Phanerochaete chrysosporium est un champignon ligninolytique largement étudié pour ses capacités à dégrader la lignine et certains xénobiotiques grâce à un important système d'enzymes extracellulaires. Son génome est entièrement séquencé et constitue un inventaire de séquences protéiques prédites qui a permis la description de nombreuses superfamilles de protéines. Parmi elles, les Glutathion S-transférases sont essentiellement impliquées dans le métabolisme secondaire du champignon. Cependant, malgré les nombreux travaux montrant l'implication de ces enzymes dans la réponse aux stress, le développement cellulaire et plus globalement dans certaines fonctions métaboliques, leurs réelles fonctions restent inconnues à cause de leur grande diversité et le manque de données concernant leurs spécificités catalytiques. P. chrysosporium possède 27 isoformes de GSTs qui se regroupent en 7 classes. Parmi elles, 3 sont étendues chez les champignons saprophytes : les classes Omega, Ure2p et ethérase. Deux membres de la classe Omega ont été caractérisés au niveau biochimique et montrent desspécificités de substrat. En effet, PcGTO1 fait partie d'une nouvelle classe appelée S-glutathionyl-phydroquinone reductase, alors que PcGTO3 est plutôt active avec le phenylacetophenone. La structure tridimensionnelle de PcGTO1 suggère que l'enzyme appartient également à une nouvelle classe structurale que nous avons appelée xi. La deuxième classe majoritaire que nous avons étudiée est la classe des Ure2p qui est composée de 9 isoformes et se regroupent en 2 sous-classes. Trois isoformes ont été étudiées au niveau transcriptionnel, biochimique et physiologique. PcUre2p4 et PcUre2p6 appartenant à la première sous-classe sont spécifiquement exprimés dans des cultures fongiques en présence d'hydrocarbures aromatiques polycycliques et l'activité des protéines recombinantes correspondantes est classique des GSTs à savoir le transfert de glutathion sur un substrat hydrophobe. A l'inverse, PcUre2p1 qui appartient à la deuxième sous-classe est exprimé de manière constitutive au niveau transcriptionnel et la protéine présente une activité thiol transférase comparable aux protéines de la classe Omega. Les analyses physiologiques menées grâce à la complémentation de souche déficience de Saccharomyces cerevisiae ont montré que PcUre2p1, PcUre2p4 et PcUre2p6 n'avaient pas la même fonction que l'isoforme de la levure puisqu'aucune complémentation n'a été détectée en ce qui concerne la résistance au stress ou la régulation du métabolisme azoté. Ces résultats suggèrent que leschampignons, en particulier ceux qui présentent des propriétés saprophytes ont développé des spécificités de fonction de leur GSTs probablement en réponse à des contraintes environnementales. / Phanerochaete chrysosporium is a ligninolytic fungus widely studied because of its capacities to degrade wood and xenobiotics through an extracellular enzymatic system. Its genome has been sequenced and has provided researchers with a complete inventory of the predicted proteins produced by this organism. This has allowed the description of many protein superfamilies. Among them, Glutathione S-transferases (GSTs) constitute a complex and widespread superfamily classified as enzymes of secondary metabolism. However, despite the numerous associations of GSTs with stress responses, cell development and metabolism in various organisms, the functions of these enzymes remain usually evasive mainly due to their high diversity and also to the lack of knowledge about their catalytic specificities. In P. chrysosporium 27 GST isoforms have been highlighted and clustered into seven classes. Among them three are extended in saprophytic fungi: the Omega, the Ure2p and the etherase classes. Two members of the Omega class have been characterized at the biochemical level showing difference in substrate specificities. Indeed, PcGTO1 is member of a new class of Sglutathionyl- p-hydroquinone reductase, while PcGTO3 is rather active with phenylacetophenone. The three-dimensional structure of PcGTO1 confirms the hypothesis not only of a new biological class, but also of a new