SC3 and SC4 hydrophobins have distinct roles in formation of aerial structures in dikaryons of Schizophyllum commune

Two monokaryons of Schizophyllum commune can form a fertile dikaryon when the mating-type genes differ. Monokaryons form sterile aerial hyphae, while dikaryons also form fruiting bodies that function in sexual reproduction. The SC3 hydrophobin gene is expressed both in monokaryons and in dikaryons. The SC4 hydrophobin is dikaryon specific. In the monokaryon, SC3 lowers the water surface tension, coats aerial hyphae with a hydrophobic layer and mediates attachment of hyphae to hydrophobic surfaces. The SC4 protein lines gas channels within fruiting bodies with a hydrophobic membrane. Using gene disruptions, in this study, we show that in dikaryons SC3 fulfils the same roles as in monokaryons. SC4, on the other hand, has a role within fruiting bodies. In contrast to gas channels in fruiting bodies of the wild type, those of a DeltaSC4 strain easily filled with water. Thus, SC4 prevents gas channels filling with water under wet conditions, probably serving uninterrupted gas exchange. Other dikaryon-specific hydrophobin genes, SC1 and SC6, apparently do not substitute for the SC4 gene. In addition, by expressing the SC4 gene behind the SC3 promoter in a DeltaSC3 monokaryon, it was shown that SC4 cannot fully substitute for SC3, indicating that both hydrophobins evolved to fulfil specific functions.     

Hydrophobins, from molecular structure to multiple functions in fungal development

Mycelial fungi secrete small, cysteine-rich, proteins, called hydrophobins, that self-assemble at hydrophilic-hydrophobic interfaces into amphipathic membranes, highly insoluble in case of Class I hydrophobins. By self-assembly at the culture medium-air interface they greatly lower the surface tension enabling emergent structures to grow into the air. By self-assembly at the interface between the hydrophilic cell wall and the air or any other hydrophobic environment, these emergent structures are coated with a hydrophobin membrane. These properties allow hydrophobins to fulfil a broad spectrum of functions in fungal development. They are involved in formation of aerial (reproductive) structures, in aerial dispersion of spores, and they line air channels within fruiting bodies with a hydrophobic coating, probably serving gas exchange. Hydrophobins also mediate hyphal attachment to hydrophobic surfaces such as those of plants. Moreover, they appear involved in complex interhyphal interactions, and in interactions with algae in lichens. Their resistance towards chemical and enzymatic treatments suggests that assembled hydrophobins also protect fungal emergent structures against adverse environmental conditions.     

Probing the self-assembly and the accompanying structural changes of hydrophobin SC3 on a hydrophobic surface by mass spectrometry

The fungal class I hydrophobin SC3 self-assembles into an amphipathic membrane at hydrophilic-hydrophobic interfaces such as the water-air and water-Teflon interface. During self-assembly, the water-soluble state of SC3 proceeds via the intermediate alpha-helical state to the stable end form called the beta-sheet state. Self-assembly of the hydrophobin at the Teflon surface is arrested in the alpha-helical state. The beta-sheet state can be induced at elevated temperature in the presence of detergent. The structural changes of SC3 were monitored by various mass spectrometry techniques. We show that the so-called second loop of SC3 (C39-S72) has a high affinity for Teflon. Binding of this part of SC3 to Teflon was accompanied by the formation of alpha-helical structure and resulted in low solvent accessibility. The solvent-protected region of the second loop extended upon conversion to the beta-sheet state. In contrast, the C-terminal part of SC3 became more exposed to the solvent. The results indicate that the second loop of class I hydrophobins plays a pivotal role in self-assembly at the hydrophilic-hydrophobic interface. Of interest, this loop is much smaller in case of class II hydrophobins, which may explain the differences in their assembly.     

