondria go through during the life cycle of the aquatic fungus Blastocladiella emersonii, the gene encoding the alpha subunit of the mitochondrial processing peptidase (alpha-MPP) was isolated. Nucleotide sequence analysis revealed that the predicted alpha-MPP polypeptide comprises 474 amino acids with a calculated molecular mass of 51,900 Da, presenting a characteristic mitochondrial signal sequence. Northern blot analysis indicated a single 1.4-kb transcript encoding the B. emersonii alpha-MPP, whose levels decrease during sporulation, becoming very low in the zoospore, and increase again during germination. Despite these variations in mRNA concentration, B. emersonii alpha-MPP protein levels do not change significantly during the life cycle of the fungus, as observed in Western blots. Experiments to investigate the submitochondrial localization of B. emersonii alpha-MPP and beta-MPP were also carried out, and the results indicated that both subunits are associated with the mitochondrial inner membrane, possibly as part of the bc1 complex, as described for plants. PMID:10400583

  13. Oriented growth of Blastocladiella emersonii in gradients of ionophores and inhibitors.

    PubMed Central

    Harold, R L; Harold, F M

    1980-01-01

    To investigate whether ion currents help to localize growth and development of Blastocladiella emersonii, we grew the organisms in gradients of various ionophores and inhibitors. Gradients were generated by placing into the culture fine glass fibers coated with insoluble inhibitors; in some cases, inhibitors were adsorbed onto beads of ion-exchange resin. Organisms growing in many of these gradients exhibited a striking tendency for the thalli to grow toward the fiber. This proved to be misleading; the cells grew not toward the source of the ionophore but into the unoccupied zone of inhibition adjacent to the fiber. Fibers coated with gramicidin-D induced marked effects on the growth of the rhizoids, which were greatly enlarged and grew toward and onto the fiber. None of the other inhibitors produced such effects, except for beads coated with the proton conductors tetrachlorosalicylanilide and compound 1799. The results suggest that orientation of rhizoid growth results from enhancement of proton flux across the plasma membrane. Growth of the rhizoids was also strongly oriented by gradients of inorganic phosphate and an amino acid mixture; gradients of glucose, K+, Ca2+, and glutamate were ineffective. We propose that a major physiological function of the rhizoid is to transport nutrients to the thallus. Finally, we examined the effects of a series of benzimidazole antitubulins as well as the cytochalasins. These did not orient growth but grossly perturbed the pattern of cellular organization, producing small spherical cells with multiple stunted rhizoids. The findings are interpreted in terms of the interaction of an endogenous transcellular proton current with elements of the cytoskeleton in the determination of form. Images PMID:6160142

  14. Structural and thermodynamic studies of two centrin isoforms from Blastocladiella emersonii upon calcium binding.

    PubMed

    Camargo, Ana I; Wiggers, Helton J; Damalio, Julio C P; Araujo, Ana P U; Ribichich, Karina F; de Camargo, Paulo C

    2013-12-01

    Centrins are calcium-binding proteins associated with microtubules organizing centers. Members of two divergent subfamilies of centrins were found in the aquatic fungus Blastocladiella emersonii, contrasting with the occurrence of only one member known for the better explored terrestrial fungi. BeCen1 shows greatest identity with human centrins HsCen1, HsCen2 and green algae centrin CrCenp, while BeCen3 records largest identity with human centrin HsCen3 and yeast centrin Cdc31p. Following the discovery of this unique feature, BeCen1 and BeCen3 centrins were produced to study whether these proteins had distinct features upon calcium binding. Circular dichroism showed opposite calcium binding effects on the α-helix arrangement of the secondary structure. The spectra indicated a decrease in α-helix signal for holo-BeCen1 contrasting with an increase for holo-BeCen3. In addition, only BeCen1 refolds after being de-natured. The fluorescence emission of the hydrophobic probe ANS increases for both proteins likely due to hydrophobic exposure, however, only BeCen1 presents a clear blue shift when calcium is added. ITC experiments identified four calcium binding sites for both proteins. In contrast to calcium binding to BeCen1, which is mainly endothermic, binding to BeCen3 is mainly exothermic. Light-scattering evidenced the formation of large particles in solution for BeCen1 and BeCen3 at temperatures above 30°C and 40°C, respectively. Atomic force microscopy confirmed the presence of supramolecular structures, which differ in the compactness and branching degree. Binding of calcium leads to different structural changes in BeCen1 and BeCen3 and the thermodynamic characteristics of the interaction also differ. PMID:24157662

  15. Selective transport of nutrients via the rhizoids of the water mold Blastocladiella emersonii.

    PubMed

    Kropf, D L; Harold, F M

    1982-07-01

    Previous work in this laboratory demonstrated that the rhizoids of Blastocladiella emersonii grow chemotropically toward a source of Pi and thus provided preliminary evidence that, in addition to serving as a holdfast, the rhizoids absorb nutrients. To further examine the role of the rhizoids in nutrient uptake, we devised a technique to introduce a barrier between the rhizoids and the thallus to that these cell compartments could be studied independently. Cells were grown on polycarbonate membrane filters in such a way that all of the thalli were on one side of the filter and essentially all of the rhizoids were on the opposite side. Nutrient uptake into the rhizoids and the thallus was measured by floating the filters bearing cells on radioactive medium so that only one side of the filter contacted the label. Mineral oil was used to block the diffusion of the label through the unfilled pores in the filter. This technique permitted us to establish clearly that the rhizoids absorb all seven of the nutrients tested. In addition, we found that some nutrients, specifically Pi and amino acids, appeared to be preferentially taken up via the rhizoids, whereas K+, Rb+, and Ca2+ entered the thallus and rhizoids equally. Cells grown in the presence of the microtubule synthesis inhibitors nocodazole and carbendazim elaborated only a stunted rhizoid system, so we examined their ability to accumulate the two classes of compounds. As expected, these cells were severely inhibited in Pi and amino acid uptake but retained normal uptake of K+, Rb+, and Ca2+. PMID:7085568

  16. Hexosamine and cell wall biogenesis in the aquatic fungus Blastocladiella emersonii.

    PubMed

    Maia, J C

    1994-08-01

    Chitin, a beta-(1-->4) polymer of N-acetyl-glucosamine, is an important constituent of fungal cell walls. This polymer is synthesized by the incorporation of N-acetyl-D-glucosamine units from the precursor UDP-N-acetyl-D-glucosamine (UDP-GlcNAc) in a reaction catalyzed by chitin synthase. In the aquatic fungus Blastocladiella emersonii, chitin, the major component of the cell wall, is synthesized and incorporated in the cell surface of the free-swimming zoospore during the abrupt transition from this wall-less cell to the sessile, wall-containing cyst. Studies with cycloheximide indicate that chitin synthesis occurs in the apparent absence of protein synthesis, and thus posttranslational controls presumably regulate the cell wall biogenesis during encystment. Glutamine: fructose 6-phosphate amidotransferase, first enzyme of the hexosamine biosynthetic pathway, was found to play a central role in the regulation of chitin synthesis in this fungus. This enzyme exists in two forms, which are interconvertible by phosphorylation or dephosphorylation of serine residues. It is allosterically inhibited in the phosphorylated form, as it is in the zoospore, by UDP-GlcNAc. In addition, UDP-GlcNAc inhibits the dephosphorylation of amidotransferase catalyzed by protein phosphatases 2A and 2C. Thus, UDP-GlcNAc plays a dual role in hexosamine and chitin synthesis in zoospore: it not only inhibits the phosphorylated form of the enzyme but also prevents its dephosphorylation. The available data suggest that substrate availability plays a role in the control of chitin synthesis during zoospore differentiation. PMID:8070634

  17. Circulation of potassium across the plasma membrane of Blastocladiella emersonii: K+ channel.

    PubMed

    Van Brunt, J; Caldwell, J H; Harold, F M

    1982-06-01

    A previous paper reported that the water mold Blastocladiella emersonii generates a transcellular electrical current, such that positive charges enter the rhizoid and leave from the thallus (Stump et al., Proc. Natl. Acad. Sci. U.S.A. 77: 6673-6677, 1980). To begin to understand the genesis of this current we investigated ionic relationships in this organism by use of intracellular microelectrodes. In cells suspended in buffered CaCl2, the membrane potential could be accounted for as a K+ diffusion potential; no evidence for an electrogenic pump was obtained. Potassium ions diffuse outward by a pathway that also carries Rb+ and Ba2+, but excludes both smaller and larger ions (Li+, Na+, Cs+, Mg2+, Ca2+, and choline). Chloride and other anions make little contribution to the potential, but the presence of Ca2+ in the external medium is required for successful potential measurements. In growing cells, the internal K+ concentration is generally somewhat higher than would be expected if the K+ distribution were determined entirely by the membrane potential. Under certain conditions, net uptake of K+ against the electrochemical potential gradient was observed. We suggest that K+ is actively accumulated by a primary transport system that may exchange K+ for H+, and that K+ leaks passively outward through the K+ channel. The K+ circulation across the membrane amounts to about 2% of the K+ pool per min, or 4.5 microA/cm2 of surface area. We propose that this K+ circulation is one arm of the transcellular current, carrying positive charge out of the thallus. PMID:6281245

  18. Autophosphorylation and rapid dephosphorylation of the cAMP-dependent protein kinase from Blastocladiella emersonii zoospores.

    PubMed

    Gomes, S L; Juliani, M H; da Costa Maia, J C; Rangel-Aldao, R

    1983-06-10

    The photoaffinity label 8-azido[32P]adenosine 3':5'-monophosphate and affinity chromatography on N6-(2-aminoethyl)-cAMP-Sepharose were used to analyze the cAMP-binding proteins present in cell-free extracts of Blastocladiella emersonii zoospores. In the presence of a mixture of protease inhibitors, 8-azido[32P]cAMP was specifically and quantitatively incorporated into a major protein band of Mr = 58,000, and three minor protein bands of Mr = 50,000, Mr = 43,000, and Mr = 36,000 respectively, after autoradiography following sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. In the absence of the protease inhibitors, the Mr = 58,000 protein band was converted into the lower molecular weight cAMP-binding proteins, indicating a high sensitivity of the intact Mr = 58,000 protein band to endogenous proteases. The Mr = 58,000 protein corresponded to the regulatory subunit (R), of the cAMP-dependent protein kinase of zoospores, as shown by their identical behavior on DEAE-cellulose chromatography. The partially purified protein kinase incorporated 32P from [gamma-32P] ATP . Mg2+ into R as demonstrated by the specific adsorption of the 32P-labeled protein with N6-(2-aminoethyl)-cAMP-Sepharose. The incorporated 32P group was rapidly removed by endogenous phosphoprotein phosphatases in the presence of cAMP, as shown by pulse-chase experiments with [gamma-32P]ATP. Dephosphorylation of R-cAMP and rapid proteolysis may indicate two other mechanisms, in addition to cAMP, for the control of this protein kinase in vivo. PMID:6304069

  19. Oriented growth of Blastocladiella emersonii in gradients of ionophores and inhibitors.

    PubMed

    Harold, R L; Harold, F M

    1980-12-01

    To investigate whether ion currents help to localize growth and development of Blastocladiella emersonii, we grew the organisms in gradients of various ionophores and inhibitors. Gradients were generated by placing into the culture fine glass fibers coated with insoluble inhibitors; in some cases, inhibitors were adsorbed onto beads of ion-exchange resin. Organisms growing in many of these gradients exhibited a striking tendency for the thalli to grow toward the fiber. This proved to be misleading; the cells grew not toward the source of the ionophore but into the unoccupied zone of inhibition adjacent to the fiber. Fibers coated with gramicidin-D induced marked effects on the growth of the rhizoids, which were greatly enlarged and grew toward and onto the fiber. None of the other inhibitors produced such effects, except for beads coated with the proton conductors tetrachlorosalicylanilide and compound 1799. The results suggest that orientation of rhizoid growth results from enhancement of proton flux across the plasma membrane. Growth of the rhizoids was also strongly oriented by gradients of inorganic phosphate and an amino acid mixture; gradients of glucose, K+, Ca2+, and glutamate were ineffective. We propose that a major physiological function of the rhizoid is to transport nutrients to the thallus. Finally, we examined the effects of a series of benzimidazole antitubulins as well as the cytochalasins. These did not orient growth but grossly perturbed the pattern of cellular organization, producing small spherical cells with multiple stunted rhizoids. The findings are interpreted in terms of the interaction of an endogenous transcellular proton current with elements of the cytoskeleton in the determination of form. PMID:6160142

  20. Potassium- and calcium-induced alterations in lipid interactions of isolated plasma membranes from blastocladiella emersonii. Evidence for an adenosine 5'-triphosphate requirement.

    PubMed

    Leonards, K S; Haug, A

    1981-03-31

    The physical-chemical properties of the isolated plasma membranes from zoospores of the chytridiomycete Blastocladiella emersonii were investigated, with electron spin resonance (ESR) spectroscopy, using the spin-label 5-nitroxystearate (5-NS). Both isolated plasma membranes and aqueous dispersion of the lipids extracted from the plasma membranes were spin-labeled and analyzed. Plots of the hyperfine splitting parameter (2T) vs. temperature indicated that the middle break point, TM, initially observed in experiments with spin-labeled zoospores in vivo [Leonards, K. S., & Haug, A. (1980) Biochim. Biophys. Acta 600, 805-816], was the result of a lipid-lipid interaction (glycolipid-glycolipid or glycolipid-neutral lipid) rather than a lipid-protein interaction. This interaction was markedly affected by Ca2+ ions, which interacted directly with the lipid components, increasing TM from 11 +/- 1 (Ca2+ removed by EDTA) to 21 +/- 1 degree C (10 mM Ca2+) in the lipid dispersions and from 12 +/- 1 to 23 +/- 1 degree C in the plasma membrane preparations. The initial ESR studies on spin-labeled zoospores in vivo had also demonstrated that the addition of K+ ions could reverse the Ca2+ ion effect, downshifting TM from 22 +/- 1 to 10 +/- 1 degree C. The addition of of K+ ions to the isolated plasma membrane had no affect on TM, indicating that K+ ions do not simply replace Ca2+ ions but exert their effect indirectly on the membrane. However, after the inclusion of ATP, K+ ions could reverse the Ca2+ ion effect. it was determined that the ATp generated an "energized membrane" state which permitted the K+ ions to reverse the Ca2+ effect. Since K+ ions have been shown to depolarize the membrane potential in both zoospores and isolated zoospore plasma membrane preparations (generated by ATP), were suggest that the K+ ion induced reversal of the Ca2+ ion effect, and therefore the change in the lipid-lipid interactions responsible for TM, is a consequence of the K+ ion induced

  1. Characterization of the major proteins in gamma particles, cytoplasmic organelles in Blastocladiella emersonii zoospores.