structural class that we propose to name GST xi. The second extended class we have studied is the Ure2p one. It is composed of nine isoforms in P. chrysosporium and clusters into two subclasses. Three Ure2p class members have been studied in more details at transcriptional, biochemical and physiological levels. PcUre2p4 and PcUre2p6 of the first subclass are specifically expressed in cultures treated with polycyclic aromatic hydrocarbons and the recombinant proteins are active as typical glutathione transferases. By contrast, PcUre2p1, which belongs to the second subclass is constitutively expressed whatever the condition tested and is active with small molecules as substrate, such as proteins from the Omega class. Physiological studies have revealed that these proteins do not have the same function than the Saccharomyce cerevisiae isoform, concerning both the response to oxidative stress and its involvement in the nitrogen catabolite repression. These results suggest that fungi, especially those with saprophytic capabilities, have developed specificities of GST function as an adaptation to environmental constraints

Phanerochaete chrysosporium est un champignon modèle étudié en particulier pour ses capacités ligninolytiques et son aptitude à dégrader un grand nombre de polymères aromatiques toxiques dérivés notamment des hydrocarbures polycycliques. Durant ces processus de dégradation, une multitude de composés hautement réactifs et toxiques pour les cellules vont dans un premier temps être générés puis dégradés ou excrétés hors de la cellule. Le séquençage complet du génome de P. chrysosporium a permis d'identifier plusieurs superfamilles d'enzymes impliquées dans les mécanismes de tolérance à ces composés toxiques. Parmi elles, les glutathion transférases sont présentes au sein de tous les règnes du vivant et constituent une superfamille multigénique jouant un rôle dans la protection cellulaire, le métabolisme secondaire et la détoxication. Cependant, en dépit de nombreuses études réalisées en particulier chez les vertébrés, le rôle joué par ces enzymes dans la détoxication des dérivés aromatiques toxiques est encore inconnu chez les basidiomycètes. L'analyse comparative des séquences de GST présentes au sein des différents règnes du vivant révèle que les GST fongiques ont évolué différemment de leurs orthologues, notamment via l'extension de sous-classes très peu représentées chez les vertébrés. Parmi elles, les GST à cystéine catalytique représentent 30 % de cette superfamille d'enzymes chez P. chrysosporium. Trois isoformes fongiques ainsi qu'une protéine orthologue exprimée chez une bactérie lignivore ont été caractérisées in vitro tant au niveau biochimique que structural. Ces enzymes sont impliquées dans la déglutathionylation d'une grande variété de molécules électrophiles potentiellement toxiques et issues notamment de la dégradation de polymères aromatiques halogénés. La recherche de substrats a permis d'identifier plusieurs classes fonctionnelles, néanmoins l'activité des quatre isoformes s'effectue via l'attaque directe du conjugué glutathionylé par la cystéine catalytique, qui est dans un deuxième temps régénérée par un réducteur. L'analyse comparée prouve également l'existence d'une nouvelle classe structurale et fonctionnelle appelée glutathionyl hydroquinone réductase absente chez les vertébrés. Ces protéines présentent un mode de dimérisation original ainsi que la capacité tout à fait particulière de déglutathionyler les quinones. Ces résultats suggèrent que les champignons ont développé des mécanismes de résistance en réponse à des contraintes environnementales, notamment via l'évolution de familles multigéniques telles que les GST à cystéine catalytique qui sont impliquées dans le métabolisme et la tolérance vis-à-vis d'une grande variété de composés d'origine exogène ou endogène / Phanerochaete chrysosporium is a model fungus well studied for its lignolytic properties towards wood compounds and various toxic aromatic derivatives such as polycyclic aromatic hydrocarbons. These degradation processes lead first to the formation of highly reactive and toxic compounds, which are then catabolized or excreted outside the cell. Genomic data allowed the identification of genes coding for superfamilies of enzymes putatively involved in these tolerance mechanisms. Among them, glutathione transferases are present in all kingdoms and constitute a multigenic superfamily of enzymes involved in cell protection and detoxification. However, although numerous studies have been performed on vertebrate enzymes, the role of these enzymes in the detoxication of toxic aromatic compounds is still unknown in basidiomycetes. The comparative analysis of GST sequences from various kingdoms of life reveals that fungal GSTs have evolved differently from their orthologs, in particular through the expansion of sub-classes poorly represented in vertebrates. Among them, GSTs with a catalytic cysteine represent 30% of this superfamily of enzymes in P. chrysosporium. Three Cys containing fungal isoforms have been characterized at the biochemical and structural levels, including an orthologue from lignolytic bacteria. All these enzymes are involved in deglutathionylation processes using a wide range of aromatic halogenated electrophilic compounds, including potentially toxic derivatives arising from the degradation of halogenated aromatic polymers. This GSTs family can be organized in various functional groups based on their substrate specificities, but still the catalytic process remains the same with the direct attack of the glutathionylated compound by the catalytic cysteine which is then reduced and regenerated. The comparative analysis of three isoforms revealed a new structural and functional class called glutathionyl hydroquinone reductase absent in vertebrates. These proteins exhibit a new mode of dimerization as well as the ability to deglutathionylate quinones. These results suggest that fungi have developed resistance mechanisms in response to environmental stresses, notably through the evolution of multigenic families such as catalytic cysteine bearing GSTs which are likely involved in the metabolism and tolerance towards a wide range of exogenous or endogenous compounds

In this study, the aim was enzymatic xylooligosaccharide production from cotton and sunflower stalks, two of main agricultural residues in Turkey. In first two parts of the study, alkali extracted xylan from both of the stalks was hydrolyzed by commercial xylanases Veron and Shearzyme. The effect of temperature, pH, enzyme and substrate concentrations were investigated to determine optimum enzymatic hydrolysis conditions of xylan. Sunflower and cotton stalk xylans were hydrolyzed by Shearzyme more efficiently than Veron under the conditions studied. Shearzyme produced different product profiles containing xylobiose (X2), xylotriose (X3), xylotetrose (X4) and xylopentose (X5) from cotton and sunflower stalk xylan. On the other hand, Veron hydrolyzed both xylan types to produce X2, X3, X5, X6 and larger xylooligosaccharides without any change in product profiles. In the third part of the study, home produced xylanase from Bacillus pumilus SB-M13, was also investigated for the production of xylooligosaccharides from both cotton and sunflower stalk xylan. The main products obtained by hydrolysis of both substrates by pure B. pumilus xylanase were X5 and X6, while crude B. pumilus xylanase generated X4 and X5 as the main products. Xylooligosaccharide production from pretreated cotton stalk without alkali extraction of xylan was the final part of the study. Three different pretreatment methods including biomass pretreatment by Phanerochaete chrysosporium fermentation, cellulase pretreatment and hydrothermal pretreatment were investigated to break down complex lignocellulosic structure of cotton stalk to improve the subsequent enzymatic hydrolysis of xylan in pretreated cotton stalk for xylooligosaccharide production. However, xylooligosaccharide was not effectively produced from pretreated cotton stalk. Shearzyme inhibiton was observed after all the pretreatment methods during further hydrolysis of pretreated cotton stalk probably due to production of inhibitory compounds of the enzyme.

