Six Hydrophobins Are Involved in Hydrophobin Rodlet Formation in Aspergillus nidulans and Contribute to Hydrophobicity of the Spore Surface

Hydrophobins are amphiphilic proteins able to self-assemble at water-air interphases and are only found in filamentous fungi. In Aspergillus nidulans two hydrophobins, RodA and DewA, have been characterized, which both localize on the conidiospore surface and contribute to its hydrophobicity. RodA is the constituent protein of very regularly arranged rodlets, 10 nm in diameter. Here we analyzed four more hydrophobins, DewB-E, in A. nidulans and found that all six hydrophobins contribute to the hydrophobic surface of the conidiospores but only deletion of rodA caused loss of the rodlet structure. Analysis of the rodlets in the dewB-E deletion strains with atomic force microscopy revealed that the rodlets appeared less robust. Expression of DewA and DewB driven from the rodA promoter and secreted with the RodA secretion signal in a strain lacking RodA, restored partly the hydrophobicity. DewA and B were able to form rodlets to some extent but never reached the rodlet structure of RodA. The rodlet-lacking rodA-deletion strain opens the possibility to systematically study rodlet formation of other natural or synthetic hydrophobins.     

MPG1 Encodes a Fungal Hydrophobin Involved in Surface Interactions during Infection-Related Development of Magnaporthe grisea

The rice blast fungus expresses a pathogenicity gene, MPG1, during appressorium formation, disease symptom development, and conidiation. The MPG1 gene sequence predicts a small protein belonging to a family of fungal proteins designated hydrophobins. Using random ascospore analysis and genetic complementation, we showed that MPG1 is necessary for infection-related development of Magnaporthe grisea on rice leaves and for full pathogenicity toward susceptible rice cultivars. The protein product of MPG1 appears to interact with hydrophobic surfaces, where it may act as a developmental sensor for appressorium formation. Ultrastructural studies revealed that MPG1 directs formation of a rodlet layer on conidia composed of interwoven ~5-nm rodlets, which contributes to their surface hydrophobicity. Using combined genetic and biochemical approaches, we identified a 15-kD secreted protein with characteristics that establish it as a class I hydrophobin. The protein is able to form detergent-insoluble high molecular mass complexes, is soluble in trifluoroacetic acid, and exhibits mobility shifts after treatment with performic acid. The production of this protein is directed by MPG1.     

Assembly of the Fungal SC3 Hydrophobin into Functional Amyloid Fibrils Depends on Its Concentration and Is Promoted by Cell Wall Polysaccharides

Class I hydrophobins function in fungal growth and development by self-assembling at hydrophobic-hydrophilic interfaces into amyloid-like fibrils. SC3 of the mushroom-forming fungus Schizophyllum commune is the best studied class I hydrophobin. This protein spontaneously adopts the amyloid state at the water-air interface. In contrast, SC3 is arrested in an intermediate conformation at the interface between water and a hydrophobic solid such as polytetrafluoroethylene (PTFE; Teflon). This finding prompted us to study conditions that promote assembly of SC3 into amyloid fibrils. Here, we show that SC3 adopts the amyloid state at the water-PTFE interface at high concentration (300 μg ml⁻¹) and prolonged incubation (16 h). Moreover, we show that amyloid formation at both the water-air and water-PTFE interfaces is promoted by the cell wall components schizophyllan (β(1–3),β(1–6)-glucan) and β(1–3)-glucan. Hydrophobin concentration and cell wall polysaccharides thus contribute to the role of SC3 in formation of aerial hyphae and in hyphal attachment.Hydrophobins are a class of surface active proteins that play diverse roles in fungal growth and development. For instance, they allow fungi to escape an aqueous environment, confer hydrophobicity to fungal surfaces in contact with air, and mediate attachment of fungi to hydrophobic surfaces (1, 2). They also play a role in the architecture of the cell wall (3).Hydrophobins share eight conserved cysteine residues, but otherwise their sequences are diverse (4). Class I and II hydrophobins are distinguished on the basis of differences in hydropathy patterns and biophysical properties (5). SC3 of Schizophyllum commune is the best characterized class I hydrophobin. It self-assembles at interfaces between water and air, water and oil, and water and hydrophobic solids (6–8). The four disulfide bridges of SC3 prevent spontaneous self-assembly in solution and thus account for the controlled assembly at hydrophobic-hydrophilic interfaces (9).The water-soluble form of SC3 is oligomeric (10) and rich in β-sheet (11). Upon assembly at the water-air interface, SC3 proceeds via an intermediate form that has increased α-helical structure (α-helical state) to a stable end form that has increased β-sheet structure (β-sheet state) (11–13). SC3 in the β-sheet state initially has no clear ultrastructure (β-sheet I state) (12), but after prolonged incubation, the protein forms 10-nm wide amyloid-like fibrils (β-sheet II state) (12–14) that are called rodlets (6, 15). Like other amyloid fibrils (16), rodlets of the hydrophobins SC3 of S. commune and EAS of Neurospora crassa increase fluorescence of thioflavin T and bind Congo red (14, 17, 18). Moreover, x-ray diffraction of rodlets of EAS showed reflections at 4.8 Å (distance between strands in a β-sheet) and 10–12 Å (spacing between β-sheets stacked perpendicular to the fibril long axis) (19), which are indicative for amyloid fibrils.Notably, SC3 does not spontaneously self-assemble into amyloid fibrils at an interface between water and a hydrophobic solid. Instead, SC3 is arrested in the intermediate α-helical state. Transition to the β-sheet state is observed only by heating the sample in the presence of detergent (11, 12). These observations prompted us to study conditions that promote assembly of SC3 into amyloid fibrils. Here, we show that amyloid formation of SC3 is promoted by increasing its concentration or by the presence of cell wall polysaccharides.     