    PubMed

    Hohn, T M; Lovett, J S; Bracker, C E

    1984-04-01

    The gamma particles of Blastocladiella emersonii are 0.5-micron (diameter), electron dense, membrane-enclosed organelles in the cytoplasm of zoospores that have been reported (E.C. Cantino and G.L. Mills, in P. Lemke, ed., Viruses and Plasmids in Fungi, 1979, and R.B. Myers and E.C. Cantino, in A. Wolsky (ed.), Monographs in Developmental Biology, 1974) to store the enzyme chitin synthetase. These particles were isolated from zoospores, and the two major proteins were purified for an analysis of their composition and function. The lower-molecular-weight protein (apparent molecular weight, 41,000) was insoluble in aqueous buffers, had an unusual, very basic amino acid composition, and comprised the characteristic electron-dense inclusions seen in micrographs of sections of fixed and stained gamma particles. After dispersal of the gamma particle membranes with detergent, the higher-molecular-weight protein (apparent molecular weight, 43,000) and a third minor protein (apparent molecular weight, 45,000) sedimented through sucrose cushions with the 41 kilodalton inclusion body protein but were dissociated from it by sonication in buffer containing 7 M urea. Together, the two major proteins represent 60 to 70% of the total protein in the gamma particle and 2.9% of the total protein in zoospores. Tests with specific antisera showed that the two major proteins were not antigenically related, a result consistent with the differences in amino acid composition. When zoospore lysates were centrifuged in sucrose density gradients, the major gamma particle proteins and chitin synthetase activity migrated to regions of different density. Proteins from sporulating thalli and germinating zoospores were separated by gel electrophoresis, and the two major gamma particle proteins were detected by reaction with specific antisera after electrophoretic transfer to nitrocellulose filters. Neither protein could be found in growth phase cells; the appearance and disappearances of both

  2. Developmental changes in translatable RNA species and protein synthesis during sporulation in the aquatic fungus Blastocladiella emersonii.

    PubMed

    da Silva, A M; da Costa Maia, J C; Juliani, M H

    1986-06-01

    Protein synthesis during sporulation in Blastocladiella emersonii is developmentally regulated as revealed using [35S]methionine pulse labeling and two-dimensional gel electrophoresis. A large increase in the synthesis of several proteins is associated with particular stages. A large number of basic proteins are synthesized exclusively during late sporulation. Changes in translatable mRNA species were also detected by two-dimensional gel electrophoresis of the polypeptides produced in a cell-free rabbit reticulocyte lysate primed with RNA prepared at different stages of sporulation. The synthesis of several proteins during sporulation seems to be transcriptionally controlled. Most of the sporulation-specific messages are not present in the mature zoospores. PMID:3719699

  3. The rhodopsin-guanylyl cyclase of the aquatic fungus Blastocladiella emersonii enables fast optical control of cGMP signaling.

    PubMed

    Scheib, Ulrike; Stehfest, Katja; Gee, Christine E; Körschen, Heinz G; Fudim, Roman; Oertner, Thomas G; Hegemann, Peter

    2015-08-11

    Blastocladiomycota fungi form motile zoospores that are guided by sensory photoreceptors to areas of optimal light conditions. We showed that the microbial rhodopsin of Blastocladiella emersonii is a rhodopsin-guanylyl cyclase (RhGC), a member of a previously uncharacterized rhodopsin class of light-activated enzymes that generate the second messenger cyclic guanosine monophosphate (cGMP). Upon application of a short light flash, recombinant RhGC converted within 8 ms into a signaling state with blue-shifted absorption from which the dark state recovered within 100 ms. When expressed in Xenopus oocytes, Chinese hamster ovary cells, or mammalian neurons, RhGC generated cGMP in response to green light in a light dose-dependent manner on a subsecond time scale. Thus, we propose RhGC as a versatile tool for the optogenetic analysis of cGMP-dependent signaling processes in cell biology and the neurosciences. PMID:26268609

  4. Isolation, characterization, and expression of the gene encoding the beta subunit of the mitochondrial processing peptidase from Blastocladiella emersonii.

    PubMed

    Costa Rocha, C R; Lopes Gomes, S

    1998-08-01

    A 2.3-kb BamHI-KpnI fragment was isolated from a partial genomic library and shown by nucleotide sequence analysis to contain the entire coding region of the gene encoding the beta subunit of the Blastocladiella mitochondrial processing peptidase (beta-MPP). The predicted beta-MPP protein has 465 amino acids and a calculated molecular mass of 50.8 kDa. S1 nuclease protection assays revealed an intron, 209 bp in size, interrupting the coding region between the putative signal sequence and the mature protein. Northern blot analysis showed that beta-MPP mRNA levels decrease significantly during B. emersonii sporulation, reaching basal levels in the zoospore stage. The amount of beta-MPP protein, determined in Western blots, unlike its mRNA, does not vary significantly throughout the fungal life cycle. PMID:9683495

  5. A P-type ATPase from the aquatic fungus Blastocladiella emersonii similar to animal Na,K-ATPases.

    PubMed

    de Souza, F S; Gomes, S L

    1998-04-01

    We have cloned a P-type ATPase gene from the aquatic fungus Blastocladiella emersonii (BePAT1) using a probe obtained with degenerate oligonucleotides, corresponding to two amino acid sequences highly conserved among all P-type ATPase isoforms, and the polymerase chain reaction technique. Nucleotide sequence analysis revealed a 3.4 kb open reading frame encoding a putative peptide of 1080 amino acid residues with a calculated molecular mass of 119 kDa, which presents all diagnostic features of P-type transporting ATPases. Comparison to other members of the family and phylogenetic analyses have shown that the BePAT1 protein belongs to the subfamily of Na,K- and H,K-ATPases, indicating that the divergence between the alpha-subunit of the Na,K-ATPase and other members of the P-type ATPase family has occurred before the divergence between the animal and fungal lineages in evolution. PMID:9602120

  6. Evidence of a Ca(2+)-(*)NO-cGMP signaling pathway controlling zoospore biogenesis in the aquatic fungus Blastocladiella emersonii.

    PubMed

    Vieira, André L G; Linares, Edlaine; Augusto, Ohara; Gomes, Suely L

    2009-08-01

    The sporulation stage of the aquatic fungus Blastocladiella emersonii culminates with the formation and release to the medium of a number of zoospores, which are motile cells responsible for the dispersal of the fungus. The presence in the sporulation solution of 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a potent and selective inhibitor of nitric oxide-sensitive guanylyl cyclases, completely prevented biogenesis of the zoospores. In addition, this compound was able to significantly reduce cGMP levels, which increase drastically during late sporulation, suggesting the existence of a nitric oxide-dependent mechanism for cGMP synthesis. Furthermore, increased levels of nitric oxide-derived products were detected during sporulation by fluorescence assays using DAF-2 DA, whose signal was drastically reduced in the presence of the nitric oxide synthase inhibitor Nomega-Nitro-L-arginine methyl ester (L-NAME). These results were confirmed by quantitative chemiluminescent determination of the intracellular levels of nitric oxide-derived products. A putative nitric oxide synthase (NOS) activity was detected throughout sporulation, and this enzyme activity decreased significantly when L-NAME and 1-[2-(Trifluoromethyl)phenyl]imidazole (TRIM) were added to the assays. NOS assays carried out in the presence of EGTA showed decreased enzyme activity, suggesting the involvement of calcium ions in enzyme activation. Additionally, expressed sequence tags (ESTs) encoding putative guanylyl cyclases and a cGMP-phosphodiesterase were found in B. emersonii EST database (http://blasto.iq.usp.br), and the mRNA levels of the corresponding genes were observed to increase during sporulation. Altogether, data presented here revealed the presence and expression of guanylyl cyclase and cGMP phosphodiesterase genes in B. emersonii and provided evidence of a Ca(2+)-(*)NO-cGMP signaling pathway playing a role in zoospore biogenesis. PMID:19393757

  7. Fructose 2,6-bisphosphate and carbohydrate metabolism during the life cycle of the aquatic fungus Blastocladiella emersonii.

    PubMed

    Vandercammen, A; François, J M; Torres, B B; Maia, J C; Hers, H G

    1990-01-01

    Removal of the growth medium and resuspension of Blastocladiella emersonii vegetative cells in a sporulation medium resulted in an abrupt fall of fructose 2,6-bisphosphate concentration to about 2% of its initial value within 10 min. The concentrations of hexose 6-phosphate and of fructose 1,6-bisphosphate also decreased by, respectively, three and tenfold over the same period. All these values remained at their low level throughout the sporulation phase and during the subsequent germination of zoospores when performed in the absence of glucose. In contrast, the concentration of cyclic AMP was low during the sporulation period and exhibited a transient increase a few minutes after the initiation of germination. Other biochemical events occurring during sporulation were a 70% reduction in glycogen content and the complete disappearance of trehalose. The remaining glycogen was degraded upon subsequent germination of the zoospores. B. emersonii phosphofructo 2-kinase (PFK-2) and fructose-2,6-bisphosphatase (FBPase-2) could not be separated from each other by various chromatographic procedures, suggesting that they were part of a single bifunctional protein. On anion-exchange chromatography, two peaks of PFK-2 and FBPase-2 were resolved. Upon incubation of fractions from the two peaks or of a crude extract in the presence of [2-32P]fructose 2,6-bisphosphate, two radiolabelled subunits with molecular masses close to 90 and 54 kDa were obtained. The labelling of the subunit of higher molecular mass was greater than that of the lower one in extracts prepared in the presence of protease inhibitors and in the first peak of the Mono Q column. PFK-2 and FBPase-2 displayed kinetic properties comparable to those of mammalian enzymes, but no indication of a cyclic AMP-dependent regulation could be obtained. Phosphofructo 1-kinase and fructose-1,6-bisphosphatase from B. emersonii were, respectively, stimulated and inhibited by micromolar concentrations of fructose 2

  8. Stage-specific synthesis of proteins complexed to ribonucleoprotein particles and ribosomes in zoospores of Blastocladiella emersonii.

    PubMed

    Jaworski, A J; Stumhofer, P

    1981-04-01

    In Blastocladiella emersonii zoospores, a set of proteins was found associated with the ribosomes and free ribonucleoprotein particles distinct from the ribosomes and polyribosomes. These proteins were designated P120, P105, P64, P56, and P42 based on their molecular weights determined by gel electrophoresis. Synthesis of these proteins was detected only during late sporulation just before the time polyadenylated ribonucleic acid accumulates in the sporangia. These proteins banded in isopycnic metrizamide gradients at densities of 1.31 and 1.27 g/cm3, which corresponded to the densities of the ribosomes and free ribonucleoprotein particles, respectively. Comparison of the distribution of the proteins in sucrose versus metrizamide gradients suggested that P105 was removed from the free ribonucleoprotein particles before complexing with the ribosomes. During germination, these proteins disappeared from the ribosomal fractions, with kinetics corresponding to the resumption of protein synthesis. Another protein (P178) was observed to bind to the ribosomes before the onset of protein synthesis during germination. Cycloheximide did not block the addition of this protein to the monoribosomes. PMID:6086010

  9. Developmental regulation of expression of the regulatory subunit of the cAMP-dependent protein kinase of Blastocladiella emersonii.

    PubMed

    Marques, M do V; Juliani, M H; Maia, J C; Gomes, S L

    1989-01-01

    A monospecific polyclonal antiserum to the regulatory subunit (R) of the cAMP-dependent protein kinase of Blastocladiella emersonii has been developed by immunization with purified regulatory subunit. In Western blots, the antiserum displays high affinity and specificity for the intact R monomer of Mr = 58,000, as well as for its proteolytic products of Mr = 43,000 and Mr = 36,000, even though the antiserum has been raised against the Mr = 43,000 fragment. Western blots of cell extracts prepared at different times during the life cycle of the fungus indicate that the increase in cAMP-binding activity occurring during sporulation, as well as its decrease during germination, are associated with the accumulation of the regulatory subunit during sporulation and its disappearance during germination, respectively. Pulse labeling with [35S]methionine and immunoprecipitation indicate that the accumulation of R is due to its increased synthesis during sporulation. Two-dimensional gel electrophoresis of affinity purified cell extracts obtained after [35S]methionine pulse labeling during sporulation confirms de novo synthesis of R during this stage and furthermore shows that the protein is rapidly phosphorylated after its synthesis. In vitro translation studies using RNA isolated from different stages of the life cycle followed by immunoprecipitation have shown that the time course of expression of the mRNA coding for the regulatory subunit parallels the rate of its synthesis in vivo. PMID:2912735

  10. Differential expression of heat-shock proteins and spontaneous synthesis of HSP70 during the life cycle of Blastocladiella emersonii.

    PubMed

    Bonato, M C; Silva, A M; Gomes, S L; Maia, J C; Juliani, M H

    1987-02-16

    The heat-shock response in Blastocladiella emersonii is dependent on the developmental stage. Cells exposed to elevated temperatures at different stages of the life cycle (sporulation, germination or growth) show a differential synthesis of heat-shock proteins (hsps). Of a total of 22 polypeptides induced, particular subsets of hsps appear in each phase, demonstrating a non-coordinate heat-shock gene expression. In contrast, heat-shock-related proteins (hsp76, hsp70, hsp39a) are spontaneously expressed at a high level during sporulation. By the criteria of two-dimensional gel electrophoresis and partial proteolysis mapping, the 70,000-Da protein, whose synthesis is induced spontaneously during sporulation, is indistinguishable from the heat-inducible hsp70. The techniques of in vitro translation, and Northern analysis using a Drosophila hsp70 probe, demonstrated that enhanced synthesis of hsp70, which occurs during heat-shock treatment and spontaneously during sporulation, is associated with an accumulation of hsp70 mRNA. These observations suggest that hsp70 gene expression is induced during sporulation. PMID:3816799

  11. Cyclic nucleotide-independent protein kinases from ribosomes and phosphorylation of a single 40S ribosomal subunit protein in zoospores of Blastocladiella emersonii.

    PubMed

    Bonato, M C; da Costa Maia, J C; Juliani, M H

    1983-06-01

    Cyclic nucleotide-independent protein kinase (EC 2.7.1.37) activity was found in the nuclear cap organelle, within which ribosomes of zoospores of Blastocladiella emersonii are sequestered. Two protein kinase activities were resolved from the high-salt wash fraction of zoospore ribosomes by selective adsorption to DEAE-cellulose. Both enzymes phosphorylated in vitro a 32,000 Mr protein of the 40S ribosomal subunit. Phosphorylation of this ribosomal protein, which exhibits electrophoretic properties similar to those of mammalian ribosomal protein S6, was also observed in vivo in 32P-labeled zoospores. PMID:6853450

  12. PEST sequences in cAMP-dependent protein kinase subunits of the aquatic fungus Blastocladiella emersonii are necessary for in vitro degradation by endogenous proteases.

    PubMed

    Borges, A C; Gomes, S L

    2000-05-01

    During Blastocladiella emersonii germination, the regulatory (R) and the catalytic (C) subunits of the cAMP-dependent protein kinase (PKA) are rapidly and concurrently degraded, after PKA activation in response to a transient increase in intracellular cAMP levels. The possibility that PEST sequences could be acting as proteolytic recognition signals in this process was investigated, and high score PEST sequences were found in both B. emersonii R and C subunits. Deletions in the PEST sequences were obtained by site-directed mutagenesis and the different PKA subunits were independently expressed in Escherichia coli. Proteolysis assays of the various R and C recombinant forms, using B. emersonii cell extracts as the source of proteases, showed a strong correlation between the presence of high score PEST sequences and susceptibility to degradation. Furthermore, the amino-terminal sequence of the proteolytic fragments indicated that the cleavage sites in both subunits are located at or near the PEST regions. The PEST sequence in B. emersonii C subunit, which when deleted or disrupted leads to resistance to proteolysis, is entirely contained in the 72-amino-acid extension located in the N-terminus of the protein. C subunit mutants carrying deletions in this region displayed little difference in their kinetic properties or enzyme thermostability. These results suggest that the N-terminal extension may only play a role in C subunit degradation. PMID:10844679

  13. Cloning and structural analysis of the gene for the regulatory subunit of cAMP-dependent protein kinase in Blastocladiella emersonii.

    PubMed

    Marques, M do V; Gomes, S L

    1992-08-25

    We have isolated and characterized cDNA and genomic DNA clones encoding the regulatory subunit of cAMP-dependent protein kinase in the aquatic fungus Blastocladiella emersonii. Nucleotide sequence analysis has shown that the predicted protein comprises 403 amino acids with a calculated molecular mass of 44,263 Da and an overall 40% identity to mammalian RII subunits, including a serine in the phosphorylation site, which confirms the Blastocladiella protein as a type II regulatory subunit. The B. emersonii R gene presents two introns, one located in the 5'-noncoding region, whereas the other interrupts the coding region, just after the dimerization domain of the protein. The promoter region does not contain recognizable TATA or CCAAT sequences and is very GC rich, a characteristic shared by mammalian cAMP-dependent protein kinase subunit genes previously analyzed. S1 mapping and primer extension experiments revealed multiple transcription initiation sites. Several sequence motifs were identified in the 5'-flanking region which could be responsible for the regulation of this gene. PMID:1512258

  14. Cloning and characterization of the gene for the catalytic subunit of cAMP-dependent protein kinase in the aquatic fungus Blastocladiella emersonii.

    PubMed

    de Oliveira, J C; Borges, A C; Marques, M do V; Gomes, S L

    1994-01-15

    We have isolated and characterized cDNA and genomic DNA clones encoding the catalytic subunit (C) of cAMP-dependent protein kinase in the aquatic fungus Blastocladiella emersonii. The C-subunit amino acid sequence derived from the nucleotide sequence predicts a basic polypeptide of 424 residues, excluding the initiator methionine, which by amino-terminal sequence analysis has been shown to be absent from the mature protein. The Blastocladiella C presents a 70-amino-acid extension at the amino terminus, when aligned to the mouse C alpha subunit, being one of the largest C subunits already characterized. The B. emersonii C-gene-coding region is interrupted by three introns, ranging in size over 57-69 bp. The positions of the introns are quite different from those found in other species, suggesting a considerable amount of evolutionary drift in the gene structure. The 5'-flanking region lacks recognizable TATA or CCAAT sequences, is remarkably high in GC content (70%), and primer extension experiments indicate that transcription initiates from multiple sites. Several sequence motifs were identified in the promoter region which could be involved in the developmental control of this gene. PMID:8307021

  15. Protein factors in Blastocladiella emersonii cell extracts recognize similar sequence elements in the promoters of the genes encoding cAMP-dependent protein kinase subunits.

    PubMed

    de Oliveira, J C; Marques, M V; Gomes, S L

    1997-08-01

    Blastocladiella emersonii contains a single cAMP-dependent protein kinase (PKA), which is similar to the mammalian type II isoforms. Its activity is regulated during development by changes in the levels of the catalytic (C) and regulatory (R) subunits, which occur in parallel with changes in levels of the corresponding mRNAs, suggesting coordinate transcriptional control of the expression of both subunits. Both R and C mRNA levels are low in vegetative cells, rise sharply during sporulation and decrease to basal levels again after germination. To investigate sequence elements common to both Blastocladiella R and C gene promoters, which might be involved in the coordinate regulation of these genes, their 5'-flanking regions were analyzed by gel mobility shift and DNase I footprinting assays. We determined that different DNA-protein complexes are generated when fragments of the R and C gene promoters are incubated with extracts from cells expressing (sporulating cells) or not expressing (vegetative cells) both subunits, and competition experiments suggested that similar protein factors bind to both promoters. DNase I footprinting experiments have indicated that a sequence common to both R and C promoters, and similar to mammalian E-boxes, binds factors present in extracts from vegetative and sporulating cells, whereas sequences flanking the E-boxes in both promoters showed a change in the pattern of DNase I digestion only when the vegetative cell extract was used. This result suggests that the composition of the protein complexes binding to these regions changes during sporulation. PMID:9294034

  16. A unique intron-containing hsp70 gene induced by heat shock and during sporulation in the aquatic fungus Blastocladiella emersonii.

    PubMed

    Stefani, R M; Gomes, S L

    1995-01-11

    We have isolated and characterized cDNA and genomic DNA clones encoding the 70-kDa heat-shock protein (Hsp70) from the aquatic fungus Blastocladiella emersonii (Be). Nucleotide (nt) sequence analysis predicts an acidic protein containing 650 amino acids, with a calculated molecular mass of 70.8 kDa. The Be hsp70 gene is induced by heat shock (HS), as well as during sporulation of the fungus, and its coding region is interrupted by a single intron. All the evidence seems to indicate that this is the only hsp70 in Be. S1 nuclease protection assays revealed that splicing of the hsp70 intron is highly thermoresistant; at the lethal temperature of 42 degrees C, only 30% of the hsp70 mRNAs have not been processed. A single transcription start point (tsp), localized about 30 nt downstream from a putative TATA box, was determined both during HS and at normal temperatures. The promoter region presented several NGAAN repeats (where N is any nucleotide) characteristic of HS elements, as well as putative binding sites for ATF, Sp1 and two metal-responsive elements. PMID:7828923

  17. Transcriptional Response to Hypoxia in the Aquatic Fungus Blastocladiella emersonii▿†

    PubMed Central

    Camilo, César M.; Gomes, Suely L.

    2010-01-01

    Global gene expression analysis was carried out with Blastocladiella emersonii cells subjected to oxygen deprivation (hypoxia) using cDNA microarrays. In experiments of gradual hypoxia (gradual decrease in dissolved oxygen) and direct hypoxia (direct decrease in dissolved oxygen), about 650 differentially expressed genes were observed. A total of 534 genes were affected directly or indirectly by oxygen availability, as they showed recovery to normal expression levels or a tendency to recover when cells were reoxygenated. In addition to modulating many genes with no putative assigned function, B. emersonii cells respond to hypoxia by readjusting the expression levels of genes responsible for energy production and consumption. At least transcriptionally, this fungus seems to favor anaerobic metabolism through the upregulation of genes encoding glycolytic enzymes and lactate dehydrogenase and the downregulation of most genes coding for tricarboxylic acid (TCA) cycle enzymes. Furthermore, genes involved in energy-costly processes, like protein synthesis, amino acid biosynthesis, protein folding, and transport, had their expression profiles predominantly downregulated during oxygen deprivation, indicating an energy-saving effort. Data also revealed similarities between the transcriptional profiles of cells under hypoxia and under iron(II) deprivation, suggesting that Fe2+ ion could have a role in oxygen sensing and/or response to hypoxia in B. emersonii. Additionally, treatment of fungal cells prior to hypoxia with the antibiotic geldanamycin, which negatively affects the stability of mammalian hypoxia transcription factor HIF-1α, caused a significant decrease in the levels of certain upregulated hypoxic genes. PMID:20418381

  18. Decreased activity of Blastocladiella emersonii zoospore ribosomes: correlation with developmental changes in ribosome-associated proteins.

    PubMed

    Jaworski, A J; Wilson, J B

    1989-10-01

    Ribosomal proteins isolated from dormant zoospores were compared to the ribosomal proteins found in the active growth phase by two-dimensional polyacrylamide gel electrophoresis. Zoospore ribosomes were found to contain a set of five proteins, designated Z1 to Z5, which were not present in growth phase ribosomes. The Z1-Z5 proteins were not removed by high-salt washes using either 1 M KCl or 1 M NH4 Cl. The Z1 protein is found associated with zoospore 60 S subunits while Z2-Z5 are bound to 40 S subunits. Zoospore monoribosomes and polyribosomes contain comparable levels of each of the five proteins. Approximately 60 min. after sporulation is induced, the Z1-Z5 proteins begin to accumulate on the ribosomes with the highest levels of these proteins found associated with ribosomes at the zoospore stage. During germination, the proteins gradually disappear and are not detectable on the ribosomes after 4 hr of germination. The presence of the Z1-Z5 proteins correlates with a decrease in in vitro protein synthetic activity of the fungal ribosomes. The data are consistent with the hypothesis that the proteins regulate translation by completely blocking protein synthesis on a subset of ribosomes while the remainder of the ribosomes function at normal rates. PMID:2776972

  19. NUTRIENT COMPOSITION DEGRADATION OF DAPHNIA PULICARIA BY A HIGHLY PREVALENT CHYTRIDIOMYCETE FUNGAL PATHOGEN (POLYCARYUM LEAVE) DURING NATURALLY OCCURRING LAKE-WIDE EPIDEMICS.

    EPA Science Inventory

    Despite evidence illustrating that chytridiomycete fungal infection can be highly prevalent in Daphnia (>80%) and that infected individuals are preferentially consumed by fish, no studies have measured the nutritional consequences of using chytrid-infected Daphnia as a food sourc...

  20. Gromochytrium mamkaevae gen. & sp. nov. and two new orders: Gromochytriales and Mesochytriales (Chytridiomycetes).

    PubMed

    Karpov, S A; Kobseva, A A; Mamkaeva, M A; Mamkaeva, K A; Mikhailov, K V; Mirzaeva, G S; Aleoshin, V V

    2014-06-01

    During the last decade several new orders were established in the class Chytridiomycetes on the basis of zoospore ultrastructure and molecular phylogeny. Here we present the ultrastructure and molecular phylogeny of strain x-51 CALU - a parasite of the alga Tribonema gayanum, originally described as Rhizophydium sp. based on light microscopy. Detailed investigation revealed that the zoospore ultrastructure of this strain has unique characters not found in any order of Chytridiomycetes: posterior ribosomal core unbounded by the endoplasmic reticulum and detached from the nucleus or microbody-lipid complex, and kinetosome composed of microtubular doublets. An isolated phylogenetic position of x-51 is further confirmed by the analysis of 18S and 28S rRNA sequences, and motivates the description of a new genus and species Gromochytrium mamkaevae. The sister position of G. mamkaevae branch relative to Mesochytrium and a cluster of environmental sequences, as well as the ultrastructural differences between Gromochytrium and Mesochytrium zoospores prompted us to establish two new orders: Gromochytriales and Mesochytriales. PMID:25264386

  1. Festering food: chytridiomycete pathogen reduces quality of Daphnia host as a food resource.

    PubMed

    Forshay, Kenneth J; Johnson, Pieter T J; Stock, Melanie; Peñalva, Carolina; Dodson, Stanley I

    2008-10-01

    When parasitic infections are severe or highly prevalent among prey, a significant component of the predator's diet may consist of parasitized hosts. However, despite the ubiquity of parasites in most food webs, comparisons of the nutritional quality of prey as a function of infection status are largely absent. We measured the nutritional consequences of chytridiomycete infections in Daphnia, which achieve high prevalence in lake ecosystems (>80%), and tested the hypothesis that Daphnia pulicaria infected with Polycaryum laeve are diminished in food quality relative to uninfected hosts. Compared with uninfected adults, infected individuals were smaller, contained less nitrogen and phosphorus, and were lower in several important fatty acids. Infected zooplankton had significantly shorter carapace lengths (8%) and lower mass (8-20%) than uninfected individuals. Parasitized animals contained significantly less phosphorus (16-18% less by dry mass) and nitrogen (4-6% less) than did healthy individuals. Infected individuals also contained 26-34% less saturated fatty acid and 31-42% less docosahexaenoic acid, an essential fatty acid that is typically low in cladocera, but critical to fish growth. Our results suggest that naturally occurring levels of chytrid infections in D. pulicaria populations reduce the quality of food available to secondary consumers, including planktivorous fishes, with potentially important effects for lake food webs. PMID:18959307

  2. Cloning, Overexpression in Escherichia coli, and Characterization of a Thermostable Fungal Acetylxylan Esterase from Talaromyces emersonii

    PubMed Central

    Murray, Patrick G.; Miki, Yuta; Martínez, Angel T.; Tuohy, Maria G.; Faulds, Craig B.

    2012-01-01

    The gene encoding an acetylxylan esterase (AXE1) from the thermophilic ascomycete Talaromyces emersonii was cloned, expressed in Escherichia coli, and characterized. This form of AXE1, rTeAXE1, exhibits increased thermostability and activity at a higher temperature than other known fungal acetyl esterases, thus having huge potential application in biomass bioconversion to high value chemicals or biofuels. PMID:22407679

  3. Cloning, overexpression in Escherichia coli, and characterization of a thermostable fungal acetylxylan esterase from Talaromyces emersonii.