The properties of citrate transport catalyzed by CitP of Lactococcus lactis ssp. lactis biovar diacetylactis

Abstract The SC3 hydrophobin gene of Schizophyllum commune was disrupted by homologous integration of an SC3 genomic fragment interrupted by a phleomycin resistance cassette. The phenotype of the mutant was particularly clear in sealed plates in which formation of aerial hyphae was blocked. In non-sealed plates aerial hyphae did form but these were hydrophilic and not hydrophobic as in wild-type strains. Complementation with a genomic SC3 clone restored formation of hydrophobic aerial hyphae in sealed plates. In a dikaryon homozygous for the SC3 mutation normal sporulating fruiting bodies were produced but aerial hyphae were hydrophilic.     

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.     

Four conserved intramolecular disulphide linkages are required for secretion and cell wall localization of a hydrophobin during fungal morphogenesis

Hydrophobins are morphogenetic proteins produced by fungi during assembly of aerial hyphae, sporulation, mushroom development and pathogenesis. Eight cysteine residues are present in hydrophobins and form intramolecular disulphide bonds. Here, we show that expressing eight cysteine-alanine substitution alleles of the MPG1 hydrophobin gene from Magnaporthe grisea causes severe defects in development of aerial hyphae and spores. Immunolocalization revealed that Mpg1 hydrophobin variants, lacking intact disulphide bonds, retain the capacity to self-assemble, but are not secreted to the cell surface. This provides the first genetic evidence that disulphide bridges in a hydrophobin are dispensable for aggregation, but essential for secretion.     

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.     

Complementation of the mpg1 mutant phenotype in Magnaporthe grisea reveals functional relationships between fungal hydrophobins.

The functional relationship between fungal hydrophobins was studied by complementation analysis of an mpg1(-) gene disruption mutant in Magnaporthe grisea. MPG1 encodes a hydrophobin required for full pathogenicity of the fungus, efficient elaboration of its infection structures and conidial rodlet protein production. Seven heterologous hydrophobin genes were selected which play distinct roles in conidiogenesis, fruit body development, aerial hyphae formation and infection structure elaboration in diverse fungal species. Each hydrophobin was introduced into an mpg1(-) mutant by transformation. Only one hydrophobin gene, SC1 from Schizophyllum commune, was able partially to complement mpg1(-) mutant phenotypes when regulated by its own promoter. In contrast, six of the transformants expressing hydrophobin genes controlled by the MPG1 promoter (SC1 and SC4 from S.commune, rodA and dewA from Aspergillus nidulans, EAS from Neurospora crassa and ssgA from Metarhizium anisopliae) could partially complement each of the diverse functions of MPG1. Complementation was always associated with partial restoration of a rodlet protein layer, characteristic of the particular hydrophobin being expressed, and with hydrophobin surface assembly during infection structure formation. This provides the first genetic evidence that diverse hydrophobin-encoding genes encode functionally related proteins and suggests that, although very diverse in amino acid sequence, the hydrophobins constitute a closely related group of morphogenetic proteins.     

Behavior of Trichoderma reesei hydrophobins in solution: interactions, dynamics, and multimer formation

Filamentous fungi utilize small amphiphilic proteins called hydrophobins in their adaptation to the environment. The hydrophobins are used to form coatings on various fungal structures, lower the surface tension of water, and to mediate surface attachment. Hydrophobins function through self-assembly at interfaces, for example, at the air-water interface, and at fungal cellular structures. Despite their high tendency to self assemble at interfaces, hydrophobins can be very soluble in water. To understand the mechanism of hydrophobin self-assembly, in this work, we have studied the behavior of two Trichoderma reesei hydrophobins, HFBI and HFBII in aqueous solution. The main methods used were F?rster resonance energy transfer (FRET) and size exclusion chromatography. A genetically engineered HFBI variant, NCys-HFBI, was utilized for the site-specific labeling of dyes for the FRET experiments. We observed the multimerization of HFBI in a concentration-dependent manner. A change from monomers to tetramers was seen when the hydrophobin concentration was increased. Interaction studies between HFBI and HFBII suggested that at low concentrations homodimers are preferred, and at higher concentrations, the heterotetramers of HFBI and HFBII are formed. In conclusion, the results support the model where hydrophobins in aqueous solutions form multimers by hydrophobic interactions. In contrast to micelles formed by detergents, the hydrophobin multimers are defined in size and involve specific protein-protein interactions.     

