Han A. B. Wösten

Genome sequencing and analysis of the versatile cell factory Aspergillus niger CBS 513.88

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis.

Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis

Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant–microbe interactions. This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development. Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis, no ‘true’ virulence factors had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens.

Hydrophobins, the fungal coat unravelled

Hydrophobins are among the most surface active molecules and self-assemble at any hydrophilic-hydrophobic interface into an amphipathic film. These small secreted proteins of about 100 amino acids can be used to make hydrophilic surfaces hydrophobic and hydrophobic surfaces hydrophilic. Although differences in the biophysical properties of hydrophobins have not yet been related to differences in primary structure it has been established that the N-terminal part, at least partly, determines wettability of the hydrophilic side of the assemblage, while the eight conserved cysteine residues that form four disulphide bridges prevent self-assembly of the hydrophobin in the absence of a hydrophilic-hydrophobic interface. Three conformations of class I hydrophobins have been identified: the monomeric state, which is soluble in water, the alpha-helical state, which is the result of self-assembly at a hydrophobic solid, and the beta-sheet state, which is formed during self-assembly at the water-air interface. Experimental evidence strongly indicates that the alpha-helical state is an intermediate and that the beta-sheet state is the end form of assembly. The latter state has a typical ultrastructure of a mosaic of 10 nm wide rodlets, which have been shown to resemble the amyloid fibrils.

Genome sequence of the model mushroom Schizophyllum commune

Much remains to be learned about the biology of mushroom-forming fungi, which are an important source of food, secondary metabolites and industrial enzymes. The wood-degrading fungus Schizophyllum commune is both a genetically tractable model for studying mushroom development and a likely source of enzymes capable of efficient degradation of lignocellulosic biomass. Comparative analyses of its 38.5-megabase genome, which encodes 13,210 predicted genes, reveal the speciess unique wood-degrading machinery. One-third of the 471 genes predicted to encode transcription factors are differentially expressed during sexual development of S. commune. Whereas inactivation of one of these, fst4, prevented mushroom formation, inactivation of another, fst3, resulted in more, albeit smaller, mushrooms than in the wild-type fungus. Antisense transcripts may also have a role in the formation of fruiting bodies. Better insight into the mechanisms underlying mushroom formation should affect commercial production of mushrooms and their industrial use for producing enzymes and pharmaceuticals.

Interfacial self-assembly of a hydrophobin into an amphipathic protein membrane mediates fungal attachment to hydrophobic surfaces.

The SC3p hydrophobin of Schizophyllum commune is a small hydrophobic protein (100‐101 amino acids with eight cysteine residues) that self‐assembles at a water/air interface and coats aerial hyphae with an SDS‐insoluble protein membrane, at the outer side highly hydrophobic and with a typical rodlet pattern. SC3p monomers in water also self‐assemble at the interfaces between water and oils or hydrophobic solids. These materials are then coated with a 10 nm thick SDS‐insoluble assemblage of SC3p making their surfaces hydrophilic. Hyphae of S. commune growing on a Teflon surface became firmly attached and SC3p was shown to be present between the fungal cell wall and the Teflon. Decreased attachment of hyphae to Teflon was observed in strains not expressing SC3, i.e. a strain containing a targeted mutation in this gene and a regulatory mutant thn. These findings indicate that hydrophobins, in addition to forming hydrophobic wall coatings, play a role in adherence of fungal hyphae to hydrophobic surfaces.

How a fungus escapes the water to grow into the air

Fungi are well known to the casual observer for producing water-repelling aerial moulds and elaborate fruiting bodies such as mushrooms and polypores. Filamentous fungi colonize moist substrates (such as wood) and have to breach the water-air interface to grow into the air. Animals and plants breach this interface by mechanical force. Here, we show that a filamentous fungus such as Schizophyllum commune first has to reduce the water surface tension before its hyphae can escape the aqueous phase to form aerial structures such as aerial hyphae or fruiting bodies. The large drop in surface tension (from 72 to 24 mJ m-2) results from self-assembly of a secreted hydrophobin (SC3) into a stable amphipathic protein film at the water-air interface. Other, but not all, surface-active molecules (that is, other class I hydrophobins and streptofactin from Streptomyces tendae) can substitute for SC3 in the medium. This demonstrates that hydrophobins not only have a function at the hyphal surface but also at the medium-air interface, which explains why fungi secrete large amounts of hydrophobin into their aqueous surroundings.

Interfacial Self-Assembly of a Fungal Hydrophobin into a Hydrophobic Rodlet Layer.

The Sc3p hydrophobin of the basidiomycete Schizophyllum commune is a small hydrophobic protein (100 to 101 amino acids) containing eight cysteine residues. Large amounts of the protein are excreted into the culture medium as monomers, but in the walls of aerial hyphae, the protein is present as an SDS-insoluble complex. In this study, we show that the Sc3p hydrophobin spontaneously assembles into an SDS-insoluble protein membrane on the surface of gas bubbles or when dried down on a hydrophilic surface. Electron microscopy of the assembled hydrophobin shows a surface consisting of rodlets spaced 10 nm apart, which is similar to those rodlets seen on the surface of aerial hyphae. When the purified Sc3p hydrophobin assembles on a hydrophilic surface, a surface is exposed with high hydrophobicity, similar to that of aerial hyphae. The rodlet layer, assembled in vivo and in vitro, can be disassembled by dissolution in trifluoroacetic acid and, after removal of the acid, reassembled into a rodlet layer. We propose, therefore, that the hydrophobic rodlet layer on aerial hyphae arises by interfacial self-assembly of Sc3p hydrophobin monomers, involving noncovalent interactions only. Submerged hyphae merely excrete monomers because these hyphae are not exposed to a water-air interface. The generally observed rodlet layers on fungal spores may arise in a similar way.

Localization of growth and secretion of proteins in Aspergillus niger

Hyphal growth and secretion of proteins in Aspergillus niger were studied using a new method of culturing the fungus between perforated membranes which allows visualization of both parameters. At the colony level the sites of occurrence of growth and general protein secretion were correlated. In 4-d-old colonies both growth and secretion were localized at the periphery of the colony, whereas in a 5-d-old colony growth and secretion also occurred in a more central zone of the colony where conidiophore differentiation was observed. However, in both cases glucoamylase secretion was mainly detected at the periphery of the colonies. At the hyphal level immunogold labelling showed glucoamylase secretion at the tips of leading hyphae only. Microautoradiography after labelling with N-acetylglucosamine showed that these hyphae were probably all growing. Glucoamylase secretion could not be demonstrated immediately after a temperature shock which stopped growth. These results indicate that glucoamylase secretion is located at the tips of growing hyphae only.

Amyloids - A functional coat for microorganisms

Amyloids are filamentous protein structures ∼10 nm wide and 0.1–10 μm long that share a structural motif, the cross-β structure. These fibrils are usually associated with degenerative diseases in mammals. However, recent research has shown that these proteins are also expressed on bacterial and fungal cell surfaces. Microbial amyloids are important in mediating mechanical invasion of abiotic and biotic substrates. In animal hosts, evidence indicates that these protein structures also contribute to colonization by activating host proteases that are involved in haemostasis, inflammation and remodelling of the extracellular matrix. Activation of proteases by amyloids is also implicated in modulating blood coagulation, resulting in potentially life-threatening complications.

Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche

Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the “button mushroom” forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost and during mushroom formation. The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation are more highly expressed in compost. The striking expansion of heme-thiolate peroxidases and β-etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor. A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics.