Browsing by Author "Vicuna, Rafael"

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    Cloning and functional characterization of the gene encoding the transcription factor Ace1 in the basidiomycete Phanerochaete chrysosporium
    (2006) Polanco, Ruben; Canessa, Paulo; Rivas, Alexis; Larrondo, Luis F.; Lobos, Sergio; Vicuna, Rafael
    In this report we describe the isolation and characterization of a gene encoding the transcription factor Ace 1 (Activation protein of cup 1 Expression) in the white rot fungus Phanerochaete chrysosporium. Pc-ace 1 encodes a predicted protein of 633 amino acids containing the copper-fist DNA binding domain typically found in fungal transcription factors such as Ace 1 Mac 1 and Haa 1 from Saccharomyces cerevisiae. The Pc-ace 1 gene is localized in Scaffold 5, between coordinates 220841 and 222983. A S. cerevisiae ace 1 null mutant strain unable to grow in high-copper medium was fully complemented by transformation with the cDNA of Pc-ace 1. Moreover, Northern blot hybridization studies indicated that Pc-ace 1 cDNA restores copper inducibility of the yeast cup 1 gene, which encodes the metal-binding protein metallothionein implicated in copper resistance. To our knowledge, this is first report describing an Ace 1 transcription factor in basidiomycetes.
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    Effect of manganese on the secretion of manganese-peroxidase by the basidiomycete Ceriporiopsis subvermispora
    (2010) Mancilla, Rodrigo A.; Canessa, Paulo; Manubens, Augusto; Vicuna, Rafael
    The ligninolytic machinery of the widely used model fungus Ceriporiopsis subvermispora includes the enzymes manganese-peroxidase (MnP) and laccase (Lcs). In this work the effect of Mn(II) on the secretion of MnP was studied. Cultures grown in the absence of Mn(II) showed high levels of mnp transcripts. However, almost no MnP enzyme was detected in the extracellular medium, either by enzymatic activity assays or Western blot hybridizations. In the corresponding mycelia, immuno-electron microscopy experiments showed high levels of MnP enzyme within intracellular compartments. These results suggest that in addition to its well-known effect on transcription regulation of mnp genes, manganese influences secretion of MnP to the extracellular medium. Experiments carried out in the presence of cycloheximide confirmed that the metal is required to secrete MnP already synthesized and retained within the cell. (C) 2010 Elsevier Inc. All rights reserved.
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    Expression of genes encoding laccase and manganese-dependent peroxidase in the fungus Ceriporiopsis subvermispora is mediated by an ACE1-like copper-fist transcription factor
    (2009) Miguel Alvarez, Jose; Canessa, Paulo; Mancilla, Rodrigo A.; Polanco, Ruben; Santibanez, Paulina A.; Vicuna, Rafael
    The effect of copper on the expression of genes encoding the ligninolytic enzymes laccase (ICS) and manganese peroxidase (mnp) in Ceriporiopsis subvermispora was evaluated. This metal increased transcript levels of lcs, mnp1 and mnp2. This finding was not unexpected in the case of lcs, since its promoter contains a putative ACE element. Originally characterized in the yeast Saccharomyces cerevisiae, ACE is the target sequence of the ACE1 copper-responsive transcription factor in this microorganism. Analysis of the promoter regions of mnp genes revealed the presence of formerly unnoticed ACE elements. Based on the ace1 gene from Phanerochaete chrysosporium, we isolated and characterized an ACE1-like transcription factor from C. subvermispora (Cs-ACE1) through complementation of a S. cerevisiae ace1 Delta strain. Surprisingly, ACE1 factors from both basidiomycetes exhibit substantial differences, not only structurally but also in their ability to complement the aforementioned yeast strain. Specific binding of Cs-ACE1 to its cognate DNA sequence was confirmed by electrophoretic mobility-shift assays. (C) 2008 Elsevier Inc. All rights reserved.
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    Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion
    (2009) Martinez, Diego; Challacombe, Jean; Morgenstern, Ingo; Hibbett, David; Schmoll, Monika; Kubicek, Christian P.; Ferreira, Patricia; Ruiz-Duenas, Francisco J.; Martinez, Angel T.; Kersten, Phil; Hammel, Kenneth E.; Wymelenberg, Amber Vanden; Gaskell, Jill; Lindquist, Erika; Sabat, Grzegorz; BonDurant, Sandra Splinter; Larrondo, Luis F.; Canessa, Paulo; Vicuna, Rafael; Yadav, Jagjit; Doddapaneni, Harshavardhan; Subramanian, Venkataramanan; Pisabarro, Antonio G.; Lavin, Jose L.; Oguiza, Jose A.; Master, Emma; Henrissat, Bernard; Coutinho, Pedro M.; Harris, Paul; Magnuson, Jon Karl; Baker, Scott E.; Bruno, Kenneth; Kenealy, William; Hoegger, Patrik J.; Kuees, Ursula; Ramaiya, Preethi; Lucash, Susan; Salamov, Asaf; Shapiro, Harris; Tu, Hank; Chee, Christine L.; Misra, Monica; Xie, Gary; Teter, Sarah; Yaver, Debbie; James, Tim; Mokrejs, Martin; Pospisek, Martin; Grigoriev, Igor V.; Brettin, Thomas; Rokhsar, Dan; Berka, Randy; Cullen, Dan
    Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome, and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exo-cellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding to many hemicellulases and to a single putative beta-1-4 endoglucanase were expressed at high levels relative to glucose-grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Also up-regulated during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H2O2. These observations are consistent with a biodegradative role for Fenton chemistry in which Fe( II) and H2O2 react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons with the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin was lost.
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    Hydroquinone and H2O2 differentially affect the ultrastructure and expression of ligninolytic genes in the basidiomycete Ceriporiopsis subvermispora
    (2009) Amoroso, Alejandro; Mancilla, Rodrigo A.; Gonzalez, Bernardo; Vicuna, Rafael
    The biodegradation of lignin is a highly oxidative process in which various oxidases and peroxidases play a major role. During lignin decay, the generation of aromatic compounds and reactive oxygen species leads to oxidative stress. In this work, the effect of the oxidative compounds H2O2 and hydroquinone in the ligninolytic fungus Ceriporiopsis subvermispora was studied, both at the ultrastructural and at the transcriptional level. Transmission electron microscopy revealed the presence of microvesicles and extensive cytoplasm degeneration after incubation with hydroquinone, but not with H2O2. Studies of the intracellular redox state of the fungus showed that hydroquinone causes a transient decrease in the reduced glutathione/oxidized glutathione (GSH/GSSG) ratio and an increase in the glutathione-S-transferase mRNA levels. These results suggest that hydroquinone produces oxidative stress in this microorganism. On the other hand, it was observed that hydroquinone, but not H2O2, affects Mn-dependent peroxide and laccase transcripts levels. We propose that the mechanism by which the fungus reacts against oxidative stress contributes to its selectivity toward lignin during wood decay.
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    Hypolithic Cyanobacteria Supported Mainly by Fog in the Coastal Range of the Atacama Desert
    (2011) Azua-Bustos, Armando; Gonzalez-Silva, Carlos; Mancilla, Rodrigo A.; Salas, Loreto; Gomez-Silva, Benito; McKay, Christopher P.; Vicuna, Rafael
    The Atacama Desert is one of the driest places on Earth, with an arid core highly adverse to the development of hypolithic cyanobacteria. Previous work has shown that when rain levels fall below similar to 1 mm per year, colonization of suitable quartz stones falls to virtually zero. Here, we report that along the coast in these arid regions, complex associations of cyanobacteria, archaea, and heterotrophic bacteria inhabit the undersides of translucent quartz stones. Colonization rates in these areas, which receive virtually no rain but mainly fog, are significantly higher than those reported inland in the hyperarid zone at the same latitude. Here, hypolithic colonization rates can be up to 80%, with all quartz rocks over 20 g being colonized. This finding strongly suggests that hypolithic microbial communities thriving in the seaward face of the Coastal Range can survive with fog as the main regular source of moisture. A model is advanced where the development of the hypolithic communities under quartz stones relies on a positive feedback between fog availability and the higher thermal conductivity of the quartz rocks, which results in lower daytime temperatures at the quartz-soil interface microenvironment.
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    Structure and transcriptional impact of divergent repetitive elements inserted within Phanerochaete chrysosporium strain RP-78 genes
    (2007) Larrondo, Luis F.; Canessa, Paulo; Vicuna, Rafael; Stewart, Philip; Vanden Wymelenberg, Amber; Cullen, Dan
    We describe the structure, organization, and transcriptional impact of repetitive elements within the lignin-degrading basidiomycete, Phanerochaete chrysosporium. Searches of the P. chrysosporium genome revealed five copies of pce1, a similar to 1,750-nt non-autonomous, class II element. Alleles encoding a putative glucosyltransferase and a cytochrome P450 harbor pce insertions and produce incomplete transcripts. Class I elements included pcret1, an intact 8.14-kb gypsy-like retrotransposon inserted within a member of the multicopper oxidase gene family. Additionally, we describe a complex insertion of nested transposons within another putative cytochrome P450 gene. The disrupted allele lies within a cluster of > 14 genes, all of which encode family 64 cytochrome P450s. Components of the insertion include a disjoint copia-like element, pcret3, the pol domain of a second retroelement, pcret2, and a duplication of an extended ORF of unknown function. As in the case of the pce elements, pcret1 and pcret2/3 insertions are confined to single alleles, transcripts of which are truncated. The corresponding wild-type alleles are apparently unaffected. In aggregate, P. chrysosporium harbors a complex array of repetitive elements, at least five of which directly influence expression of genes within families of structurally related sequences.
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    The copper-dependent ACE1 transcription factor activates the transcription of the mco1 gene from the basidiomycete Phanerochaete chrysosporium
    (2008) Canessa, Paulo; Alvarez, Jose Miguel; Polanco, Ruben; Bull, Paulina; Vicuna, Rafael
    We have previously identified and functionally characterized the transcription factor ACE1 (Pc-ACE1) from Phanerochaete chrysosporium. In Saccharomyces cerevisiae, ACE1 activates the copper-dependent transcription of target genes through a DNA sequence element named ACE. However, the possible target gene(s) of Pc-ACE1 were unknown. An in silico search led to the identification of putative ACE elements in the promoter region of mco1, one of the four clustered genes encoding multicopper oxidases (MCOs) in P. chrysosporium. Since copper exerts an effect at the transcriptional level in MCOs from several organisms, in this work we analysed the effect of copper on transcript levels of the clustered MCO genes from P. chrysosporium, with the exception of the transcriptionally inactive mco3. Copper supplementation of cultures produced an increment in transcripts from genes mco1 and mco2, but not from mco4. Electrophoretic mobility-shift assays revealed that Pc-ACE1 binds specifically to a probe containing one of the putative ACE elements found in the promoter of mco1. In addition, using a cell-free transcription system, we showed that in the presence of cuprous ion, Pc-ACE1 induces activation of the promoter of mco1, but not that of mco2.