RNA-binding proteins (RBP) shuttle between cellular compartments either constitutively or in response to stress and regulate localization, translation and turn over of mRNAs. In our laboratory, cytosolic proteome map of Phanerochaete chrysosporium was established and upon Pb exposure, the changes in cytosolic protein expressions were determined. The identified RBPs were a newly induced polyadenylate-binding protein (RRM superfamily) as well as two up-regulated proteins, namely splicing factor RNPS1 and ATP-dependent RNA helicase, all being very important candidates of post-transcriptional control in response to stress. This finding inspired us to conduct Real Time PCR studies in order to have a better understanding of the changes in the expression of corresponding genes at mRNA level in response to Pb exposure, thus the present study aims at examining the effect of lead exposure on the transcript levels of the genes coding for ATP-dependent RNA helicase, splicing factor RNPS1 and polyadenylate binding protein. As shown via expression analysis based on Real Time PCR, the mRNA level of splicing factor RNPS1 showed 2.68, 2.62 and 4.86 fold increases in a dose-dependent manner when the cells were grown for 40 h in the presence of 25, 50 and 100 &micro / M Pb, repectively. ATP-dependent RNA helicase mRNA level showed no significant increase in response to 25 &micro / M Pb exposure while increased 2 and 1.84 fold in response to 50 and 100 &micro / M Pb, respectively. Polyadenylate binding protein mRNA levels revealed no significant increase when exposed to 25, 50 and 100 &micro / M Pb. As to the mRNA dynamics as a function of duration of lead exposure, the mRNA level of this protein showed 2.54-fold increase upon 1 h exposure to 100 &micro / M Pb. Splicing factor RNPS1 mRNA level showed a significant increase of 19.22 fold at 2nd h of 50 &micro / M Pb exposure. Expression level of ATP-dependent RNA helicase was not affected by the time of exposure to Pb.

Biosorption is a phenomenon involving the mechanisms that basically mediate heavy metal tolerance of microorganisms as well as sequestration of heavy metals from environment. Different classes of microorganisms have different biosorption capacities, as a result of the differences in composition and types of functional groups found on cell surfaces. The present study was undertaken to identify the molecular mechanisms for lead [Pb(II)] biosorption in the white-rot fungus, Phanerochaete chrysosporium. The methodology involved selective blocking of the functional groups known to participate in heavy metal biosorption and allowed us to determine their relative roles in Pb (II) biosorption in this organism. The relative concentrations of the Pb (II) sorbed from the aqueous environment and Mg2+ and Ca2+ ions released to the aqueous environment were measured and compared with both native and chemically-modified biomasses by using atomic absorption spectroscopy. Fourier-Transform Infrared (FTIR) spectroscopy technique was used to monitor and analyze the molecular-level changes in both native and chemically modified cell surfaces upon Pb (II) exposure. Interactions of Pb (II) with the biomass surface was determined by observing the changes in wavenumber and absorbance of NH stretching and Amide I bands arising from the amine groups and C=O stretching band arising from the carboxyl groups. The roles of phosphate groups and lipids were also investigated. Carboxyl groups seemed to be the most important functional groups for Pb (II) biosorption in P. chrysosporium, since the biosorption capacity dramatically decreased (by 92.8 %) in carboxyl groups-blocked biomass. Amine groups were found to play a secondary and minor role in Pb (II) biosorption, only a slight decrease (6 %) in Pb (II) biosorption was detected with amine groups-blocked biomass. Blocking of phosphate groups provided a small increase in biosorptive capacity and did not appear to have much significant role in biosorption. Upon chemical treatment with acetone to extract lipids of the cell surfaces, an increase of 20.3 % in the Pb (II) biosorptive capacity was determined. It was concluded that carbonyl and carboxyl groups of chitin and glucan are the major sites and ion exchange via these groups is the main mechanism for Pb (II) biosorption in P. chrysosporium.

Thesis (MTech (Chemical Engineering))--Cape Technikon, 2000. / The large quantities of wood chips produced at mines from damaged underground timber contain gold that cannot be completely recovered by cyanidation. A fungus that can degrade a portion of the wood matrix will allow the gold that was previously locked up, to come into contact with the cyanide solution during beneficiation, thereby improving recoveries. The fungus Phanerochaete chrysosporium produces enzymes that use the organic compounds found in lignin as substrate. Consequently, the fungus is able to selectively break down lignin, which is one of the major components of wood. Chips sampled from Vaal Reef Mine contained between 2 and 5 mg/kg gold. The main source of gold in the chips was determined to be impregnated gold-bearing ore and discrete gold particles. Direct cyanidation resulted in around 60 per cent recovery prior to biological treatment. Despite relatively high weight losses caused to the chips as a result of treatment with Phanerochaete chrysosporium gold recovery only increased 10 per cent after 4 weeks treatment compared to direct recovery without treatment.

Le sol est une interface complexe entre tous les compartiments de l'environnement. Leur pollution participe à la diffusion de nombreux polluants. Les polychlorobiphényles sont des molécules toxiques persistantes dans l'environnement. Largement utilisés notamment dans les huiles diélectriques, ils contaminent aujourd'hui de nombreux sols industriels. Le traitement thermique de ces sols est très onéreux et peut entraîner l'émission de dioxines. L'objectif de ce travail est d'étudier un procédé de traitement biologique pour la dégradation des PCB dans les sols. Une dégradation biologique de PCB a été observée en présence de deux organismes cultivés, Burkholderia xenovorans et Phanerochaete chrysosporium, confirmant leur potentiel technologique en condition aérobie. En condition anaérobie, une communauté microbienne présentant la capacité de dégrader les PCB a été développé. Une étude de la diversité du gène ADNr 16S au sein de cette communauté a permis d'identifier les espèces présentes dans cette communauté. Une analyse de cycle de vie évalue les performances environnementales de deux procédés de traitement de sols contaminés par des PCB, l'un thermique, l'autre biologique. Cette analyse permet de quantifier l’avantage environnemental du procédé biologique sur son concurrent thermique. / Soil is a complex interface between all compartments of the environment. Their pollution contributes to the spread of many pollutants. PCBs are persistent toxic compounds in the environment. Widely used especially in dielectric oils, they now contaminate many industrial floors. Heat treatment of these soils is very expensive and can cause the emission of dioxins. The objective of this work is to study a biological treatment process for the degradation of PCBs in soils. Biological degradation of PCBs has been observed in the presence of two cultured organisms, Burkholderia xenovorans and Phanerochaete chrysosporium, confirming their technological potential under aerobic conditions. Under anaerobic conditions, a microbial community with the ability to degrade PCBs was developed. A study of the diversity of 16S rDNA gene within this community has identified the species in this community. An analysis of life cycle assess the environmental performance of two methods for treating soils contaminated with PCBs, one thermal and one biological. This analysis quantifies the environmental benefit of the biological process compared with the heat treatment

To gain a better understanding of the mechanisms by which the white-rot decay fungus Phanerochaete chrysosporium regulates selective delignification versus simultaneous decay, differential gene expression of its two key enzymes were measured over the course of aspen and birch wood decay tests. The type of decay was determined by differential staining and scanning electron microscopy. Real-time qPCR assessed gene expression of four lignin peroxidase genes and two manganese peroxidase genes at each stage of decay. Differential expression was significant in the mnp2 gene between aspen and birch decay tests during incipient decay; abundant expression of mnp2 in aspen corresponded to early initiation of selective delignification, whereas birch underwent initial simultaneous decay in the absence of abundant mnp2 expression. The lipC gene was the most abundantly expressed lip gene at all time points in both wood species and likely plays an important role in regulating wood decay.