Expression of a Fungal Hydrophobin in the Saccharomyces cerevisiae Cell Wall: Effect on Cell Surface Properties and Immobilization

The aim of this work was to modify the cell surface properties of Saccharomyces cerevisiae by expression of the HFBI hydrophobin of the filamentous fungus Trichoderma reesei on the yeast cell surface. The second aim was to study the immobilization capacity of the modified cells. Fusion to the Flo1p flocculin was used to target the HFBI moiety to the cell wall. Determination of cell surface characteristics with contact angle and zeta potential measurements indicated that HFBI-producing cells are more apolar and slightly less negatively charged than the parent cells. Adsorption of the yeast cells to different commercial supports was studied. A twofold increase in the binding affinity of the hydrophobin-producing yeast to hydrophobic silicone-based materials was observed, while no improvement in the interaction with hydrophilic carriers could be seen compared to that of the parent cells. Hydrophobic interactions between the yeast cells and the support are suggested to play a major role in attachment. Also, a slight increase in the initial adsorption rate of the hydrophobin yeast was observed. Furthermore, due to the engineered cell surface, hydrophobin-producing yeast cells were efficiently separated in an aqueous two-phase system by using a nonionic polyoxyethylene detergent, C_12-18EO₅.     

Spontaneous Self-Assembly of Engineered Armadillo Repeat Protein Fragments into a Folded Structure

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The Entry of Ammonia into Fungal Cells

When Scopulariopsis brevicaulis grown on an ammonia-free mediumis supplied with an ammonium salt ammonia enters the cells morerapidly than it is removed by assimilation, until an equilibriumlevel of ammonia is reached in the cells. The equilibrium concentrationin the cells is independent of metabolism and depends on theexternal concentration over a wide range. The internal concentra--tionof ammonia can be higher than the external under suitable conditionsof pH. The cells are shown to be permeable to ammonia also inthe outward direction, and the rate of entry or loss dependson the concentration difference between external and internalenvironment. The results support the view that ammonia enters the cells mainlyby the free diffusion of the undissociated molecule.     

Hydrophobic Interactions Involved in Attachment of a Baculovirus to Hydrophobic Surfaces

The hydrophobic interactions of Trichoplusia ni nuclear polyhedrosis virus were characterized by hydrophobic interaction chromatography. The determination of the hydrophobic force and some of the factors that influence its size is discussed in relation to the attachment to leaf surfaces of polyhedra during their use as biological control agents against insect pests.     