Spontaneous self-assembly of SC3 hydrophobins into nanorods in aqueous solution

Hydrophobins are small surface active proteins secreted by filamentous fungi. Because of their ability to self-assemble at hydrophilic-hydrophobic interfaces, hydrophobins play a key role in fungal growth and development. In the present work, the organization in aqueous solution of SC3 hydrophobins from the fungus Schizophyllum commune was assessed using Dynamic Light Scattering, Atomic Force Microscopy and fluorescence spectroscopy. These complementary approaches have demonstrated that SC3 hydrophobins are able not only to spontaneously self-assemble at the air-water interface but also in pure water. AFM experiments evidenced that hydrophobins self-assemble in solution into nanorods. Fluorescence assays with thioflavin T allowed establishing that the mechanism governing SC3 hydrophobin self-assembly into nanorods involves β-sheet stacking. SC3 assembly was shown to be strongly influenced by ionic strength and solution pH. The presence of a very low ionic strength significantly favoured the protein self-assembly but a further increase of ions in solution disrupted the protein assembly. It was assessed that solution pH had a significant effect on the SC3 hydrophobins organization. In peculiar, the self-assembly process was considerably reduced at acidic pH. Our findings demonstrate that the self-assembly of SC3 hydrophobins into nanorods of well-defined length can be directly controlled in solution. Such control allows opening the way for the development of new smart self-assembled structures for targeted applications.     

The SC15 protein of Schizophyllum commune mediates formation of aerial hyphae and attachment in the absence of the SC3 hydrophobin

Disruption of the SC3 gene in the basidiomycete Schizophyllum commune affected not only formation of aerial hyphae but also attachment to hydrophobic surfaces. However, these processes were not completely abolished, indicating involvement of other molecules. We here show that the SC15 protein mediates formation of aerial hyphae and attachment in the absence of SC3. SC15 is a secreted protein of 191 aa with a hydrophilic N-terminal half and a highly hydrophobic C-terminal half. It is not a hydrophobin as it lacks the eight conserved cysteine residues found in these proteins. Besides being secreted into the medium, SC15 was localized in the cell wall and the mucilage that binds aerial hyphae together. In a strain in which the SC15 gene was deleted (DeltaSC15) formation of aerial hyphae and attachment were not affected. However, these processes were almost completely abolished when the SC15 gene was deleted in the DeltaSC3 background. The absence of aerial hyphae in the DeltaSC3DeltaSC15 strain can be explained by the inability of the strain to lower the water surface tension and to make aerial hyphae hydrophobic.     

Hydrophobin Genes Involved in Formation of Aerial Hyphae and Fruit Bodies in Schizophyllum

Fungi typically grow by apical extension of hyphae that penetrate moist substrates. After establishing a branched feeding mycelium, the hyphae differentiate and grow away from the substrate into the air where they form various structures such as aerial hyphae and mushrooms. In the basidiomycete species Schizophyllum commune, we previously identified a family of homologous genes that code for small cysteine-rich hydrophobic proteins. We now report that the encoded hydrophobins are excreted in abundance into the culture medium by submerged feeding hyphae but form highly insoluble complexes in the walls of emerging hyphae. The Sc3 gene encodes a hydrophobin present in walls of aerial hyphae. The homologous Sc1 and Sc4 genes, which are regulated by the mating-type genes, encode hydrophobins present in walls of fruit body hyphae. The hydrophobins are probably instrumental in the emergence of these aerial structures.     

