The role of fungal nitrogen cycling in soil systems

Pilze spielen eine zentrale Rolle im Ökosystem Erde. Dennoch ist nach wie vor wenig über die Pilzdiversität in Böden und die spezifischen Funktionen einzelner phylogenetischer Gruppen bekannt. Vier verschiedene Ackerböden (Maissau, Niederschleinz, Purkersdorf, Tulln) sowie eine Graslanderde (Riederberg) wurden auf ihre Pilzdiversität untersucht. Hierfür wurden ein Kultivierungsansatz sowie eine direkte molekularbiologische Methode verwendet. Die Pilze wurden aufgrund ihrer DNS-Sequenz der ITS/LSU ribosomalen RNS Genregion identifiziert. Es wurden 61 verschiedene Pilzarten isoliert, die hauptsächlich zum Phylum Ascomycota gehören, darunter zB Fusarium spp., Penicillium spp., Trichoderma spp., sowie bislang unbeschriebene Arten. Im Rahmen des kultivierungsunabhängigen Ansatzes wurde eine Klon-Bibliothek erstellt. Danach wurde eine RFLP-Analyse durchgeführt. Auch hier dominierten Spezies der Ascomycota, die Artenzusammensetzung unterschied sich jedoch erheblich vom Kultivierungsansatz. Vertreter der Basidiomycota traten hierbei öfter und in allen fünf Bibliotheken auf. Die Zusammensetzung der Pilzgemeinschaften wies deutliche Unterschiede innerhalb der einzelnen Klon-Bibliotheken auf. Die Artenzahl (species richness) wurde mit verschiedenen Methoden berechnet. Chao2 zeigte für die untersuchten Böden eine gute Abdeckung der erwarteten Pilz-Artenzahl. Lediglich 8,6% der identifizierten Arten konnten mit beiden Verfahren detektiert werden. Die Mehrzahl der Arten (62,6%) wurde mittels des kultivierungs-unabhängigen Verfahrens ermittelt, während 28,8% ausschließlich mit Hilfe des Kultivierungsansatzes identifiziert wurden. Hierdurch wird deutlich, dass beide Methoden sich vielmehr ergänzen, als überlappen. Die vom Grasland Riederberg entstammende Klon-Bibliothek enthielt die größte Diversität und wies als einzige das neu beschriebene Ascomycota Subphylum Soil Clone Group I (SCGI) auf, was eventuell darauf hindeuten könnte, dass SCGI-Pilze bevorzugt an unberührten Standorten vorkommen. Die Maissau Klon-Bibliothek zeigte den geringsten Artenreichtum und auch andere spezifische Eigenschaften grenzen diesen Standort von den anderen ab. Der zweite Teil dieser Studie befasste sich mit der Erforschung der Rolle von Pilzen in N-Transformationsprozessen. Hierfür wurden die Gene, welche für die pilzliche Nitratreduktase kodieren (niaD) untersucht. Dabei stand die Erstellung eines Sequenz-Sets im Vordergrund, welches partielle niaD Sequenzen von Isolaten sowie unkultivierten Pilzen enthält. Auf Grund dieser Erkenntnis soll es möglich werden, transkriptionelle Aktivitäten von Pilz-Populationen, welche dieses Gen exprimieren, unter verschiedenen Umweltbedingungen zu beobachten. Zusätzlich wurde Fluoreszenz in situ Hybridisierung (FISH) mit dem Modelorganismus Aspergillus nidulans durchgeführt. Im Rahmen eines Vorversuchs wurden die FISH Parameter optimiert und die Detektion der niaD mRNA angestrebt. Unsere viel versprechenden Ergebnisse stellen die Basis für zukünftige Studien dar, welche die Expression der Pilz-Nitratreduktase unter verschiedenen Umweltbedingungen erforschen sollen.Fungi fulfil a range of important ecological functions. However, there is poor understanding of soil fungal community diversity and the specific roles of individual phylogenetic groups present in the environment. Fungal diversity of four different agricultural soils (Maissau, Niederschleinz, Purkersdorf, Tulln) and one grassland soil (Riederberg) was examined by a culture-dependent and a culture-independent approach. Identification of fungi was accomplished by DNA sequence analysis of the ITS/LSU of the ribosomal RNA gene region. A diverse set of 61 different species of mainly Ascomycota was cultivated, including Fusarium spp., Penicillium spp., Trichoderma spp., as well as previously undescribed species. In the course of the culture-independent approach, clone libraries were constructed followed by RFLP-analysis. Again, Ascomycota predominated all libraries, whereas a highly different list of species was obtained compared to the cultivation approach. Basidiomycota occured more often and were distributed over all libraries. Generally, all clone libraries differed in their fungal community composition from each other. Species richness estimator Chao2 analysis of all clone libraries revealed a good coverage of the expected fungal species richness from agricultural soils. Only 8.6 % of the identified species were detected by both methods. The majority of species (62.6 %) was detected by the culture-independent procedure, while 28.8 % were exclusively found by the culturing-approach. The analysis clearly demonstrates that both methods are complementary rather than overlapping. The clone library derived from the Riederberg grassland soil harboured the highest diversity and exclusively contained the newly described ascomycetous subphylum Soil Clone Group I (SCGI), maybe suggesting a preference of SCGI-fungi for undisturbed sites. The Maissau clone library showed the lowest species richness, and other specific characteristics contrasting with the other clone libraries. The second part of the study dealt with the exploration of the role of fungi in N-transformation processes. Therefore, fungal nitrate reductase genes (niaD) were investigated by the generation of a set of partial niaD sequences from isolates as well as from uncultured soil fungi. This knowledge will facilitate the monitoring of transcriptional activities of fungal populations expressing the niaD gene under different environmental settings. Additionally, fluorescence in situ hybridisation (FISH) was performed using the model organism Aspergillus nidulans. During a preliminary test, the optimisation of FISH conditions for the detection of niaD mRNA was attempted. Our promising results form the basis for future studies, dedicated to the investigation of fungal niaD expression under different conditions

