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.     

The SC3 hydrophobin self-assembles into a membrane with distinct mass transfer properties

Hydrophobins are a class of small proteins that fulfill a wide spectrum of functions in fungal growth and development. They do so by self-assembling into an amphipathic membrane at hydrophilic-hydrophobic interfaces. The SC3 hydrophobin of Schizophyllum commune is the best-studied hydrophobin. It assembles at the air-water interface into a membrane consisting of functional amyloid fibrils that are called rodlets. Here we examine the dynamics of SC3 assembly at an oil-water and air-water interface and the permeability characteristics of the assembled layer. Hydrophobin assembled at an oil-water interface is a dynamic system capable of emulsifying oil. It accepts soluble-state SC3 oligomers from water in a unidirectional process and sloughs off SC3 vesicles back into the water phase enclosing a portion of the oil phase in their hydrophobic interior. The assembled layer is impermeable to solutes >200 Da from either the water phase or the oil phase; however, due to the emulsification process, oil and the hydrophobic marker molecules in the oil phase can be transferred into the water phase, thus giving the impression that the assembled layer is permeable to the marker molecules. By contrast, the layer assembled at an air-water interface is permeable to water vapor from either the hydrophobic or hydrophilic side.     

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.     

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.     

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.     

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.     

Surface modifications created by using engineered hydrophobins

Hydrophobins are small (ca. 100 amino acids) secreted fungal proteins that are characterized by the presence of eight conserved cysteine residues and by a typical hydropathy pattern. Class I hydrophobins self-assemble at hydrophilic-hydrophobic interfaces into highly insoluble amphipathic membranes, thereby changing the nature of surfaces. Hydrophobic surfaces become hydrophilic, while hydrophilic surfaces become hydrophobic. To see whether surface properties of assembled hydrophobins can be changed, 25 N-terminal residues of the mature SC3 hydrophobin were deleted (TrSC3). In addition, the cell-binding domain of fibronectin (RGD) was fused to the N terminus of mature SC3 (RGD-SC3) and TrSC3 (RGD-TrSC3). Self-assembly and surface activity were not affected by these modifications. However, physiochemical properties at the hydrophilic side of the assembled hydrophobin did change. This was demonstrated by a change in wettability and by enhanced growth of fibroblasts on Teflon-coated with RGD-SC3, TrSC3, or RGD-TrSC3 compared to bare Teflon or Teflon coated with SC3. Thus, engineered hydrophobins can be used to functionalize surfaces.     

Surface Modifications Created by Using Engineered Hydrophobins

Hydrophobins are small (ca. 100 amino acids) secreted fungal proteins that are characterized by the presence of eight conserved cysteine residues and by a typical hydropathy pattern. Class I hydrophobins self-assemble at hydrophilic-hydrophobic interfaces into highly insoluble amphipathic membranes, thereby changing the nature of surfaces. Hydrophobic surfaces become hydrophilic, while hydrophilic surfaces become hydrophobic. To see whether surface properties of assembled hydrophobins can be changed, 25 N-terminal residues of the mature SC3 hydrophobin were deleted (TrSC3). In addition, the cell-binding domain of fibronectin (RGD) was fused to the N terminus of mature SC3 (RGD-SC3) and TrSC3 (RGD-TrSC3). Self-assembly and surface activity were not affected by these modifications. However, physiochemical properties at the hydrophilic side of the assembled hydrophobin did change. This was demonstrated by a change in wettability and by enhanced growth of fibroblasts on Teflon-coated with RGD-SC3, TrSC3, or RGD-TrSC3 compared to bare Teflon or Teflon coated with SC3. Thus, engineered hydrophobins can be used to functionalize surfaces.     

New insights into the structure and hydration of a stratum corneum lipid model membrane by neutron diffraction