Self-Assembly of the Bacterial Cytoskeleton-Associated RNA Helicase B Protein into Polymeric Filamentous Structures

The Escherichia coli RNA degradosome proteins are organized into a helical cytoskeletal-like structure within the cell. Here we describe the ATP-dependent assembly of the RhlB component of the degradosome into polymeric filamentous structures in vitro, which suggests that extended polymers of RhlB are likely to comprise a basic core element of the degradosome cytoskeletal structures.The RNA degradosome plays an essential role in normal RNA processing and degradation. Within the cell, the degradosome proteins (RNA helicase B [RhlB], RNase E, polynucleotide phosphorylase [PNPase], and enolase) (4, 13, 15, 16) are organized into coiled structures that resemble the pole-to-pole helical structures of the MreB and MinCDE bacterial cytoskeletal systems (4, 12, 13). However, the degradosomal structures are also present in cells that lack the MreB and MinCDE cytoskeletal elements, suggesting that the degradosomal structures may be part of an independent class of prokaryotic cytoskeletal elements (19-21).One of the degradosomal proteins, RhlB, is organized into similar helical cellular structures in cells that lack the other degradosome proteins (Fig. ​(Fig.11 A). In addition, RhlB recruits PNPase to the helical framework in the absence of other degradosome proteins, suggesting that the RhlB structures are core elements of the degradosomal cytoskeletal-like elements of the cell (Fig. ​(Fig.1B)1B) (20). The cellular RhlB structures could be generated in two ways: (i) individual RhlB molecules may bind to an as-yet-undefined underlying track, or (ii) RhlB may polymerize to form the filamentous helical structures independent of any underlying template.Open in a separate windowFIG. 1.The RhlB filamentous cytoskeletal-like structures. (A) Cellular organization of RhlB based on immunofluorescence microscopy using purified anti-RhlB antibody in the absence of RNase E filamentous elements in AT8 cells (rne^1-417), which fail to generate RNase E coiled structures because of the absence of the RNase E cytoskeletal localization domain (20). (B) Proposed model for the cytoskeletal-like organization of the RNA degradosome (modified from reference 20). Arcs depict the RNase E (blue) and RhlB (red) helical strands. It is not known whether the RNase E helical strand is formed by RNase E polymerization or by the association of RNase E with an unknown underlying cytoskeletal structure. Enolase (Eno) and PNPase are shown in gray. Molecular dimensions and stoichiometry of the proteins were arbitrarily chosen to simplify the figure. (C to H) Electron micrographs of uranyl acetate-stained RhlB filaments (C, E, and H) and RhlB sheets (D). Unless otherwise indicated, the sample contained 9 μM RhlB, 2 mM ATP, 5 mM MgCl₂, and 5 mM CaCl₂. (E) Calcium was omitted. (F) ATP was omitted. (G and H) ATP was replaced by ATPγS (G) or AMP-PNP (H). Samples were loaded on glow-discharged 300-mesh carbon-coated copper grids and then stained. Images were taken with a JEOL 100CX transmission electron microscope. Magnification, ×10,000 to 50,000.Here we report that RhlB can self-assemble into extended polymeric structures in vitro in a process that requires ATP binding but not ATP hydrolysis. It is likely that extended RhlB polymers such as those described here are the basic components of the RhlB filamentous helical elements that comprise the core of the degradosomal cytoskeletal structures of the Escherichia coli cell.Evidence that RhlB can self-assemble into filamentous polymeric structures came from electron microscopic studies of purified His-tagged RhlB negatively stained with 2% uranyl acetate. This staining showed large numbers of long uniform filamentous structures when the purified protein was incubated in the presence of ATP and Ca²⁺ (Fig. ​(Fig.1C).1C). The filaments were 25 ± 1.8 nm wide (n = 91; mean ± standard deviation) and were generally more than 10 μm long. Some wider sheets were also observed (Fig. ​(Fig.1D).1D). Optimal assembly of the RhlB filamentous structures required ATP and Ca²⁺, as shown by the observation that only occasional single structures were