The Genomes of the Fungal Plant Pathogens Cladosporium fulvum and Dothistroma septosporum Reveal Adaptation to Different Hosts and Lifestyles But Also Signatures of Common Ancestry.

We sequenced and compared the genomes of the Dothideomycete fungal plant pathogensCladosporium fulvum (Cfu) (syn. Passalora fulva) and Dothistroma septosporum (Dse) that are closely related phylogenetically, but have different lifestyles and hosts. Although both fungi grow extracellularly in close contact with host mesophyll cells, Cfu is a biotroph infecting tomato, while Dse is a hemibiotroph infecting pine. The genomes of these fungi have a similar set of genes (70% of gene content in both genomes are homologs), but differ significantly in size (Cfu \u3e61.1-Mb; Dse 31.2-Mb), which is mainly due to the difference in repeat content (47.2% in Cfu versus 3.2% in Dse). Recent adaptation to different lifestyles and hosts is suggested by diverged sets of genes. Cfu contains an α-tomatinase gene that we predict might be required for detoxification of tomatine, while this gene is absent in Dse. Many genes encoding secreted proteins are unique to each species and the repeat-rich areas in Cfu are enriched for these species-specific genes. In contrast, conserved genes suggest common host ancestry. Homologs of Cfu effector genes, including Ecp2 and Avr4, are present in Dse and induce a Cf-Ecp2- and Cf-4-mediated hypersensitive response, respectively. Strikingly, genes involved in production of the toxin dothistromin, a likely virulence factor for Dse, are conserved in Cfu, but their expression differs markedly with essentially no expression by Cfu in planta. Likewise, Cfu has a carbohydrate-degrading enzyme catalog that is more similar to that of necrotrophs or hemibiotrophs and a larger pectinolytic gene arsenal than Dse, but many of these genes are not expressed in planta or are pseudogenized. Overall, comparison of their genomes suggests that these closely related plant pathogens had a common ancestral host but since adapted to different hosts and lifestyles by a combination of differentiated gene content, pseudogenization, and gene regulation

Community profiling and gene expression of fungal assimilatory nitrate reductases in agricultural soil

Although fungi contribute significantly to the microbial biomass in terrestrial ecosystems, little is known about their contribution to biogeochemical nitrogen cycles. Agricultural soils usually contain comparably high amounts of inorganic nitrogen, mainly in the form of nitrate. Many studies focused on bacterial and archaeal turnover of nitrate by nitrification, denitrification and assimilation, whereas the fungal role remained largely neglected. To enable research on the fungal contribution to the biogeochemical nitrogen cycle tools for monitoring the presence and expression of fungal assimilatory nitrate reductase genes were developed. To the ∼100 currently available fungal full-length gene sequences, another 109 partial sequences were added by amplification from individual culture isolates, representing all major orders occurring in agricultural soils. The extended database led to the discovery of new horizontal gene transfer events within the fungal kingdom. The newly developed PCR primers were used to study gene pools and gene expression of fungal nitrate reductases in agricultural soils. The availability of the extended database allowed affiliation of many sequences to known species, genera or families. Energy supply by a carbon source seems to be the major regulator of nitrate reductase gene expression for fungi in agricultural soils, which is in good agreement with the high energy demand of complete reduction of nitrate to ammonium