    PubMed

    Waters, Deborah M; Murray, Patrick G; Miki, Yuta; Martínez, Angel T; Tuohy, Maria G; Faulds, Craig B

    2012-05-01

    The gene encoding an acetylxylan esterase (AXE1) from the thermophilic ascomycete Talaromyces emersonii was cloned, expressed in Escherichia coli, and characterized. This form of AXE1, rTeAXE1, exhibits increased thermostability and activity at a higher temperature than other known fungal acetyl esterases, thus having huge potential application in biomass bioconversion to high value chemicals or biofuels. PMID:22407679

  4. Cellulose hydrolysis by the cellulases produced by Talaromyces emersonii when grown on different inducing substrates

    SciTech Connect

    Moloney, A.P.; Considine, P.J.; Coughlan, M.P.

    1983-04-01

    Pulp obtained from the processing of sugar beet at a local factory is mixed with molasses and sold as cattle food. However, the value of the pulp would be increased considerably if its constituent cellulose and hemicellulose fractions could be converted to fermentable sugars. To this end we are investigating the enzymic hydrolysis of beet pulp using the cellulase system produced by the thermophilic fungus, Talaromyces emersonii. In this Communication, we report on the initial results of studies. (Refs. 21).

  5. Characterisation of a Talaromyces emersonii thermostable enzyme cocktail with applications in wheat dough rheology.

    PubMed

    Waters, Deborah M; Ryan, Liam A M; Murray, Patrick G; Arendt, Elke K; Tuohy, Maria G

    2011-07-10

    In this paper, we report new sequence data for secreted thermostable fungal enzymes from the un-sequenced xylanolytic filamentous fungus Talaromyces emersonii and reveal novel insights on the potential role of enzymes relevant as wheat dough improvers. The presence of known and de novo enzyme sequences were confirmed through NanoLC-ESI-MS/MS and resultant peptide sequences were identified using SWISS PROT databases. The de novo protein sequences were assigned identity based on homology to known fungal proteins. Other proteins were assigned function based on the limited T. emersonii genome coverage. This approach allowed the identification of enzymes with relevance as wheat dough improvers. Rheological examination of wheat dough and wheat flour components treated with the thermostable fungal enzyme cocktail revealed structural alterations that can be extrapolated to the baking process. Thermoactive amylolytic, xylanolytic, glucanolytic, proteolytic and lipolytic enzyme activities were observed. Previously characterized T. emersonii enzymes present included; β-glucosidase, xylan-1,4-β-xyloxidase, acetylxylan esterase, acid trehalase, avenacinase, cellobiohydrolase and endo-glucanase. De novo sequence analysis confirmed peptides as being; α-glucosidase, endo-1,4-β-xylanase, endo-arabinase, endo-glucanase, exo-β-1,3-glucanase, glucanase/cellulase, endopeptidase and lipase/acylhydrolase. Rheology tests using wheat dough and fractioned wheat flour components in conjunction with T. emersonii enzymes show the role of these novel biocatalysts in altering properties of wheat substrates. Enzyme treated wheat flour fractions showed the effects of particular enzymes on appropriate substrates. This proteomic approach combined with rheological characterization is the first such report to the authors' knowledge. PMID:22112414

  6. An enigmatic fossil fungus from the 410 Ma Rhynie chert that resembles Macrochytrium (Chytridiomycota) and Blastocladiella (Blastocladiomycota).

    PubMed

    Krings, Michael; Taylor, Thomas N; Martin, Helmut

    2016-01-01

    Litter layers in the Lower Devonian (~ 410 Ma) Rhynie chert were inhabited by a wide variety of saprotrophic fungi, however, only a few of these organisms have been described formally. A new microfungus, Trewinomyces annulifer gen. et sp. nov., occurs as tufts on decaying land plant axes from the Rhynie chert. The fungus consists of an intramatrical rhizoidal system and an erect extramatrical hypha (stalk) that bears a single, terminal sporangium. One or two successive rings often are present in the stalk immediately below the sporangium base. Overall morphology of T. annulifer resembles the extant genera Macrochytrium (Chytridiomycota) and Blastocladiella (Blastocladiomycota). However, the rhizoids are septate or pseudoseptate, a feature not known in extant zoosporic fungi, and thus render the systematic affinities of T. annulifer unresolved. Trewinomyces annulifer offers a rare view of the morphology of a distinctive Early Devonian saprotrophic microfungus. PMID:26740543

  7. Characterization of a multimeric, eukaryotic prolyl aminopeptidase: an inducible and highly specific intracellular peptidase from the non-pathogenic fungus Talaromyces emersonii.

    PubMed

    Mahon, Cathal S; O'Donoghue, Anthony J; Goetz, David H; Murray, Patrick G; Craik, Charles S; Tuohy, Maria G

    2009-11-01

    Fungi are capable of degrading proteins in their environment by secreting peptidases. However, the link between extracellular digestion and intracellular proteolysis has scarcely been investigated. Mycelial lysates of the filamentous fungus Talaromyces emersonii were screened for intracellular peptidase production. Five distinct proteolytic activities with specificity for the p-nitroanilide (pNA) peptides Suc-AAPF-pNA, Suc-AAA-pNA, K-pNA, F-pNA and P-pNA were identified. The native enzyme responsible for the removal of N-terminal proline residues was purified to homogeneity by ammonium sulfate fractionation followed by five successive chromatographic steps. The enzyme, termed Talaromyces emersonii prolyl aminopeptidase (TePAP), displayed a 50-fold specificity for cleaving N-terminal Pro-X (k(cat)/K(m)=2.1 x 10(6) M(-1) s(-1)) compared with Ala-X or Val-X bonds. This intracellular aminopeptidase was optimally active at pH 7.4 and 50 degrees C. Peptide sequencing facilitated the design of degenerate oligonucleotides from homologous sequences encoding putative fungal proline aminopeptidases, enabling subsequent cloning of the gene. TePAP was shown to be relatively uninhibited by classical serine peptidase inhibitors and to be sensitive to selected cysteine- and histidine-modifying reagents, yet gene sequence analysis identified the protein as a serine peptidase with an alpha/beta hydrolase fold. Northern analysis indicated that Tepap mRNA levels were regulated by the composition of the growth medium. Highest Tepap transcript levels were observed when the fungus was grown in medium containing glucose and the protein hydrolysate casitone. Interestingly, both the induction profile and substrate preference of this enzyme suggest potential co-operativity between extracellular and intracellular proteolysis in this organism. Gel filtration chromatography suggested that the enzyme exists as a 270 kDa homo-hexamer, whereas most bacterial prolyl aminopeptidases (PAPs) are

  8. Characterization of a multimeric, eukaryotic prolyl aminopeptidase: an inducible and highly specific intracellular peptidase from the non-pathogenic fungus Talaromyces emersonii

    PubMed Central

    Mahon, Cathal S.; O'Donoghue, Anthony J.; Goetz, David H.; Murray, Patrick G.; Craik, Charles S.; Tuohy, Maria G.

    2009-01-01

    Fungi are capable of degrading proteins in their environment by secreting peptidases. However, the link between extracellular digestion and intracellular proteolysis has scarcely been investigated. Mycelial lysates of the filamentous fungus Talaromyces emersonii were screened for intracellular peptidase production. Five distinct proteolytic activities with specificity for the p-nitroanilide (pNA) peptides Suc-AAPF-pNA, Suc-AAA-pNA, K-pNA, F-pNA and P-pNA were identified. The native enzyme responsible for the removal of N-terminal proline residues was purified to homogeneity by ammonium sulfate fractionation followed by five successive chromatographic steps. The enzyme, termed Talaromyces emersonii prolyl aminopeptidase (TePAP), displayed a 50-fold specificity for cleaving N-terminal Pro–X (kcat/Km=2.1×106 M−1 s−1) compared with Ala–X or Val–X bonds. This intracellular aminopeptidase was optimally active at pH 7.4 and 50 °C. Peptide sequencing facilitated the design of degenerate oligonucleotides from homologous sequences encoding putative fungal proline aminopeptidases, enabling subsequent cloning of the gene. TePAP was shown to be relatively uninhibited by classical serine peptidase inhibitors and to be sensitive to selected cysteine- and histidine-modifying reagents, yet gene sequence analysis identified the protein as a serine peptidase with an α/β hydrolase fold. Northern analysis indicated that Tepap mRNA levels were regulated by the composition of the growth medium. Highest Tepap transcript levels were observed when the fungus was grown in medium containing glucose and the protein hydrolysate casitone. Interestingly, both the induction profile and substrate preference of this enzyme suggest potential co-operativity between extracellular and intracellular proteolysis in this organism. Gel filtration chromatography suggested that the enzyme exists as a 270 kDa homo-hexamer, whereas most bacterial prolyl aminopeptidases (PAPs) are

  9. Comparison of wild-type and UV-mutant beta-glucanase-producing strains of Talaromyces emersonii with potential in brewing applications.

    PubMed

    McCarthy, Tracey C; Lalor, Eoin; Hanniffy, Orla; Savage, Angela V; Tuohy, Maria G

    2005-04-01

    A screen of 46 UV-mutant strains of the moderately thermophilic fungus Talaromyces emersonii yielded two mutants (TC2, TC5) that displayed gross morphological differences to the parent strain and enhanced activity against mixed linkage cereal beta-glucans. Activity against beta-(1, 3)(1, 4)-D: -glucan from barley (BBGase) was measured during growth of the mutant and wild-type strains on a variety of carbon sources, ranging from solka floc to crude cereal fractions. In liquid culture, TC2 and TC5 secreted 1.2- to 8.6-fold more BBGase than the parent strain and markedly less beta-glucosidase (exo-activity); enzyme levels were dependent on the carbon source. Cellulose induced high BBGase. However, beet pulp, wheat bran, carob and tea-leaves were cheap and effective inducers. T. emersonii wild-type, TC2 and TC5 crude enzyme preparations achieved similar end-points during the hydrolysis of commercial barley beta-glucan (13.0-16.9%), but were more active against crude beta-glucan from barley (16.0-24.2% hydrolysis). The products of hydrolysis were quantified by high-performance anion-exchange chromatography. Mash trials indicated that enzyme preparations from all three organisms effected a significant reduction in wort viscosity and residual mash beta-glucan. Finally, TC2 and TC5 produce more efficient beta-glucan-depolymerizing enzymes; and wheat bran and solka floc can be used to provide inexpensive and potent enzyme cocktails with potential in brewing applications. PMID:15856354

  10. Structural and functional studies of the glycoside hydrolase family 3 β-glucosidase Cel3A from the moderately thermophilic fungus Rasamsonia emersonii.

    PubMed

    Gudmundsson, Mikael; Hansson, Henrik; Karkehabadi, Saeid; Larsson, Anna; Stals, Ingeborg; Kim, Steve; Sunux, Sergio; Fujdala, Meredith; Larenas, Edmund; Kaper, Thijs; Sandgren, Mats

    2016-07-01

    The filamentous fungus Hypocrea jecorina produces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. β-Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the β-glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the β-glucosidase from H. jecorina (HjCel3A) with the β-glucosidase Cel3A from the thermophilic fungus Rasamsonia emersonii (ReCel3A) in enzyme mixtures results in increased efficiency in the saccharification of lignocellulosic materials. Biochemical characterization of ReCel3A, heterologously produced in H. jecorina, reveals a preference for disaccharide substrates over longer gluco-oligosaccharides. Crystallographic studies of ReCel3A revealed a highly N-glycosylated three-domain dimeric protein, as has been observed previously for glycoside hydrolase family 3 β-glucosidases. The increased thermal stability and saccharification yield and the superior biochemical characteristics of ReCel3A compared with HjCel3A and mixtures containing HjCel3A make ReCel3A an excellent candidate for addition to enzyme mixtures designed to operate at higher temperatures. PMID:27377383

  11. A thermophilic endo-1,4-β-glucanase from Talaromyces emersonii CBS394.64 with broad substrate specificity and great application potentials.

    PubMed

    Wang, Kun; Luo, Huiying; Bai, Yingguo; Shi, Pengjun; Huang, Huoqing; Xue, Xianli; Yao, Bin

    2014-08-01

    Thermophilic cellulases are of significant interest to the efficient conversion of plant cell wall polysaccharides into simple sugars. In this study, a thermophilic and thermostable endo-1,4-β-glucanase, TeEgl5A, was identified in the thermophilic fungus Talaromyces emersonii CBS394.64 and functionally expressed in Pichia pastoris. Purified recombinant TeEgl5A exhibits optimal activity at pH 4.5 and 90 °C. It is highly stable at 70 °C and over a broad pH range of 1.0-10.0, and shows strong resistance to most metal ions, sodium dodecyl sulfate (SDS), and proteases. TeEgl5A has broad substrate specificity and exhibits high activity on substrates containing β-1,4-glycosidic bonds and β-1,3-glycosidic bonds (barley β-glucan, laminarin, lichenan, CMC-Na, carob bean gum, and birchwood xylan). Under simulated mashing conditions, addition of 60 U TeEgl5A reduced more viscosity (10.0 vs.7.6 %) than 80 U of Ultraflo XL from Novozymes. These properties make TeEgl5A a good candidate for extensive application in the detergent, textile, feed, and food industries. PMID:24668246

  12. Structural and functional studies of the glycoside hydrolase family 3 β-glucosidase Cel3A from the moderately thermophilic fungus Rasamsonia emersonii

    PubMed Central

    Gudmundsson, Mikael; Hansson, Henrik; Karkehabadi, Saeid; Larsson, Anna; Stals, Ingeborg; Kim, Steve; Sunux, Sergio; Fujdala, Meredith; Larenas, Edmund; Kaper, Thijs; Sandgren, Mats

    2016-01-01

    The filamentous fungus Hypocrea jecorina produces a number of cellulases and hemicellulases that act in a concerted fashion on biomass and degrade it into monomeric or oligomeric sugars. β-Glucosidases are involved in the last step of the degradation of cellulosic biomass and hydrolyse the β-glycosidic linkage between two adjacent molecules in dimers and oligomers of glucose. In this study, it is shown that substituting the β-glucosidase from H. jecorina (HjCel3A) with the β-glucosidase Cel3A from the thermophilic fungus Rasamsonia emersonii (ReCel3A) in enzyme mixtures results in increased efficiency in the saccharification of lignocellulosic materials. Biochemical characterization of ReCel3A, heterologously produced in H. jecorina, reveals a preference for disaccharide substrates over longer gluco-oligosaccharides. Crystallographic studies of ReCel3A revealed a highly N-glycosylated three-domain dimeric protein, as has been observed previously for glycoside hydrolase family 3 β-glucosidases. The increased thermal stability and saccharification yield and the superior biochemical characteristics of ReCel3A compared with HjCel3A and mixtures containing HjCel3A make ReCel3A an excellent candidate for addition to enzyme mixtures designed to operate at higher temperatures. PMID:27377383

  13. Tensile Strength of Cell Walls of Living Cells 1

    PubMed Central

    Carpita, Nicholas C.