The structure and hydration of a stratum corneum (SC) lipid model membrane composed of N-(-hydroxyoctadecanoyl)-phytosphingosine (CER6)/cholesterol (Ch)/palmitic acid (PA)/cholesterol sulfate (ChS) were characterized by neutron diffraction. The neutron scattering length density across the SC lipid model membrane was calculated from measured diffraction peak intensities. The internal membrane structure and water distribution function across the bilayer were determined. The low hydration of the intermembrane space is a major feature of the SC lipid model membrane. The thickness of the water layer in the SC lipid model membrane is about 1 Å at full hydration. For the composition 55% CER6/25% Ch/15% PA/5% ChS, in a partly dehydrated state (60% humidity) and at 32°C, the lamellar repeat distance and the membrane thickness have the same value of 45.6 Å . The hydrophobic region of the membrane has a thickness of 31.2 Å . A decrease of the Ch content increases the membrane thickness. The water diffusion through the SC lipid model multilamellar membrane is a considerably slow process relative to that through phospholipid membranes. In excess water, the membrane hydration follows an exponential law with two characteristic times of 93 and 44 min. At 81°C and 97% humidity, the membrane separates into two phases with repeat distances of 45.8 and 40.5 Å . Possible conformations of CER6 molecules in the dry and hydrated multilayers are discussed.     

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.     

Structural changes and molecular interactions of hydrophobin SC3 in solution and on a hydrophobic surface

The hydrophobin SC3 belongs to a class of small proteins functioning in the growth and development of fungi. Its unique amphipathic property and remarkable surface activity make it interesting not only for biological studies but also for medical and industrial applications. Biophysical studies have revealed that SC3 possesses at least three distinct conformations, named "soluble-state SC3" for the protein in solution, and "alpha-helical-state SC3" and "beta-sheet-state SC3" for the different states of the protein associated at a hydrophobic-water interface. The present fluorescence study shows that the microenvironment of the dansyl-labeled N terminus of soluble-state SC3 is relatively hydrophobic, whereas it is hydrophilic for alpha-helical-state and beta-sheet-state SC3. Fluorescence collisional quenching indicates that the N terminus of soluble-state SC3 is more solvent-accessible than those of alpha-helical-state and beta-sheet-state SC3, with Stern-Volmer constants for acrylamide of 4.63, 0.02, and 0.2 M(-1) for the different states, respectively. Fluorescence resonance energy transfer measurements show that soluble-state SC3 tends to associate in solution but dissociates in TFA. Fluorescence energy transfer was eliminated by conversion of soluble-state SC3 to alpha-helical-state SC3 on a hydrophobic surface, indicating a spatial separation of the molecules in this state. By inducing the beta-sheet state, structural changes were observed, both by CD and by fluorescence, that could be fit to two exponentials with lifetimes of about 10 min and 4 h. Molecules in the beta-sheet state also underwent a slow change in spatial proximity on the hydrophobic surface, as revealed by the reappearance of fluorescence resonance energy transfer in time.     

SUMO modification of septin-interacting proteins in Candida albicans

The initiation of bud and hyphal growth in the opportunistic fungal pathogen Candida albicans both involve polarized morphogenesis. However, there are many differences including the function of the septin proteins, a family of proteins involved in membrane organization in a wide range of organisms. Septins form a characteristic ring on the inner surface of the plasma membrane at the bud neck, whereas the septins are diffusely localized across emerging hyphal tips. In addition, septin rings are maintained at sites of septum formation in hyphae rather than being disassembled immediately after cytokinesis. The possibility that C. albicans septins are regulated by the small ubiquitin-like protein SUMO was examined in this study because the Saccharomyces cerevisiae septins were shown previously to be modified by SUMO (Smt3p). However, SUMO conjugation to septins was not detected during budding or hyphal morphogenesis in C. albicans. These results are supported by the lack of conserved SUMO consensus motifs between septins from the two organisms even after adjusting the predicted Cdc3p and Cdc12p septin sequences to account for mRNA splicing in C. albicans. Interestingly, a homolog of the Smt3p SUMO was identified in the C. albicans genome, and an epitope tagged version of Smt3p was conjugated to a variety of proteins. Immunofluorescence analysis showed prominent Smt3p SUMO localization at bud necks and sites of septum formation in hyphae similar to the septins. However, Smt3p was primarily detected on the mother cell side of the septin ring. A subset of these Smt3p-modified proteins co-immunoprecipitated with the septin Cdc11p. These results indicate that septin-associated proteins and not the septins themselves are the key target of SUMO modification at the bud neck in C. albicans.     