Building filaments in the air: aerial morphogenesis in bacteria and fungi

To disperse their spores to new sites, filamentous fungi and bacteria need to erect aerial filaments, which develop into fruiting bodies and spore-bearing structures. The first challenge to aerial development is breaking surface tension at an aqueous-air interface, and in both groups of microorganisms, surface-active proteins take part in the initiation of aerial morphogenesis. Comparative analysis of fungi and bacteria is providing new insights into the means by which aerial filamentation is accomplished.     

Structural characterization of the hydrophobin SC3, as a monomer and after self-assembly at hydrophobic/hydrophilic interfaces.

Hydrophobins are small fungal proteins that self-assemble at hydrophilic/hydrophobic interfaces into amphipathic membranes that, in the case of Class I hydrophobins, can be disassembled only by treatment with agents like pure trifluoroacetic acid. Here we characterize, by spectroscopic techniques, the structural changes that occur upon assembly at an air/water interface and upon assembly on a hydrophobic solid surface, and the influence of deglycosylation on these events. We determined that the hydrophobin SC3 from Schizophyllum commune contains 16-22 O-linked mannose residues, probably attached to the N-terminal part of the peptide chain. Scanning force microscopy revealed that SC3 adsorbs specifically to a hydrophobic surface and cannot be removed by heating at 100 degrees C in 2% sodium dodecyl sulfate. Attenuated total reflection Fourier transform infrared spectroscopy and circular dichroism spectroscopy revealed that the monomeric, water-soluble form of the protein is rich in beta-sheet structure and that the amount of beta-sheet is increased after self-assembly on a water-air interface. Alpha-helix is induced specifically upon assembly of the protein on a hydrophobic solid. We propose a model for the formation of rodlets, which may be induced by dehydration and a conformational change of the glycosylated part of the protein, resulting in the formation of an amphipathic alpha-helix that forms an anchor for binding to a substrate. The assembly in the beta-sheet form seems to be involved in lowering of the surface tension, a potential function of hydrophobins.     

Interaction and comparison of a class I hydrophobin from Schizophyllum commune and class II hydrophobins from Trichoderma reesei

Hydrophobins fulfill a wide spectrum of functions in fungal growth and development. These proteins self-assemble at hydrophilic-hydrophobic interfaces into amphipathic membranes. Hydrophobins are divided into two classes based on their hydropathy patterns and solubility. We show here that the properties of the class II hydrophobins HFBI and HFBII of Trichoderma reesei differ from those of the class I hydrophobin SC3 of Schizophyllum commune. In contrast to SC3, self-assembly of HFBI and HFBII at the water-air interface was neither accompanied by a change in secondary structure nor by a change in ultrastructure. Moreover, maximal lowering of the water surface tension was obtained instantly or took several minutes in the case of HFBII and HFBI, respectively. In contrast, it took several hours in the case of SC3. Oil emulsions prepared with HFBI and SC3 were more stable than those of HFBII, and HFBI and SC3 also interacted more strongly with the hydrophobic Teflon surface making it wettable. Yet, the HFBI coating did not resist treatment with hot detergent, while that of SC3 remained unaffected. Interaction of all the hydrophobins with Teflon was accompanied with a change in the circular dichroism spectra, indicating the formation of an alpha-helical structure. HFBI and HFBII did not affect self-assembly of the class I hydrophobin SC3 of S. commune and vice versa. However, precipitation of SC3 was reduced by the class II hydrophobins, indicating interaction between the assemblies of both classes of hydrophobins.     