    1985-01-01

    A gas decompression technique was used to determine the breaking strength of cell walls of single cells. Breaking strengths of the bacterium Salmonella typhimurium and the unicellular green alga Chlamydomonas eugametos were 100 and 95 atmospheres, respectively, while those of sporophytes of the water mold Blastocladiella emersonii were 65 atmospheres, and those of suspension cultured cells of carrot were only 30 atmospheres. Estimation of wall tensile stress based on breaking pressures, cell radii, and estimation of wall thickness, indicates that microfibrillar walls are not necessarily stronger than walls of primitive organisms. Hence, alternative hypotheses for their evolution must be considered. PMID:16664436

  14. Searching for the role of protein phosphatases in eukaryotic microorganisms.

    PubMed

    da-Silva, A M; Zapella, P D; Andrioli, L P; Campanhã, R B; Fiorini, L C; Etchebehere, L C; da-Costa-Maia, J C; Terenzi, H F

    1999-07-01

    Preference for specific protein substrates together with differential sensitivity to activators and inhibitors has allowed classification of serine/threonine protein phosphatases (PPs) into four major types designated types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C, respectively). Comparison of sequences within their catalytic domains has indicated that PP1, PP2A and PP2B are members of the same gene family named PPP. On the other hand, the type 2C enzyme does not share sequence homology with the PPP members and thus represents another gene family, known as PPM. In this report we briefly summarize some of our studies about the role of serine/threonine phosphatases in growth and differentiation of three different eukaryotic models: Blastocladiella emersonii, Neurospora crassa and Dictyostelium discoideum. Our observations suggest that PP2C is the major phosphatase responsible for dephosphorylation of amidotransferase, an enzyme that controls cell wall synthesis during Blastocladiella emersonii zoospore germination. We also report the existence of a novel acid- and thermo-stable protein purified from Neurospora crassa mycelia, which specifically inhibits the PP1 activity of this fungus and mammals. Finally, we comment on our recent results demonstrating that Dictyostelium discoideum expresses a gene that codes for PP1, although this activity has never been demonstrated biochemically in this organism. PMID:10454741

  15. FESTERING FOOD: CHYTRIDIOMYCETE PATHOGEN REDUCES QUALITY OF DAPHNIA HOST AS A FOOD RESOURCE

    EPA Science Inventory

    When parasitic infections are severe or highly prevalent among prey, a significant component of the predator’s diet may consist of parasitized hosts. However, despite the ubiquity of parasites in most food webs, comparisons of the nutritional quality of prey as a function of inf...

  16. Cyclopsomyces plurioperculatus: a new genus and species of Lobulomycetales (Chytridiomycota, Chytridiomycetes) from Japan.

    PubMed

    Seto, Kensuke; Degawa, Yousuke

    2015-01-01

    Lobulomycetales is one of the smallest orders of Chytridiomycota, containing only four genera and five species. In a survey in Japan we isolated a chytrid from a soil sample collected in a broadleaf forest, which grouped in Lobulomycetales by BLAST query. To identify this chytrid and determine its taxonomic position, thallus development and morphology were observed by light microscopy and zoospore ultrastructure was examined using a transmission electron microscopy. We conducted a phylogenetic analysis using nuc 28S rDNA sequences. Thallus morphology was characterized by a spherical zoosporangium with multiple operculate discharge papillae, which is different from that of any other species in Lobulomycetales. This chytrid is similar to Chytriomyces multioperculatus in having multiple operculate discharge papillae, but these are distinguished by characters of the discharge papillae and rhizoidal systems. Zoospores of this chytrid had electron-dense material in the kinetosome, a unique character in the order. Our 28S phylogeny placed it in a distinct clade, sister to all described species in Lobulomycetaceae. Based on these results, we propose a new genus and species of Lobulomycetales, Cyclopsomyces plurioperculatus. PMID:25800251

  17. Effect of acute heat stress on heat shock protein 70 messenger RNA and on heat shock protein expression in the liver of broilers.

    PubMed

    Gabriel, J E; Ferro, J A; Stefani, R M; Ferro, M I; Gomes, S L; Macari, M

    1996-05-01

    1. The synthesis of heat shock protein 70 (Hsp70) mRNA and the expression of Hsp70 in the liver of broiler chickens submitted to acute heat stress (35 degrees C for 5 h) was investigated. 2. Hsp70 expression was detected by SDS-PAGE and Western blot analysis using a polyclonal antiserum against Hsp70 of Blastocladiella emersonii. The specific signal of Hsp70 mRNA was analysed by Northern blot using as probe a Hsp70 cDNA of B. emersonii. 3. An increase in the amount of Hsp70 was detected from the first up to the fifth hour of acute heat exposure. This increase in the amount of Hsp70 was accompanied by an increase in Hsp70 mRNA which peaked at 3 h. 4. This study shows that the heat induced increase in Hsp70 mRNA and protein in broiler liver, in vivo, are time dependent, similar to that in mammals. PMID:8773853

  18. Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp

    PubMed Central

    Gao, Shiqiang; Nagpal, Jatin; Schneider, Martin W.; Kozjak-Pavlovic, Vera; Nagel, Georg; Gottschalk, Alexander

    2015-01-01

    Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s−1). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals. PMID:26345128

  19. A Rhodopsin-Guanylyl Cyclase Gene Fusion Functions in Visual Perception in a Fungus

    PubMed Central

    Avelar, Gabriela M.; Schumacher, Robert I.; Zaini, Paulo A.; Leonard, Guy; Richards, Thomas A.; Gomes, Suely L.

    2014-01-01

    Summary Sensing light is the fundamental property of visual systems, with vision in animals being based almost exclusively on opsin photopigments [1]. Rhodopsin also acts as a photoreceptor linked to phototaxis in green algae [2, 3] and has been implicated by chemical means as a light sensor in the flagellated swimming zoospores of the fungus Allomyces reticulatus [4]; however, the signaling mechanism in these fungi remains unknown. Here we use a combination of genome sequencing and molecular inhibition experiments with light-sensing phenotype studies to examine the signaling pathway involved in visual perception in the closely related fungus Blastocladiella emersonii. Our data show that in these fungi, light perception is accomplished by the function of a novel gene fusion (BeGC1) of a type I (microbial) rhodopsin domain and guanylyl cyclase catalytic domain. Photobleaching of rhodopsin function prevents accumulation of cGMP levels and phototaxis of fungal zoospores exposed to green light, whereas inhibition of guanylyl cyclase activity negatively affects fungal phototaxis. Immunofluorescence microscopy localizes the BeGC1 protein to the external surface of the zoospore eyespot positioned close to the base of the swimming flagellum [4, 5], demonstrating this is a photoreceptive organelle composed of lipid droplets. Taken together, these data indicate that Blastocladiomycota fungi have a cGMP signaling pathway involved in phototaxis similar to the vertebrate vision-signaling cascade but composed of protein domain components arranged as a novel gene fusion architecture and of distant evolutionary ancestry to type II rhodopsins of animals. PMID:24835457

  20. A rhodopsin-guanylyl cyclase gene fusion functions in visual perception in a fungus.

    PubMed

    Avelar, Gabriela M; Schumacher, Robert I; Zaini, Paulo A; Leonard, Guy; Richards, Thomas A; Gomes, Suely L

    2014-06-01

    Sensing light is the fundamental property of visual systems, with vision in animals being based almost exclusively on opsin photopigments [1]. Rhodopsin also acts as a photoreceptor linked to phototaxis in green algae [2, 3] and has been implicated by chemical means as a light sensor in the flagellated swimming zoospores of the fungus Allomyces reticulatus [4]; however, the signaling mechanism in these fungi remains unknown. Here we use a combination of genome sequencing and molecular inhibition experiments with light-sensing phenotype studies to examine the signaling pathway involved in visual perception in the closely related fungus Blastocladiella emersonii. Our data show that in these fungi, light perception is accomplished by the function of a novel gene fusion (BeGC1) of a type I (microbial) rhodopsin domain and guanylyl cyclase catalytic domain. Photobleaching of rhodopsin function prevents accumulation of cGMP levels and phototaxis of fungal zoospores exposed to green light, whereas inhibition of guanylyl cyclase activity negatively affects fungal phototaxis. Immunofluorescence microscopy localizes the BeGC1 protein to the external surface of the zoospore eyespot positioned close to the base of the swimming flagellum [4, 5], demonstrating this is a photoreceptive organelle composed of lipid droplets. Taken together, these data indicate that Blastocladiomycota fungi have a cGMP signaling pathway involved in phototaxis similar to the vertebrate vision-signaling cascade but composed of protein domain components arranged as a novel gene fusion architecture and of distant evolutionary ancestry to type II rhodopsins of animals. PMID:24835457

  1. SpotWhatR: a user-friendly microarray data analysis system.

    PubMed

    Koide, Tie; Salem-Izacc, Silvia M; Gomes, Suely L; Vêncio, Ricardo Z N

    2006-01-01

    SpotWhatR is a user-friendly microarray data analysis tool that runs under a widely and freely available R statistical language (http://www.r-project.org) for Windows and Linux operational systems. The aim of SpotWhatR is to help the researcher to analyze microarray data by providing basic tools for data visualization, normalization, determination of differentially expressed genes, summarization by Gene Ontology terms, and clustering analysis. SpotWhatR allows researchers who are not familiar with computational programming to choose the most suitable analysis for their microarray dataset. Along with well-known procedures used in microarray data analysis, we have introduced a stand-alone implementation of the HTself method, especially designed to find differentially expressed genes in low-replication contexts. This approach is more compatible with our local reality than the usual statistical methods. We provide several examples derived from the Blastocladiella emersonii and Xylella fastidiosa Microarray Projects. SpotWhatR is freely available at http://blasto.iq.usp.br/~tkoide/SpotWhatR, in English and Portuguese versions. In addition, the user can choose between "single experiment" and "batch processing" versions. PMID:16755501

  2. Developmentally regulated interconversions between end product-inhibitable and noninhibitable forms of a first pathway-specific enzyme activity can be mimicked in vitro by protein dephosphorylation-phosphorylation reactions.

    PubMed

    Frisa, P S; Sonneborn, D R

    1982-10-01

    During the life cycle of Blastocladiella emersonii, dramatic shifts occur in the sensitivity of the first hexosamine biosynthetic pathway-specific enzyme [amidotransferase; 2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase (amino-transferring), EC 5.3.1.19] to end product inhibition. These shifts are developmentally correlated with changes in the utilization of the end product (uridine-5'-diphospho-N-acetylglucosamine) for chitin synthesis [Selitrennikoff, C. P., Dalley, N. E. & Sonneborn, D. R. (1980) Proc. Natl. Acad. Sci. USA 77, 5998-6002]. Alterations in amidotransferase sensitivity to end product inhibition can be mimicked by in vitro protein dephosphorylation-phosphorylation reactions, as follows: (i) Zoospore end product-inhibitable amidotransferase activity can be converted to a noninhibitable form by an endogenous (zoospore) protein phosphatase (phosphoprotein phosphohydrolase EC 3.1.3.16) reaction; this noninhibitable form can be converted back to an inhibitable form either by an endogenous cAMP-independent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) reaction or with an added cAMP-dependent protein kinase. (ii) Noninhibitable amidotransferase activity from growing cells can also be converted to the inhibitable form with added protein kinase. PMID:6959119

  3. Optogenetic manipulation of cGMP in cells and animals by the tightly light-regulated guanylyl-cyclase opsin CyclOp.

    PubMed

    Gao, Shiqiang; Nagpal, Jatin; Schneider, Martin W; Kozjak-Pavlovic, Vera; Nagel, Georg; Gottschalk, Alexander

    2015-01-01

    Cyclic GMP (cGMP) signalling regulates multiple biological functions through activation of protein kinase G and cyclic nucleotide-gated (CNG) channels. In sensory neurons, cGMP permits signal modulation, amplification and encoding, before depolarization. Here we implement a guanylyl cyclase rhodopsin from Blastocladiella emersonii as a new optogenetic tool (BeCyclOp), enabling rapid light-triggered cGMP increase in heterologous cells (Xenopus oocytes, HEK293T cells) and in Caenorhabditis elegans. Among five different fungal CyclOps, exhibiting unusual eight transmembrane topologies and cytosolic N-termini, BeCyclOp is the superior optogenetic tool (light/dark activity ratio: 5,000; no cAMP production; turnover (20 °C) ∼17 cGMP s(-1)). Via co-expressed CNG channels (OLF in oocytes, TAX-2/4 in C. elegans muscle), BeCyclOp photoactivation induces a rapid conductance increase and depolarization at very low light intensities. In O2/CO2 sensory neurons of C. elegans, BeCyclOp activation evokes behavioural responses consistent with their normal sensory function. BeCyclOp therefore enables precise and rapid optogenetic manipulation of cGMP levels in cells and animals. PMID:26345128

  4. RECONSTRUCTING THE EARLY EVOLUTION OF FUNGI USING A SIX-GENE PHYLOGENY

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ancestors of fungi are believed to be simple aquatic forms with flagellated spores, similar to modern-day Chytridiomycete fungi (chytrids). Current classifications for the fungi assume that the chytrids form an early-diverging clade of Fungi, and imply that there was a single loss of the flagel...

  5. Preliminary checklist of fungi of the Fernow Experimental Forest. Forest Service general technical report (Final)

    SciTech Connect

    Stephenson, S.L.; Kumar, A.; Bhatt, R.; Dubey, T.; Landolt, J.C.

    1994-01-01

    The report provides a checklist of fungi found on the Fernow Experimental Forest in West Virginia during 4 years of research and collecting by the authors. More than 500 fungi in seven major taxonomic groups (Acrasiomycetes, Myxomycetes, Chytridiomycetes, Oomycetes, Ascomycetes, Deuteromycetes, and Basidiomycetes) are listed alphabetically by genus and species. Also provided is a general description of the forest vegetation of the Fernow Experimental Forest.