Hydrophobin gene expression affects hyphal wall composition in Schizophyllum commune

Disruption of the SC3 hydrophobin gene of Schizophyllum commune (DeltaSC3 strain) affected the composition of the cell wall. Compared to a wild-type strain the amount of mucilage (i.e., water-soluble (1-3)beta-glucan with single glucose residues attached by (1-6)beta-linkages) increased considerably, while the amount of alkali-resistant glucan (linked to chitin) decreased. Reintroduction of the SC3 gene or other hydrophobins genes expressed behind the SC3 promotor restored wild-type cell wall composition. However, addition of purified SC3 protein to the medium or growing the DeltaSC3 strain in spent medium of the wild-type strain had no effect. In young cultures of wild-type strains of S.commune, not yet expressing SC3, the amount of mucilage was also relatively high. These data show that hydrophobins not only function at hydrophilic/hydrophobic interfaces, as shown previously, but also affect wall composition.     

Nucleotide Sequence of the Akv env Gene

The sequence of 2,191 nucleotides encoding the env gene of murine retrovirus Akv was determined by using a molecular clone of the Akv provirus. Deduction of the encoded amino acid sequence showed that a single open reading frame encodes a 638-amino acid precursor to gp70 and p15E. In addition, there is a typical leader sequence preceding the amino terminus of gp70. The locations of potential glycosylation sites and other structural features indicate that the entire gp70 molecule and most of p15E are located on the outer side of the membrane. Internal cleavage of the env precursor to generate gp70 and p15E occurs immediately adjacent to several basic amino acids at the carboxyl terminus of gp70. This cleavage generates a region of 42 uncharged, relatively hydrophobic amino acids at the amino terminus of p15E, which is located in a position analogous to the hydrophobic membrane fusion sequence of influenza virus hemagglutinin. The mature polypeptides are predicted to associate with the membrane via a region of 30 uncharged, mostly hydrophobic amino acids located near the carboxyl terminus of p15E. Distal to this membrane association region is a sequence of 35 amino acids at the carboxyl terminus of the env precursor, which is predicted to be located on the inner side of the membrane. By analogy to Moloney murine leukemia virus, a proteolytic cleavage in this region removes the terminal 19 amino acids, thus generating the carboxyl terminus of p15E. This leaves 15 amino acids at the carboxyl terminus of p15E on the inner side of the membrane in a position to interact with virion cores during budding. The precise location and order of the large RNase T(1)-resistant oligonucleotides in the env region were determined and compared with those from several leukemogenic viruses of AKR origin. This permitted a determination of how the differences in the leukemogenic viruses affect the primary structure of the env gene products.     

A surface active protein involved in aerial hyphae formation in the filamentous fungus Schizophillum commune restores the capacity of a bald mutant of the filamentous bacterium Streptomyces coelicolor to erect aerial structures

The filamentous bacterium Streptomyces coelicolor undergoes a complex process of morphological differentiation involving the formation of a dense lawn of aerial hyphae that grow away from the colony surface into the air to form an aerial mycelium. Bald mutants of S. coelicolor, which are blocked in aerial mycelium formation, regain the capacity to erect aerial structures when exposed to a small hydrophobic protein called SapB, whose synthesis is temporally and spatially correlated with morphological differentiation. We now report that SapB is a surfactant that is capable of reducing the surface tension of water from 72 mJ m^?2 to 30 mJ m^?2 at a concentration of 50 μg ml^?1. We also report that SapB, like the surface-active peptide streptofactin produced by the species S. tendae, was capable of restoring the capacity of bald mutants of S. tendae to erect aerial structures. Strikingly, a member (SC3) of the hydrophobin family of fungal proteins involved in the erection of aerial hyphae in the filamentous fungus Schizophyllum commune was also capable of restoring the capacity of S. coelicolor and S. tendae bald mutants to erect aerial structures. SC3 is unrelated in structure to SapB and streptofactin but, like the streptomycetes proteins, the fungal protein is a surface active agent. Scanning electron microscopy revealed that aerial structures produced in response to both the bacterial or the fungal proteins were undifferentiated vegetative hyphae that had grown away from the colony surface but had not commenced the process of spore formation. We conclude that the production of SapB and streptofactin at the start of morphological differentiation contributes to the erection of aerial hyphae by decreasing the surface tension at the colony surface but that subsequent morphogenesis requires additional developmentally regulated events under the control of bald genes.     