New glucuronoyl esterases for wood processing

The development of new wood-based materials is of great interest to the forest industry. Wood tissue is composed of a complex biopolymer mixture containing cellulose, hemicellulose and lignin. Covalent bonds between lignin and different polysaccharides form closely associated structures known as lignin-carbohydrate complexes (LCCs). As a result, the successful extraction and separation of wood polymers poses a major challenge for materials biorefinery concepts. Enzymes that target lignin-carbohydrate (LC) bonds are especially useful for biorefinery applications as they can facilitate the isolation of individual wood components in combination with mild chemical treatments.The main LCCs present in wood are believed to be esters, benzyl ethers and phenyl glycosides [1,2]. Glucuronoyl esterases (GEs) have been proposed to degrade ester bonds between glucuronic acids in xylans and lignin alcohols. GEs belong to the carbohydrate esterase (CE) 15 family and are present in the genomes of a wide range of fungi and bacteria. The aims of our study were to characterize new GE enzymes, to investigate their capacity in disconnecting hemicellulose from lignin and to apply them in the extraction process. Selected candidate genes encoding novel GEs from a diverse range of filamentous fungi were produced in the eukaryotic enzyme production host Pichia pastoris. Purified enzymes were tested on model substrates as well as LCC fractions and their applicability in wood processing is investigated.AcknowledgementsResearch on glucuronoyl esterases at Chalmers is funded by the Knut and Alice Wallenberg Foundation. Selection of candidate genes was performed in the context of OPTIBIOCAT (FP7 KBBE. 2013.3.3-04), which is gratefully acknowledged.Literature1. Balakshin MY, et at. (2007) MWL fraction with a high concentration of lignin-carbohydrate linkages: Isolation and 2D NMR spectroscopic analysis. Holzforschung 61:1–7.2. Watanabe T (1995) Important properties of lignin-carbohydrates complexes (LCCs) in environmentally safe paper making. Trends in Glycoscience and Glycotechnology 7:57–68

Novel glucuronoyl esterases for wood processing

Wood tissue constitutes of a tight network of biopolymers. Covalent linkages between lignin and carbohydrates hamper efficient extraction of intact wood polymers. Selective cleavage of lignin-carbohydrate (LC) bonds by specific enzymes provides an aid for wood processing using mild conditions. Glucuronoyl esterases (GE) are presumed to target LC ester bonds between lignin alcohols and glucuronic acid residues of xylan. In this study we recombinantly produced novel fungal GE enzymes and characterized them using model substrates. The application of these esterases on native LCC fractions will provide insight into their biological function and their potential use in extraction processes

Characterisation of three fungal glucuronoyl esterases on glucuronic acid ester model compounds

The glucuronoyl esterases (GEs) that have been identified so far belong to family 15 of the carbohydrate esterases in the CAZy classification system and are presumed to target ester bonds between lignin alcohols and (4-O-methyl-)d-glucuronic acid residues of xylan. Few GEs have been cloned, expressed and characterised to date. Characterisation has been done on a variety of synthetic substrates; however, the number of commercially available substrates is very limited. We identified novel putative GEs from a wide taxonomic range of fungi and expressed the enzymes originating from Acremonium alcalophilum and Wolfiporia cocos as well as the previously described PcGE1 from Phanerochaete chrysosporium. All three fungal GEs were active on the commercially available compounds benzyl glucuronic acid (BnGlcA), allyl glucuronic acid (allylGlcA) and to a lower degree on methyl glucuronic acid (MeGlcA). The enzymes showed pH stability over a wide pH range and tolerated 6-h incubations of up to 50 degrees C. Kinetic parameters were determined for BnGlcA. This study shows the suitability of the commercially available model compounds BnGlcA, MeGlcA and allylGlcA in GE activity screening and characterisation experiments. We enriched the spectrum of characterised GEs with two new members of a relatively young enzyme family. Due to its biotechnological significance, this family deserves to be more extensively studied. The presented enzymes are promising candidates as auxiliary enzymes to improve saccharification of plant biomass

A glucuronoyl esterase from Acremonium alcalophilum cleaves native lignin-carbohydrate ester bonds

The Glucuronoyl esterases (GE) have been proposed to target lignin-carbohydrate (LC) ester bonds between lignin moieties and glucuronic acid side groups of xylan, but to date, no direct observations of enzymatic cleavage on native LC ester bonds have been demonstrated. In the present investigation, LCC fractions from spruce and birch were treated with a recombinantly produced GE originating from Acremonium alcalophilum (AaGE1). A combination of size exclusion chromatography and 31P NMR analyses of phosphitylated LCC samples, before and after AaGE1 treatment provided the first evidence for cleavage of the LC ester linkages existing in wood

A novel L-arabinose-responsive regulator discovered in the rice-blast fungus Pyricularia oryzae (Magnaporthe oryzae)

In this study we identified the L-arabinose-responsive regulator of Pyricularia oryzae that regulates L-arabinose release and catabolism. Previously we identified the Zn2Cys6 transcription factor (TF), AraR, that has this role in the Trichocomaceae family (Eurotiales), but is absent in other fungi. Candidate Zn2Cys6 TF genes were selected according to their transcript profiles on L-arabinose. Deletion mutants of these genes were screened for their growth phenotype on L-arabinose. One mutant, named Delta ara1, was further analyzed. Our analysis demonstrated that Ara1 from P. oryzae is the functional analog of AraR from A. niger, while there is no significant sequence similarity between them