  6. Engineering ionic liquid-tolerant cellulases for biofuels production.

    PubMed

    Wolski, Paul W; Dana, Craig M; Clark, Douglas S; Blanch, Harvey W

    2016-04-01

    Dissolution of lignocellulosic biomass in certain ionic liquids (ILs) can provide an effective pretreatment prior to enzymatic saccharification of cellulose for biofuels production. Toward the goal of combining pretreatment and enzymatic hydrolysis, we evolved enzyme variants of Talaromyces emersonii Cel7A to be more active and stable than wild-type T. emersonii Cel7A or Trichoderma reesei Cel7A in aqueous-IL solutions (up to 43% (w/w) 1,3-dimethylimdazolium dimethylphosphate and 20% (w/w) 1-ethyl-3-methylimidazolium acetate). In general, greater enzyme stability in buffer at elevated temperature corresponded to greater stability in aqueous-ILs. Post-translational modification of the N-terminal glutamine residue to pyroglutamate via glutaminyl cyclase enhanced the stability of T. emersonii Cel7A and variants. Differential scanning calorimetry revealed an increase in melting temperature of 1.9-3.9°C for the variant 1M10 over the wild-type T. emersonii Cel7A in aqueous buffer and in an IL-aqueous mixture. We observed this increase both with and without glutaminyl cyclase treatment of the enzymes. PMID:26819239

  7. Parallels in amphibian and bat declines from pathogenic fungi.

    PubMed

    Eskew, Evan A; Todd, Brian D

    2013-03-01

    Pathogenic fungi have substantial effects on global biodiversity, and 2 emerging pathogenic species-the chytridiomycete Batrachochytrium dendrobatidis, which causes chytridiomycosis in amphibians, and the ascomycete Geomyces destructans, which causes white-nose syndrome in hibernating bats-are implicated in the widespread decline of their vertebrate hosts. We synthesized current knowledge for chytridiomycosis and white-nose syndrome regarding disease emergence, environmental reservoirs, life history characteristics of the host, and host-pathogen interactions. We found striking similarities between these aspects of chytridiomycosis and white-nose syndrome, and the research that we review and propose should help guide management of future emerging fungal diseases. PMID:23622255

  8. Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America

    PubMed Central

    Berger, Lee; Speare, Rick; Daszak, Peter; Green, D. Earl; Cunningham, Andrew A.; Goggin, C. Louise; Slocombe, Ron; Ragan, Mark A.; Hyatt, Alex D.; McDonald, Keith R.; Hines, Harry B.; Lips, Karen R.; Marantelli, Gerry; Parkes, Helen

    1998-01-01

    Epidermal changes caused by a chytridiomycete fungus (Chytridiomycota; Chytridiales) were found in sick and dead adult anurans collected from montane rain forests in Queensland (Australia) and Panama during mass mortality events associated with significant population declines. We also have found this new disease associated with morbidity and mortality in wild and captive anurans from additional locations in Australia and Central America. This is the first report of parasitism of a vertebrate by a member of the phylum Chytridiomycota. Experimental data support the conclusion that cutaneous chytridiomycosis is a fatal disease of anurans, and we hypothesize that it is the proximate cause of these recent amphibian declines. PMID:9671799

  9. Nucleotide sequences of 5S ribosomal RNA from four oomycete and chytrid water molds.

    PubMed

    Walker, W F; Doolittle, W F

    1982-09-25

    The nucleotide sequences of the 5S rRNAs of the oomycete water molds Saprolegnia ferax and Pythium hydnosporum and of the chytrid water molds Blastocladiella simplex and Phlyctochytrium irregulare were determined by chemical and enzymatic partial degradation of 3' and 5' end-labelled molecules, followed by gel sequence analysis. The two oomycete sequences differed in 24 positions and the two chytrid sequences differed in 27 positions. These pairs differed in a mean of 44 positions. The chytrid sequences clearly most resemble the sequence from the zygomycete Phycomyces, while the oomycete sequences appear to be allied with those from protozoa and slime molds. PMID:6890670

  10. Reproductive strategies and adaptations for survival among obligatory microsporidian and fungal parasites of mosquitoes: a comparative analysis of Amblyospora and Coelomomyces.

    PubMed

    Lucarotti, C J; Andreadis, T G

    1995-03-01

    Amblyospora (Microsporida: Amblyosporidae) and Coelomomyces (Chytridiomycetes: Blastocladiales) have independently evolved a diverse array of unique and highly specialized mechanisms that have allowed them to more fully exploit their mosquito hosts and the aquatic environment that their hosts inhabit. Amblyospora and Coelomomyces both have complex life cycles that include obligatory development in an intermediate microcrustacean host and 2 mosquito generations for completion. Amblyospora is polymorphic with 3 separate and distinct developmental sequences, asexual and sexual reproduction, and aspects of both vertical (transovarial) and horizontal transmission. Infective stages of Coelomomyces are motile, a temporal gating mechanism coordinates gamete release, and, even though there is no transovarial transmission, infection of primary host ovaries is important in dissemination of the fungus to new habitats. The intent of this review is to examine how these and other strategies and adaptations facilitate parasite reproduction within the host(s) and enhance transmission and survival between hosts. PMID:7616177

  11. Characterization of thermophilic fungal community associated with pile fermentation of Pu-erh tea.

    PubMed

    Zhang, Wei; Yang, Ruijuan; Fang, Wenjun; Yan, Liang; Lu, Jun; Sheng, Jun; Lv, Jie

    2016-06-16

    This study aimed to characterize the thermophilic fungi in pile-fermentation process of Pu-erh tea. Physicochemical analyses showed that the high temperature and low pH provided optimal conditions for propagation of fungi. A number of fungi, including Blastobotrys adeninivorans, Thermomyces lanuginosus, Rasamsonia emersonii, Aspergillus fumigatus, Rhizomucor pusillus, Rasamsonia byssochlamydoides, Rasamsonia cylindrospora, Aspergillus tubingensis, Aspergillus niger, Candida tropicalis and Fusarium graminearum were isolated as thermophilic fungi under combination of high temperature and acid culture conditions from Pu-erh tea pile-fermentation. The fungal communities were analyzed by PCR-DGGE. Results revealed that those fungi are closely related to Debaryomyces hansenii, Cladosporium cladosporioides, A. tubingensis, R. emersonii, R. pusillus, A. fumigatus and A. niger, and the last four presented as dominant species in the pile process. These four preponderant thermophilic fungi reached the order of magnitude of 10(7), 10(7), 10(7) and 10(6)copies/g dry tea, respectively, measured by real-time quantitative PCR (q-PCR). The results indicate that the thermophilic fungi play an important role in Pu-erh tea pile fermentation. PMID:27046629

  12. Batrachochytrium salamandrivorans sp. nov. causes lethal chytridiomycosis in amphibians

    PubMed Central

    Martel, An; Spitzen-van der Sluijs, Annemarieke; Blooi, Mark; Bert, Wim; Ducatelle, Richard; Fisher, Matthew C.; Woeltjes, Antonius; Bosman, Wilbert; Chiers, Koen; Bossuyt, Franky; Pasmans, Frank

    2013-01-01

    The current biodiversity crisis encompasses a sixth mass extinction event affecting the entire class of amphibians. The infectious disease chytridiomycosis is considered one of the major drivers of global amphibian population decline and extinction and is thought to be caused by a single species of aquatic fungus, Batrachochytrium dendrobatidis. However, several amphibian population declines remain unexplained, among them a steep decrease in fire salamander populations (Salamandra salamandra) that has brought this species to the edge of local extinction. Here we isolated and characterized a unique chytrid fungus, Batrachochytrium salamandrivorans sp. nov., from this salamander population. This chytrid causes erosive skin disease and rapid mortality in experimentally infected fire salamanders and was present in skin lesions of salamanders found dead during the decline event. Together with the closely related B. dendrobatidis, this taxon forms a well-supported chytridiomycete clade, adapted to vertebrate hosts and highly pathogenic to amphibians. However, the lower thermal growth preference of B. salamandrivorans, compared with B. dendrobatidis, and resistance of midwife toads (Alytes obstetricans) to experimental infection with B. salamandrivorans suggest differential niche occupation of the two chytrid fungi. PMID:24003137

  13. Synthesis of substrates for periodate-coupled assay of phospholipases C and sphingomyelinases.

    PubMed

    Larsen, Kira Løw; Andersen, Rokhsana J; Brask, Jesper

    2016-09-01

    A series of 4-nitrophenyl (pNP) and 4-methylumbelliferyl (4MU) substrate analogues of phosphatidyl choline (PC) and phosphatidic acid (PA) were synthesized from 4-bromo-1-butene by ether formation, olefin epoxidation and ring opening with the phosphate head group. The pNP PC analogue, 4-(4-nitrophenoxy)-2-hydroxy-butyl-1-phosphoryl choline (1) was evaluated in assays of fungal sphingomyelinases, also displaying phospholipase C activity. Reactions were terminated with a periodate-containing stop solution, leading to liberation of pNP, quantified spectrophotometrically in an end-point measurement. A kinetic evaluation of sphingomyelinases from Kionochaeta sp. and Penicillium emersonii showed relatively high KM and low kcat values for this substrate, limiting its practical applicability in assays with low sphingomyelinase concentrations. PMID:27444331

  14. Importance of algae as a potential source of biofuel.

    PubMed

    Singh, A K; Singh, M P

    2014-01-01

    Algae have a great potential source of biofuels and also have unique importance to reduce gaseous emissions, greenhouse gases, climatic changes, global warming receding of glaciers, rising sea levels and loss of biodiversity. The microalgae, like Scenedesmus obliquus, Neochloris oleabundans, Nannochloropsis sp., Chlorella emersonii, and Dunaliella tertiolecta have high oil content. Among the known algae, Scenedesmus obliquus is one of the most potential sources for biodiesel as it has adequate fatty acid (linolenic acid) and other polyunsaturated fatty acids. Bio—ethanol is already in the market of United States of America and Europe as an additive in gasoline. Bio—hydrogen is the cleanest biofuel and extensive efforts are going on to bring it to market at economical price. This review highlights recent development and progress in the field of algae as a potential source of biofuel. PMID:25535720

  15. Occurrence of fungi and fungus-like organisms in the Horodnianka River in the vicinity of Białystok, Poland.

    PubMed

    Kiziewicz, Bozena; Zdrojkowska, Ewa; Gajo, Bernadetta; Godlewska, Anna; Muszyńska, Elzbieta; Mazalska, Bozenna

    2011-01-01

    Studies of fungi and fungus- like organisms in the northeastern Poland have mainly concentrated on running waters in the vicinity of Białystok, including the Horodnianka River. The main objective was to investigate biodiversity of fungi and fungus-like organisms which take part in decomposition of organic matter commonly found in inland waters. To obtain a complete picture of species composition of fungi and fungus-like organisms in running waters we decided to explore representative sites of the Horodnianka River such as Olmonty, Hryniewicze and Horodniany with close localization of landfill. Fungal species were isolated using baiting technique. Baits of onion skin (Alium cepa), hemp-seeds (Cannabis sativa), impregnated cellophane and snake skin (Natrix natrix) were applied to isolate fungi from water of the Horodnianka River. The fungal community consists of 26 species, 10 species of fungi belonging to class Chytridiomycetes (3), anamorphic fungi (6), and Zygomycetes (1). 16 species belong to fungus-like organisms from class Oomycetes. Most of the recognized species have already been found in other running waters. From all the examined habitats the fungi belonging to 26 species of 18 genera Achlya, Alternaria, Aphanomyces, Aspergillus, Catenophlyctis, Dictyuchus, Fusarium, Karlingia, Lagenidium, Leptomitus, Olpidiopsis, Penicillium, Phlyctochytrium, Pythium, Saprolegnia, Scoliognia, Thraustotheca and Zoophagus were obtained. Certain fungal species like Aphanomyces laevis, Fusarium aqueductum, F. moniliforme, F. oxysporum, Leptomitus lacteus, Saprolegnia feax and S. parasitica were found at all the study sites. Among fungi potentially pathogenic and allergogenic for humans the genera Alternaria, Aspergillus, Fusarium, Lagenidium and Penicillium have already been described. However, the species Lagenidium giganteum and Achlya androgyna are new in the fungal biota of Poland. The greatest number of fungal species occurred in Olmonty (24), the smallest in Horodniany

  16. Structure, expression and function of Allomyces arbuscula CDP II (metacaspase) gene.

    PubMed

    Ojha, Mukti; Cattaneo, Arlette; Hugh, Séverine; Pawlowski, Jan; Cox, Jos A

    2010-06-01

    Allomyces arbuscula, a primitive chytridiomycete fungus, has two Ca(2+)-dependent cysteine proteases, the CDP I and CDP II. We have cloned and analyzed the nucleotide sequence of CDP II gene and domain structure of the protein. Blast analysis of the sequence has shown that the protein belongs to a newly described member of caspase superfamily protein, the metacaspase, a CD clan of C14 family cysteine protease, we hence-forth name it as AMca 2 (Allomyces metacaspase 2). Southern hybridization studies have shown that the gene exists in a single copy per genome. The transcriptional analysis by Northern hybridization has confirmed our previous results that the protein is developmentally regulated, i.e. present in active growth phase but disappears during nutritional stress which also induces reproductive differentiation, indicating that the protein promotes cell growth, not death. The recombinant gene product expressed in Escherichiacoli has all the catalytic properties of native enzyme, i.e. sensitivity to protease inhibitors and substrate specificity. There is an absolute requirement of Ca(2+) for the activation of catalytic activity and the presence of R residue at the cleavage site (P1 position) in the substrate. The presence of a second basic residue, either R or K, in the P2 position strongly inhibits the catalytic activity which is stimulated by the presence of P and to a lesser extent G at this site. Peptide substrates with D at the cleavage site are not recognised and therefore not cleaved. The enzyme activity is inhibited by EDTA-EGTA, cysteine protease inhibitors and a specific peptide inhibitor Ac GVRCHCL TFA, but not by E64, although a potent inhibitor of cysteine proteases. PMID:20214955

  17. Long-term disease dynamics in lakes: causes and consequences of chytrid infections in Daphnia populations.

    PubMed

    Johnson, Pieter T J; Ives, Anthony R; Lathrop, Richard C; Carpenter, Stephen R

    2009-01-01

    Understanding the drivers and consequences of disease epidemics is an important frontier in ecology. However, long-term data on hosts, their parasites, and the corresponding environmental conditions necessary to explore these interactions are often unavailable. We examined the dynamics of Daphnia pulicaria, a keystone zooplankter in lake ecosystems, to explore the long-term causes and consequences of infection by a chytridiomycete parasitoid (Polycaryum laeve). After quantifying host-pathogen dynamics from vouchered samples collected over 15 years, we used autoregressive models to evaluate (1) hypothesized drivers of infection, including host density, water temperature, dissolved oxygen, host-food availability, and lake mixing; and (2) the effects of epidemics on host populations. Infection was present in most years but varied widely in prevalence, from < 1% to 34%, with seasonal peaks in early spring and late fall. Within years, lake stratification strongly inhibited P. laeve transmission, such that epidemics occurred primarily during periods of water mixing. Development of the thermocline likely reduced transmission by spatially separating susceptible hosts from infectious zoospores. Among years, ice duration and cumulative snowfall correlated negatively with infection prevalence, likely because of reductions in spring phytoplankton and D. pulicaria density in years with extended winters. Epidemics also influenced dynamics of the host population. Infected D. pulicaria rarely (< 1%) contained eggs, and P. laeve prevalence was positively correlated with sexual reproduction in D. pulicaria. Analyses of D. pulicaria density-dependent population dynamics predicted that, in the absence of P. laeve infection, host abundance would be 11-50% higher than what was observed. By underscoring the importance of complex physical processes in controlling host-parasite interactions and of epidemic disease in influencing host populations, our results highlight the value of long

  18. Improvement of the catalytic performance of a Bispora antennata cellulase by replacing the N-terminal semi-barrel structure.