Dans les écosystèmes forestiers, la décomposition du bois est un processus majeur, notamment impliqué dans le cycle du carbone et des nutriments. Les champignons basidiomycètes saprotrophes, incluant les pourritures blanches, sont les principaux agents de cette décomposition dans les forêts tempérées. Bien que peu étudiées, des communautés bactériennes sont également présentes dans le bois en décomposition et cohabitent avec ces communautés fongiques. L'impact des interactions bactéries-champignons sur le fonctionnement d'une niche écologique a été décrit dans de nombreux environnements. Cependant, leur rôle dans le processus de décomposition du bois n'a été que très peu investigué. A partir d'expériences en microcosme et en utilisant une approche non cultivable, il a été démontré que la présence du champignon Phanerochaete chrysosporium influençait significativement la structure et la diversité des communautés bactériennes associées au processus de décomposition du hêtre (Fagus sylvatica). Par une approche cultivable, cet effet mycosphère a été confirmé, se traduisant par une augmentation de la densité des communautés bactériennes en présence du champignon ainsi que par une modification de la diversité fonctionnelle de ces communautés. Enfin, une approche polyphasique a été développée, combinant l'analyse des propriétés physico-chimiques du bois et des activités enzymatiques extracellulaires. Les résultats de cette expérience ont révélé que l'association de P. chrysosporium avec une communauté bactérienne issue de la mycosphère de ce dernier aboutissait à une dégradation plus importante du matériau bois par rapport à la dégradation par le champignon seul, démontrant pour la première fois des interactions bactéries-champignons synergiques dans le bois en décomposition / Wood decomposition is an important process in forest ecosystems in terms of their carbon and nutrient cycles. In temperate forests, saprotrophic basidiomycetes such as white-rot fungi are the main wood decomposers. While they have been less studied, bacterial communities also colonise decaying wood and coexist with these fungal communities. Although the impact of bacterial-fungal interactions on niche functioning has been highlighted in a wide range of environments, little is known about their role in wood decay. Based on microcosm experiments and using a culture-independent approach, we showed that the presence of the white-rot fungus Phanerochaete chrysosporium significantly modified the structure and diversity of the bacterial communities associated with the degradation of beech wood (Fagus sylvatica). Using a culture-dependent approach, it was confirmed that in the presence of the fungus the mycosphere effect resulted in increased bacterial abundance and modified the functional diversity of the fungal-associated bacterial communities. Lastly, a polyphasic approach simultaneously analysing wood physicochemical properties and extracellular enzyme activities was developed. This approach revealed that P. chrysosporium associated with a bacterial community isolated from its mycosphere was more efficient in degrading wood compared to the fungus on its own, highlighting for the first time synergistic bacterial-fungal interactions in decaying wood

In this study, the soluble protein fraction of P. chrysosporium grown under standard conditions was analyzed by using 2D-PAGE approach and a 2-D reference map was constructed. 910 spots could be separated and detected on Coomassie-stained 2-D gels by the help of Delta2D image analysis software. 720 spots could be cut from the master gel and were subjected to MALDI-TOF MS analysis followed by MASCOT search. A total of 517 spots out of 720 were assigned to specific accession numbers from the P. chrysosporium genome database. Further analysis of the data revealed 314 different gene products (distinct ORFs). The theoretical pI and MW values were plotted against the experimental migration distances. Results indicated the existence of 124 protein spots whose horizontal migration differed significantly from the expected migration according to the calculated pI values and 52 spots with an apparent molecular weight that is significantly different from their theoretical molecular weight. While protein modification could be predicted by these analyses, the main support was the presence of multiple spots of the same gene product. As much as 118 ORFs yielded multiple spots on the master gel, corresponding to 37.5% of the all distinct ORFs identified in this work. The relative abundance of each of the 517 identified polypeptides was calculated in terms of spot intensity. The majority of the most abundant proteins were found to be housekeeping ones. When the relative distribution of the proteins into four main functional categories was taken into consideration, &ldquo / Metabolism&rdquo / appeared the most important category with a share of 50.6% among identified proteins. However, among the functional classes, &ldquo / Posttranslational modifications, protein turnover, chaperones&rdquo / which is listed under the main category &ldquo / Cellular Processing and Signalling&rdquo / was represented by the highest number (104) of the identified proteins. Only 6 of the proteins listed in this study were assigned to hypothetical proteins. Out of the 314 identified gene products shown in P. chrysosporium, 29 were predicted to have a signal peptide sequence according to the SignalP algorithm. By making a WoLF PSORT search, subcellular localization of the proteins was predicted. Accordingly, 147 of the proteins were predicted to be located in cytoplasm. The phosphorylated proteins of P. chrysosporium were detected by ProQ phosphoprotein staining of the 2-D gel. 380 out of 910 distinct protein spots (40%) were found to be phosphorylated in exponentially growing cells of P. chrysosporium. Of these spots, 96 could be matched to the identified proteins.