Structural and functional role of the disulfide bridges in the hydrophobin SC3

Hydrophobins function in fungal development by self-assembly at hydrophobic-hydrophilic interfaces such as the interface between the fungal cell wall and the air or a hydrophobic solid. These proteins contain eight conserved cysteine residues that form four disulfide bonds. To study the effect of the disulfide bridges on the self-assembly, the disulfides of the SC3 hydrophobin were reduced with 1,4-dithiothreitol. The free thiols were then blocked with either iodoacetic acid (IAA) or iodoacetamide (IAM), introducing eight or zero negative charges, respectively. Circular dichroism and infrared spectroscopy showed that after opening of the disulfide bridges SC3 is initially unfolded. IAA-SC3 did not self-assemble at the air-water interface upon shaking an aqueous solution. Remarkably, after drying down IAA-SC3 or after exposing it to Teflon, it refolded into a structure similar to that observed for native SC3 at these interfaces. Iodoacetamide-SC3 on the other hand, which does not contain extra charges, spontaneously refolded in water in the amyloid-like beta-sheet conformation, characteristic for SC3 assembled at the water-air interface. From this we conclude that the disulfide bridges of SC3 are not directly involved in self-assembly but keep hydrophobin monomers soluble in the fungal cell or its aqueous environment, preventing premature self-assembly.     

The Cys3-Cys4 loop of the hydrophobin EAS is not required for rodlet formation and surface activity

Class I hydrophobins are fungal proteins that self-assemble into robust amphipathic rodlet monolayers on the surface of aerial structures such as spores and fruiting bodies. These layers share many structural characteristics with amyloid fibrils and belong to the growing family of functional amyloid-like materials produced by microorganisms. Although the three-dimensional structure of the soluble monomeric form of a class I hydrophobin has been determined, little is known about the molecular structure of the rodlets or their assembly mechanism. Several models have been proposed, some of which suggest that the Cys3-Cys4 loop has a critical role in the initiation of assembly or in the polymeric structure. In order to provide insight into the relationship between hydrophobin sequence and rodlet assembly, we investigated the role of the Cys3-Cys4 loop in EAS, a class I hydrophobin from Neurospora crassa. Remarkably, deletion of up to 15 residues from this 25-residue loop does not impair rodlet formation or reduce the surface activity of the protein, and the physicochemical properties of rodlets formed by this mutant are indistinguishable from those of its full-length counterpart. In addition, the core structure of the truncation mutant is essentially unchanged. Molecular dynamics simulations carried out on the full-length protein and this truncation mutant binding to an air-water interface show that, although it is hydrophobic, the loop does not play a role in positioning the protein at the surface. These results demonstrate that the Cys3-Cys4 loop does not have an integral role in the formation or structure of the rodlets and that the major determinant of the unique properties of these proteins is the amphipathic core structure, which is likely to be preserved in all hydrophobins despite the high degree of sequence variation across the family.     

Structural hierarchy in molecular films of two class II hydrophobins

Hydrophobins are highly surface-active proteins that are specific to filamentous fungi. They function as coatings on various fungal structures, enable aerial growth of hyphae, and facilitate attachment to surfaces. Little is known about their structures and structure-function relationships. In this work we show highly organized surface layers of hydrophobins, representing the most detailed structural study of hydrophobin films so far. Langmuir-Blodgett films of class II hydrophobins HFBI and HFBII from Trichoderma reesei were prepared and analyzed by atomic force microscopy. The films showed highly ordered two-dimensional crystalline structures. By combining our recent results on small-angle X-ray scattering of hydrophobin solutions, we found that the unit cells in the films have dimensions similar to those of tetrameric aggregates found in solutions. Further analysis leads to a model in which the building blocks of the two-dimensional crystals are shape-persistent supramolecules consisting of four hydrophobin molecules. The results also indicate functional and structural differences between HFBI and HFBII that help to explain differences in their properties. The possibility that the highly organized surface assemblies of hydrophobins could allow a route for manufacturing functional surfaces is suggested.     

Hydrophobins: New prospects for biotechnology

Hydrophobins are small amphipathic molecules found uniquely in fungi. They perform crucial roles in allowing filamentous species to break through interfaces during aerial hyphae formation, sporulation, fruit body production and cell penetration. Initial biotechnological applications have exploited materials coated with hydrophobins to switch hydrophobic surfaces to hydrophilic and vice versa. Recent improvements in our understanding of the biophysics of hydrophobin layer formation, including the use of mixed types of molecules, together with advances in genomics promise to extend greatly the potential for hydrophobin biotechnologies.