    PubMed

    Zheng, Fei; Huang, Huoqing; Wang, Xiaoyu; Tu, Tao; Liu, Qiong; Meng, Kun; Wang, Yuan; Su, Xiaoyun; Xie, Xiangming; Luo, Huiying

    2016-10-01

    The aim of this work was to study the contribution of the N-terminal structure to cellulase catalytic performance. A wild-type cellulase (BaCel5) of glycosyl hydrolase (GH) family 5 from Bispora antennata and two hybrid enzymes (BaCel5(127) and BaCel5(167)) with replacement of the N-terminal (βα)3 (127 residues) or (βα)4 (167 residues)-barrel with the corresponding sequences of TeEgl5A from Talaromyces emersonii were produced in Pichia pastoris and biochemically characterized. BaCel5 exhibited optimal activity at pH 5.0 and 50°C but had low catalytic efficiency (25.4±0.8mLs(-1)mg(-1)). In contrast, BaCel5(127) and BaCel5(167) showed similar enzymatic properties but improved catalytic performance. When using CMC-Na, barley β-glucan, lichenan, and cellooligosaccharides as substrates, BaCel5(127) and BaCel5(167) had increased specific activities and catalytic efficiencies by ∼1.8-6.7-fold and ∼1.0-4.7-fold, respectively. The catalytic efficiency of BaCel5(167) was even higher than that of parental proteins. The underlying mechanism was analyzed by molecular docking and molecular dynamic simulation. PMID:27372007

  19. Direct utilization of waste water algal biomass for ethanol production by cellulolytic Clostridium phytofermentans DSM1183.

    PubMed

    Fathima, Anwar Aliya; Sanitha, Mary; Kumar, Thangarathinam; Iyappan, Sellamuthu; Ramya, Mohandass

    2016-02-01

    Direct bioconversion of waste water algal biomass into ethanol using Clostridium phytofermentans DSM1183 was demonstrated in this study. Fermentation of 2% (w/v) autoclaved algal biomass produced ethanol concentration of 0.52 g L(-1) (solvent yield of 0.19 g/g) where as fermentation of acid pretreated algal biomass (2%, w/v) produced ethanol concentration of 4.6 g L(-1) in GS2 media (solvent yield of 0.26 g/g). The control experiment with 2% (w/v) glucose in GS2 media produced ethanol concentration of 2.8 g L(-1) (solvent yield of 0.25 g/g). The microalgal strains from waste water algal biomass were identified as Chlamydomonas dorsoventralis, Graesiella emersonii, Coelastrum proboscideum, Scenedesmus obliquus, Micractinium sp., Desmodesmus sp., and Chlorella sp., based on ITS-2 molecular marker. The presence of glucose, galactose, xylose and rhamnose were detected by high performance liquid chromatography in the algal biomass. Scanning Electron Microscopy observations of fermentation samples showed characteristic morphological changes in algal cells and bioaccessibility of C. phytofermentans. PMID:26705954

  20. Evaluation of certain food additives and contaminants. Eightieth report of the Joint FAO/WHO Expert Committee on Food Additives.

    PubMed

    2016-01-01

    This report represents the conclusions of a Joint FAO/WHO Expert Committee convened to evaluate the safety of various food additives and contaminants and to prepare specifications for identity and purity. The first part of the report contains a brief description of general considerations addressed at the meeting, including updates on matters of interest to the work of the Committee. A summary follows of the Committee's evaluations of technical, toxicological and/or dietary exposure data for seven food additives (benzoates; lipase from Fusarium heterosporum expressed in Ogataea polymorpha; magnesium stearate; maltotetraohydrolase from Pseudomonas stutzeri expressed in Bacillus licheniformis; mixed β-glucanase, cellulase and xylanase from Rasamsonia emersonii; mixed β-glucanase and xylanase from Disporotrichum dimorphosporum; polyvinyl alcohol (PVA)- polyethylene glycol (PEG) graft copolymer) and two groups of contaminants (non-dioxin-like polychlorinated biphenyls and pyrrolizidine alkaloids). Specifications for the following food additives were revised or withdrawn: advantame; annatto extracts (solavnt extracted bixin, ad solvent-extracted norbixin); food additives containing aluminium and/or silicon (aluminium silicate; calcium aluminium silicate; calcium silicate; silicon dioxide, amorphous; sodium aluminium silicate); and glycerol ester of gum rosin. Annexed to the report are tables or text summarizing the toxicological and dietary exposure information and information on specifications as well as the Committees recommendations on the food additives and contaminants considered at this meeting. PMID:27514183

  1. High Protein- and High Lipid-Producing Microalgae from Northern Australia as Potential Feedstock for Animal Feed and Biodiesel

    PubMed Central

    Duong, Van Thang; Ahmed, Faruq; Thomas-Hall, Skye R.; Quigley, Simon; Nowak, Ekaterina; Schenk, Peer M.

    2015-01-01

    Microalgal biomass can be used for biodiesel, feed, and food production. Collection and identification of local microalgal strains in the Northern Territory, Australia was conducted to identify strains with high protein and lipid contents as potential feedstock for animal feed and biodiesel production, respectively. A total of 36 strains were isolated from 13 samples collected from a variety of freshwater locations, such as dams, ponds, and streams and subsequently classified by 18S rDNA sequencing. All of the strains were green microalgae and predominantly belong to Chlorella sp., Scenedesmus sp., Desmodesmus sp., Chlamydomonas sp., Pseudomuriella sp., Tetraedron caudatum, Graesiella emersonii, and Mychonastes timauensis. Among the fastest growing strains, Scenedesmus sp. NT1d possessed the highest content of protein; reaching up to 33% of its dry weight. In terms of lipid production, Chlorella sp. NT8a and Scenedesmus dimorphus NT8e produced the highest triglyceride contents of 116.9 and 99.13 μg mL−1 culture, respectively, as measured by gas chromatography–mass spectroscopy of fatty acid methyl esters. These strains may present suitable candidates for biodiesel production after further optimization of culturing conditions, while their protein-rich biomass could be used for animal feed. PMID:26042215

  2. Redox-initiated hydrogel system for detection and real-time imaging of cellulolytic enzyme activity.

    PubMed

    Malinowska, Klara H; Verdorfer, Tobias; Meinhold, Aylin; Milles, Lukas F; Funk, Victor; Gaub, Hermann E; Nash, Michael A

    2014-10-01

    Understanding the process of biomass degradation by cellulolytic enzymes is of urgent importance for biofuel and chemical production. Optimizing pretreatment conditions and improving enzyme formulations both require assays to quantify saccharification products on solid substrates. Typically, such assays are performed using freely diffusing fluorophores or dyes that measure reducing polysaccharide chain ends. These methods have thus far not allowed spatial localization of hydrolysis activity to specific substrate locations with identifiable morphological features. Here we describe a hydrogel reagent signaling (HyReS) system that amplifies saccharification products and initiates crosslinking of a hydrogel that localizes to locations of cellulose hydrolysis, allowing for imaging of the degradation process in real time. Optical detection of the gel in a rapid parallel format on synthetic and natural pretreated solid substrates was used to quantify activity of T. emersonii and T. reesei enzyme cocktails. When combined with total internal reflection fluorescence microscopy and AFM imaging, the reagent system provided a means to visualize enzyme activity in real-time with high spatial resolution (<2 μm). These results demonstrate the versatility of the HyReS system in detecting cellulolytic enzyme activity and suggest new opportunities in real-time chemical imaging of biomass depolymerization. PMID:25116339

  3. Structural insights into the affinity of Cel7A carbohydrate-binding module for lignin.

    PubMed

    Strobel, Kathryn L; Pfeiffer, Katherine A; Blanch, Harvey W; Clark, Douglas S

    2015-09-11

    The high cost of hydrolytic enzymes impedes the commercial production of lignocellulosic biofuels. High enzyme loadings are required in part due to their non-productive adsorption to lignin, a major component of biomass. Despite numerous studies documenting cellulase adsorption to lignin, few attempts have been made to engineer enzymes to reduce lignin binding. In this work, we used alanine-scanning mutagenesis to elucidate the structural basis for the lignin affinity of Trichoderma reesei Cel7A carbohydrate binding module (CBM). T. reesei Cel7A CBM mutants were produced with a Talaromyces emersonii Cel7A catalytic domain and screened for their binding to cellulose and lignin. Mutation of aromatic and polar residues on the planar face of the CBM greatly decreased binding to both cellulose and lignin, supporting the hypothesis that the cellulose-binding face is also responsible for lignin affinity. Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity. Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin. Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability. This work provides insights into the structural underpinnings of lignin affinity, identifies residues amenable to mutation without compromising cellulose affinity, and informs engineering strategies for family one CBMs. PMID:26209638

  4. Genetic manipulation, a feasible tool to enhance unique characteristic of Chlorella vulgaris as a feedstock for biodiesel production.

    PubMed

    Talebi, Ahmad Farhad; Tohidfar, Masoud; Tabatabaei, Meisam; Bagheri, Abdolreza; Mohsenpor, Motahhareh; Mohtashami, Seyed Kaveh

    2013-07-01

    Developing a reliable technique to transform unicellular green algae, Chlorella vulgaris, could boost potentials of using microalgae feedstock in variety of applications such as biodiesel production. Volumetric lipid productivity (VLP) is a suitable variable for evaluating potential of algal species. In the present study, the highest VLP level was recorded for C. vulgaris (79.08 mg l(-1 )day(-1)) followed by 3 other strains studied; C. emersonii, C. protothecoides, and C. salina by 54.41, 45 and 18.22 mg l(-1)day(-1), respectively. Having considered the high productivity of C. vulgaris, it was selected for the preliminary transformation experiment through micro-particle bombardment. Plasmid pBI 121, bearing the reporter gene under the control of CaMV 35S promoter and the kanamycin marker gene, was used in cells bombardment. Primary selection was done on a medium supplemented by 50 mg l(-1) kanamycin. After several passages, the survived cells were PCR-tested to confirm the stability of transformation and then were found to exhibit β-glucuronidase (GUS) activity in comparison with the control cells. Southern hybridization of npt II probe with genomic DNA revealed stable integration of the cassette in three different positions in the genome. The whole process was successfully implemented as a pre-step to transform the algal cells by genes involved in lipid production pathway which will be carried out in our future studies. PMID:23652998

  5. Immobilization of microalgae for biosorption and degradation of butyltin chlorides.

    PubMed

    Zhang, L; Huang, G; Yu, Y

    1998-07-01

    Since the discovery of their biocidal properties in the 1950s, organotin compounds have found a large spectrum of industrial applications such as wood and textile preservatives, fungicides and pesticides, and antifouling paint on ships and fishing equipment. The fate and environmental impact of butyltins have been the subjects of a large body of research in the last decades. Biosorption and degradation of butyltin compounds by immobilized microalgae chlorella were studied in this paper, aiming to find an alternative way to solve organotin pollution problem. Chlorella emersonii cells were entrapped in a calcium aginate matrix. The cell growth rates, respiratory rate and chlorophyll a content were studied and compared. Results showed that immobilized chlorella had increased respiratory and growth rates, and almost equal chlorophyll a content when compared with free cells. Cell leakage was slight during the 20-day experimental period Cell leakage from the matrix was unrelated to cell growth within the matrix. Immobilized chlorella was applied to deal with butytin contaminated aquatic solutions. Immobilized chlorella had increased degradation rates of tri-, di-, and mono-butyltin chlorides in aquatic solutions, and lower biological accumulation factors on cells, than free cells, which indicates a potential use for tackleing organotin polluted water body. PMID:9663338

  6. Structural Insights into the Affinity of Cel7A Carbohydrate-binding Module for Lignin*

    PubMed Central

    Strobel, Kathryn L.; Pfeiffer, Katherine A.; Blanch, Harvey W.; Clark, Douglas S.

    2015-01-01

    The high cost of hydrolytic enzymes impedes the commercial production of lignocellulosic biofuels. High enzyme loadings are required in part due to their non-productive adsorption to lignin, a major component of biomass. Despite numerous studies documenting cellulase adsorption to lignin, few attempts have been made to engineer enzymes to reduce lignin binding. In this work, we used alanine-scanning mutagenesis to elucidate the structural basis for the lignin affinity of Trichoderma reesei Cel7A carbohydrate binding module (CBM). T. reesei Cel7A CBM mutants were produced with a Talaromyces emersonii Cel7A catalytic domain and screened for their binding to cellulose and lignin. Mutation of aromatic and polar residues on the planar face of the CBM greatly decreased binding to both cellulose and lignin, supporting the hypothesis that the cellulose-binding face is also responsible for lignin affinity. Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity. Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin. Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability. This work provides insights into the structural underpinnings of lignin affinity, identifies residues amenable to mutation without compromising cellulose affinity, and informs engineering strategies for family one CBMs. PMID:26209638

  7. Bioprospecting the thermal waters of the Roman baths: isolation of oleaginous species and analysis of the FAME profile for biodiesel production.