Surface modification using interfacial assembly of the Streptomyces chaplin proteins

The chaplin proteins are instrumental in the formation of reproductive aerial structures by the filamentous bacterium Streptomyces coelicolor. They lower the water surface tension thereby enabling aerial growth. In addition, chaplins provide surface hydrophobicity to the aerial hyphae by assembling on the cell surface into an amphipathic layer of amyloid fibrils. We here show that mixtures of cell wall-extracted chaplins can be used to modify a variety of hydrophilic and hydrophobic surfaces in vitro thereby changing their nature. Assembly on glass leads to a protein coating that makes the surface hydrophobic. Conversely, the assembly of chaplins on hydrophobic surfaces renders them hydrophilic. Furthermore, we show that chaplins can stabilize emulsions of oil into water and have an unprecedented surface activity at high pH. Interestingly, this high surface activity coincides with the interfacial assembly of chaplins into a semi-liquid membrane, as opposed to the rigid membrane formed at neutral pH. This semi-liquid membrane possibly represents a trapped intermediate in the assembly process towards the more rigid amyloidal conformation. Taken together, our data shows that chaplins are suitable candidate proteins for a wide range of biotechnological applications.     

Molecular dynamics simulations of the hydrophobin SC3 at a hydrophobic/hydrophilic interface

Hydrophobins are small ( approximately 100 aa) proteins that have an important role in the growth and development of mycelial fungi. They are surface active and, after secretion by the fungi, self-assemble into amphipathic membranes at hydrophobic/hydrophilic interfaces, reversing the hydrophobicity of the surface. In this study, molecular dynamics simulation techniques have been used to model the process by which a specific class I hydrophobin, SC3, binds to a range of hydrophobic/hydrophilic interfaces. The structure of SC3 used in this investigation was modeled based on the crystal structure of the class II hydrophobin HFBII using the assumption that the disulfide pairings of the eight conserved cysteine residues are maintained. The proposed model for SC3 in aqueous solution is compact and globular containing primarily beta-strand and coil structures. The behavior of this model of SC3 was investigated at an air/water, an oil/water, and a hydrophobic solid/water interface. It was found that SC3 preferentially binds to the interfaces via the loop region between the third and fourth cysteine residues and that binding is associated with an increase in alpha-helix formation in qualitative agreement with experiment. Based on a combination of the available experiment data and the current simulation studies, we propose a possible model for SC3 self-assembly on a hydrophobic solid/water interface.     

环境pH诱导出芽短梗霉细胞多形化

出芽短梗霉具有酵母状细胞、膨大细胞、菌丝、厚垣孢子、念珠状菌丝和分生组织状结构。在最适pH条件下,出芽短梗霉生长繁殖以酵母状细胞（CBS100225等4菌株）或膨大细胞（CBS249.65等4菌株）为主。pH 2.2或pH 7.0诱导全部8株出芽短梗霉形成分生组织状结构。酵母状细胞转变成膨大细胞受低pH值诱导的占75%,还受高pH诱导的占50%。膨大细胞是多形性细胞转变的中心环节,可以转变成菌丝、厚垣孢子或分生组织状结构。     

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.     

N-alkylated chitosan as a potential nonviral vector for gene transfection

Alkylated chitosans (ACSs) were prepared by modifying chitosan (CS) with alkyl bromide. The self-aggregation of ACSs in acetic acid solution was characterized by fluorescence spectroscopy and dynamic light scattering method. The results indicate that introducing alkyl side chains leads to the self-aggregation of ACSs, and CS with a 99% deacetylation degree shows no aggregation due to the electrostatic repulsion. The electrophoresis experiment demonstrates that the complex between CS and DNA was formed at a charge ratio (+/-) of 1/1; ACS/DNA complexes were formed at a lower charge ratio (+/-) of 1/4. A small amount of alkylated chitosans play the same shielding role as chitosan in protecting DNA from DNase hydrolysis. Differential scanning calorimetry (DSC) and atomic force microscopy (AFM) were employed separately to investigate the thermodynamic behavior of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/CS and DPPC/ACS mixtures and the variation in topological structure of DPPC membrane induced by CS and ACS. It is shown that CS and ACS can cause the fusion of DPPC multilamellar vesicles as well as membrane destabilization. In contrast, the perturbation effect induced by ACS is more evident due to the hydrophobic interaction. CS and ACS were used to transfer plasmid-encoding CAT into C(2)C(12) cell lines. Upon elongating the alkyl side chain, the transfection efficiency is increased and levels off after the number of carbons in the side chain exceeds 8. It is proposed that the higher transfection efficiency of ACS is attributed to the increasing entry into cells facilitated by hydrophobic interactions and easier unpacking of DNA from ACS carriers due to the weakening of electrostatic attractions between DNA and ACS.