    PubMed

    Smith-Bädorf, Holly D; Chuck, Christopher J; Mokebo, Kirsty R; Macdonald, Heather; Davidson, Matthew G; Scott, Rod J

    2013-01-01

    The extensive diversity of microalgae provides an opportunity to undertake bioprospecting for species possessing features suited to commercial scale cultivation. The outdoor cultivation of microalgae is subject to extreme temperature fluctuations; temperature tolerant microalgae would help mitigate this problem. The waters of the Roman Baths, which have a temperature range between 39°C and 46°C, were sampled for microalgae. A total of 3 green algae, 1 diatom and 4 cyanobacterial species were successfully isolated into 'unialgal' culture. Four isolates were filamentous, which could prove advantageous for low energy dewatering of cultures using filtration.Lipid content, profiles and growth rates of the isolates were examined at temperatures of 20, 30, 40°C, with and without nitrogen starvation and compared against the oil producing green algal species, Chlorella emersonii. Some isolates synthesized high levels of lipids, however, all were most productive at temperatures lower than those of the Roman Baths. The eukaryotic algae accumulated a range of saturated and polyunsaturated FAMEs and all isolates generally showed higher lipid accumulation under nitrogen deficient conditions (Klebsormidium sp. increasing from 1.9% to 16.0% and Hantzschia sp. from 31.9 to 40.5%). The cyanobacteria typically accumulated a narrower range of FAMEs that were mostly saturated, but were capable of accumulating a larger quantity of lipid as a proportion of dry weight (M. laminosus, 37.8% fully saturated FAMEs). The maximum productivity of all the isolates was not determined in the current work and will require further effort to optimise key variables such as light intensity and media composition. PMID:23369619

  8. Bioprospecting the thermal waters of the Roman baths: isolation of oleaginous species and analysis of the FAME profile for biodiesel production

    PubMed Central

    2013-01-01

    The extensive diversity of microalgae provides an opportunity to undertake bioprospecting for species possessing features suited to commercial scale cultivation. The outdoor cultivation of microalgae is subject to extreme temperature fluctuations; temperature tolerant microalgae would help mitigate this problem. The waters of the Roman Baths, which have a temperature range between 39°C and 46°C, were sampled for microalgae. A total of 3 green algae, 1 diatom and 4 cyanobacterial species were successfully isolated into ‘unialgal’ culture. Four isolates were filamentous, which could prove advantageous for low energy dewatering of cultures using filtration. Lipid content, profiles and growth rates of the isolates were examined at temperatures of 20, 30, 40°C, with and without nitrogen starvation and compared against the oil producing green algal species, Chlorella emersonii. Some isolates synthesized high levels of lipids, however, all were most productive at temperatures lower than those of the Roman Baths. The eukaryotic algae accumulated a range of saturated and polyunsaturated FAMEs and all isolates generally showed higher lipid accumulation under nitrogen deficient conditions (Klebsormidium sp. increasing from 1.9% to 16.0% and Hantzschia sp. from 31.9 to 40.5%). The cyanobacteria typically accumulated a narrower range of FAMEs that were mostly saturated, but were capable of accumulating a larger quantity of lipid as a proportion of dry weight (M. laminosus, 37.8% fully saturated FAMEs). The maximum productivity of all the isolates was not determined in the current work and will require further effort to optimise key variables such as light intensity and media composition. PMID:23369619

  9. Heterologous expression of cellulase genes in natural Saccharomyces cerevisiae strains.

    PubMed

    Davison, Steffi A; den Haan, Riaan; van Zyl, Willem Heber

    2016-09-01

    Enzyme cost is a major impediment to second-generation (2G) cellulosic ethanol production. One strategy to reduce enzyme cost is to engineer enzyme production capacity in a fermentative microorganism to enable consolidated bio-processing (CBP). Ideally, a strain with a high secretory phenotype, high fermentative capacity as well as an innate robustness to bioethanol-specific stressors, including tolerance to products formed during pre-treatment and fermentation of lignocellulosic substrates should be used. Saccharomyces cerevisiae is a robust fermentative yeast but has limitations as a potential CBP host, such as low heterologous protein secretion titers. In this study, we evaluated natural S. cerevisiae isolate strains for superior secretion activity and other industrially relevant characteristics needed during the process of lignocellulosic ethanol production. Individual cellulases namely Saccharomycopsis fibuligera Cel3A (β-glucosidase), Talaromyces emersonii Cel7A (cellobiohydrolase), and Trichoderma reesei Cel5A (endoglucanase) were utilized as reporter proteins. Natural strain YI13 was identified to have a high secretory phenotype, demonstrating a 3.7- and 3.5-fold higher Cel7A and Cel5A activity, respectively, compared to the reference strain S288c. YI13 also demonstrated other industrially relevant characteristics such as growth vigor, high ethanol titer, multi-tolerance to high temperatures (37 and 40 °C), ethanol (10 % w/v), and towards various concentrations of a cocktail of inhibitory compounds commonly found in lignocellulose hydrolysates. This study accentuates the value of natural S. cerevisiae isolate strains to serve as potential robust and highly productive chassis organisms for CBP strain development. PMID:27470141

  10. Expression and evaluation of enzymes required for the hydrolysis of galactomannan.

    PubMed

    Malherbe, A R; Rose, S H; Viljoen-Bloom, M; van Zyl, W H

    2014-08-01

    The cost-effective production of bioethanol from lignocellulose requires the complete conversion of plant biomass, which contains up to 30 % mannan. To ensure utilisation of galactomannan during consolidated bioprocessing, heterologous production of mannan-degrading enzymes in fungal hosts was explored. The Aspergillus aculeatus endo-β-mannanase (Man1) and Talaromyces emersonii α-galactosidase (Agal) genes were expressed in Saccharomyces cerevisiae Y294, and the Aspergillus niger β-mannosidase (cMndA) and synthetic Cellvibrio mixtus β-mannosidase (Man5A) genes in A. niger. Maximum enzyme activity for Man1 (374 nkat ml(-1), pH 5.47), Agal (135 nkat ml(-1), pH 2.37), cMndA (12 nkat ml(-1), pH 3.40) and Man5A (8 nkat ml(-1), pH 3.40) was observed between 60 and 70 °C. Co-expression of the Man1 and Agal genes in S. cerevisiae Y294[Agal-Man1] reduced the extracellular activity relative to individual expression of the respective genes. However, the combined action of crude Man1, Agal and Man5A enzyme preparations significantly decreased the viscosity of galactomannan in locust bean gum, confirming hydrolysis thereof. Furthermore, when complemented with exogenous Man5A, S. cerevisiae Y294[Agal-Man1] produced 56 % of the theoretical ethanol yield, corresponding to a 66 % carbohydrate conversion, on 5 g l(-1) mannose and 10 g l(-1) locust bean gum. PMID:24888762

  11. Analysis of the dynamics of fungal communities in soil via fungal-specific PCR of soil DNA followed by denaturing gradient gel electrophoresis.

    PubMed

    van Elsas, J D; Duarte, G F; Keijzer-Wolters, A; Smit, E

    2000-12-15

    A molecular method for profiling of fungal communities in soil was applied in experiments in soil microcosms, with two objectives, (1) to assess the persistence of two selected fungal species in soil, and (2) to analyze the response of the natural fungal community to a spill of sulphurous petrol in the same soil. To achieve the aims, two soil DNA extraction methods, one originally designed for the direct extraction of bacterial community DNA and the other one aimed to obtain fungal DNA, were tested for their efficiency in recovering DNA of fungal origin from soil. Both methods allowed for the efficient extraction of DNA from introduced Trichoderma harzianum spores as well as Arthrobotrys oligospora mycelial fragments, at comparable rates. Several PCR amplification systems based on primers specific for fungal 18S ribosomal RNA genes were tested to design strategies for the assessment of fungal communities in soil. The PCR systems produced amplicons of expected size with DNA of most fungi studied, which included members of the Ascomycetes, Basidiomycetes, Zygomycetes and Chytridiomycetes. On the other hand, the 18S rRNA genes of Oomycetes (including key plant pathogens) were poorly amplified. Plant (Solanum tuberosum), nematode (Meloidogyne sp.) and bacterial DNA was not amplified. For studies of soil fungal communities, a nested PCR approach was selected, in which the first PCR provided the required specificity for fungi, whereas the second (nested) PCR served to produce amplicons separable on denaturing gradient gels. Denaturing gradient gel electrophoresis (DGGE) allowed the resolution of mixtures of PCR products of several different fungi, as well as products resulting from mixed-template amplifications, into distinct banding patterns. The persistence of fungal species in soil was assessed using T. harzianum spores and A. oligospora hyphal fragments added to silt loam soil microcosms. Using PCR-DGGE, these fungi were detectable for about 14 days and 2 months

  12. Two structurally discrete GH7-cellobiohydrolases compete for the same cellulosic substrate fiber

    PubMed Central

    2012-01-01

    Background Cellulose consisting of arrays of linear beta-1,4 linked glucans, is the most abundant carbon-containing polymer present in biomass. Recalcitrance of crystalline cellulose towards enzymatic degradation is widely reported and is the result of intra- and inter-molecular hydrogen bonds within and among the linear glucans. Cellobiohydrolases are enzymes that attack crystalline cellulose. Here we report on two forms of glycosyl hydrolase family 7 cellobiohydrolases common to all Aspergillii that attack Avicel, cotton cellulose and other forms of crystalline cellulose. Results Cellobiohydrolases Cbh1 and CelD have similar catalytic domains but only Cbh1 contains a carbohydrate-binding domain (CBD) that binds to cellulose. Structural superpositioning of Cbh1 and CelD on the Talaromyces emersonii Cel7A 3-dimensional structure, identifies the typical tunnel-like catalytic active site while Cbh1 shows an additional loop that partially obstructs the substrate-fitting channel. CelD does not have a CBD and shows a four amino acid residue deletion on the tunnel-obstructing loop providing a continuous opening in the absence of a CBD. Cbh1 and CelD are catalytically functional and while specific activity against Avicel is 7.7 and 0.5 U.mg prot-1, respectively specific activity on pNPC is virtually identical. Cbh1 is slightly more stable to thermal inactivation compared to CelD and is much less sensitive to glucose inhibition suggesting that an open tunnel configuration, or absence of a CBD, alters the way the catalytic domain interacts with the substrate. Cbh1 and CelD enzyme mixtures on crystalline cellulosic substrates show a strong combinatorial effort response for mixtures where Cbh1 is present in 2:1 or 4:1 molar excess. When CelD was overrepresented the combinatorial effort could only be partially overcome. CelD appears to bind and hydrolyze only loose cellulosic chains while Cbh1 is capable of opening new cellulosic substrate molecules away from the cellulosic

  13. Engineering towards a complete heterologous cellulase secretome in Yarrowia lipolytica reveals its potential for consolidated bioprocessing

    SciTech Connect

    Wei, Hui; Wang, Wei; Alahuhta, Markus; Vander Wall, Todd; Baker, John O.; Taylor, Larry E.; Decker, Stephen R.; Himmel, Michael E.; Zhang, Min

    2014-10-16

    Background: Yarrowia lipolytica is an oleaginous yeast capable of metabolizing glucose to lipids, which then accumulate intracellularly. However, it lacks the suite of cellulolytic enzymes required to break down biomass cellulose and cannot therefore utilize biomass directly as a carbon source. Toward the development of a direct microbial conversion platform for the production of hydrocarbon fuels from cellulosic biomass, the potential for Y. lipolytica to function as a consolidated bioprocessing strain was investigated by first conducting a genomic search and functional testing of its endogenous glycoside hydrolases. Once the range of endogenous enzymes was determined, the critical cellulases from Trichoderma reesei were cloned into Yarrowia. Results: Initially, work to express T. reesei endoglucanase II (EGII) and cellobiohydrolase (CBH) II in Y. lipolytica resulted in the successful secretion of active enzymes. However, a critical cellulase, T. reesei CBHI, while successfully expressed in and secreted from Yarrowia, showed less than expected enzymatic activity, suggesting an incompatibility (probably at the post-translational level) for its expression in Yarrowia. This result prompted us to evaluate alternative or modified CBHI enzymes. Our subsequent expression of a T. reesei-Talaromyces emersonii (Tr-Te) chimeric CBHI, Chaetomium thermophilum CBHI, and Humicola grisea CBHI demonstrated remarkably improved enzymatic activities. Specifically, the purified chimeric Tr-Te CBHI showed a specific activity on Avicel that is comparable to that of the native T. reesei CBHI. Furthermore, the chimeric Tr-Te CBHI also showed significant synergism with EGII and CBHII in degrading cellulosic substrates, using either mixed supernatants or co-cultures of the corresponding Y. lipolytica transformants. The consortia system approach also allows rational volume mixing of the transformant cultures in accordance with the optimal ratio of cellulases required for efficient

  14. Engineering towards a complete heterologous cellulase secretome in Yarrowia lipolytica reveals its potential for consolidated bioprocessing

    DOE PAGESBeta

    Wei, Hui; Wang, Wei; Alahuhta, Markus; Vander Wall, Todd; Baker, John O.; Taylor, Larry E.; Decker, Stephen R.; Himmel, Michael E.; Zhang, Min

    2014-10-16

    Background: Yarrowia lipolytica is an oleaginous yeast capable of metabolizing glucose to lipids, which then accumulate intracellularly. However, it lacks the suite of cellulolytic enzymes required to break down biomass cellulose and cannot therefore utilize biomass directly as a carbon source. Toward the development of a direct microbial conversion platform for the production of hydrocarbon fuels from cellulosic biomass, the potential for Y. lipolytica to function as a consolidated bioprocessing strain was investigated by first conducting a genomic search and functional testing of its endogenous glycoside hydrolases. Once the range of endogenous enzymes was determined, the critical cellulases from Trichodermamore » reesei were cloned into Yarrowia. Results: Initially, work to express T. reesei endoglucanase II (EGII) and cellobiohydrolase (CBH) II in Y. lipolytica resulted in the successful secretion of active enzymes. However, a critical cellulase, T. reesei CBHI, while successfully expressed in and secreted from Yarrowia, showed less than expected enzymatic activity, suggesting an incompatibility (probably at the post-translational level) for its expression in Yarrowia. This result prompted us to evaluate alternative or modified CBHI enzymes. Our subsequent expression of a T. reesei-Talaromyces emersonii (Tr-Te) chimeric CBHI, Chaetomium thermophilum CBHI, and Humicola grisea CBHI demonstrated remarkably improved enzymatic activities. Specifically, the purified chimeric Tr-Te CBHI showed a specific activity on Avicel that is comparable to that of the native T. reesei CBHI. Furthermore, the chimeric Tr-Te CBHI also showed significant synergism with EGII and CBHII in degrading cellulosic substrates, using either mixed supernatants or co-cultures of the corresponding Y. lipolytica transformants. The consortia system approach also allows rational volume mixing of the transformant cultures in accordance with the optimal ratio of cellulases required for efficient