present when the polymerization reaction was carried out in the absence of Ca²⁺ (Fig. ​(Fig.1E)1E) or ATP (Fig. ​(Fig.1F).1F). The polymeric RhlB-His structures were observed with approximately similar frequencies when ATP was replaced by the nonhydrolyzable ATP analog adenosine 5′-(γ-thiotriphosphate) (ATPγS) or AMP-PNP (Fig. 1G and H).Cellular localization studies showed that the presence of the His tag did not interfere with the ability of RhlB to form the helical cellular structures. Thus, RhlB-His was present in extended helical filamentous structures that were indistinguishable from those formed by untagged RhlB (20, 21). Similarly, the RhlB-His structures recruited PNPase to the helical framework in a manner similar to untagged RhlB (20) (see Fig. S1 in the supplemental material).Immunogold staining showed that the filaments and sheets were decorated with gold particles when stained with mouse anti-His tag antibody and gold-labeled secondary antibody (Fig. ​(Fig.22 A to C), confirming that the structures were composed of RhlB. In contrast, the structures were not decorated with gold particles in the absence of the primary antibody or when mouse anti-His tag antibody was replaced by nonimmune mouse IgG (Fig. ​(Fig.2D2D and E). The polymeric structures were observed with C-terminally His-tagged RhlB, which is functional in terms of helicase activity (8), but not when the tag was present at the amino terminus of the protein, where the His tag may interfere with RhlB self-assembly.Open in a separate windowFIG. 2.Immunogold electron microscopy of RhlB structures. Samples were prepared as describe for Fig. ​Fig.1C,1C, except that the grids were stained with 2% uranyl acetate after exposure to primary and/or secondary antibodies as indicated. (A to C) RhlB structures decorated with 10-nm gold particles in samples stained with mouse anti-His tag monoclonal antibody and gold-labeled secondary antibody. (A) single filaments; (B) clustered filaments; (C) a single RhlB filament and RhlB sheet. Arrows indicate gold particles. (D) The primary mouse anti-His tag antibody was replaced by mouse IgG. (E) The primary antibody was omitted.Changes in light scattering were used to follow the course of polymerization and to compare polymerization conditions in a more quantitative way than is possible by electron microscopy. The initial rate of increase in scattering was used to estimate polymerization rate (see Table S1 in the supplemental material). Significant rates of polymerization were observed in the presence of ATP and Ca²⁺, whereas there was very little increase in light scattering in the absence of nucleotide and/or Ca²⁺ (Fig. ​(Fig.33 A). ADP was less effective than ATP, whereas AMP and cyclic AMP (cAMP) were inactive (Fig. ​(Fig.3B).3B). In the presence of Ca²⁺, the extent and rate of RhlB polymerization varied as a function of ATP concentration (Fig. ​(Fig.3C).3C). Millimolar concentrations of Ca²⁺ were required to produce a measurable rate of polymerization in the light scattering assay (Fig. ​(Fig.3A).3A). It is not known how these relatively high concentrations of Ca²⁺ promote the in vitro polymerization of RhlB and other cytoskeletal proteins, such as MreB and FtsZ (1, 11, 12, 14, 23).Open in a separate windowFIG. 3.RhlB polymerization. (A and B) RhlB polymerization as shown by 90° light scattering is indicated in arbitrary units (a.u.). RhlB polymerization was followed at room temperature in a 1-cm light path quartz cuvette using a Hitachi fluorometer (FL-2500) set to 400 V with excitation and emission wavelengths set at 455 nm and a slit width of 10 nm. The reaction (100 μl volume) was performed in a polymerization buffer (50 mM Tris, 50 mM KCl, 5 mM MgCl₂; pH 8) as indicated. (A) The sample contained 9 μM RhlB-His, 1 mM ATP, and either 10 mM CaCl₂, 7.5 mM CaCl₂, or no CaCl₂ (squares). In the lower three curves the samples lacked ATP or CaCl₂, as indicated. (B) The sample contained 9 μM RhlB-His, 7.5 mM CaCl₂, and 1 mM adenosine nucleotides: ATP, ADP, AMP, cAMP, AMP-PNP, and ATPγS. (C) The sample contained 9 μM RhlB-His, 7.5 mM CaCl₂, and ATP as indicated (1 mM, 0.75 mM, 0.5 mM, 0.25 mM, or 0.1 mM ATP). (D) Effects of Ca²⁺ concentration on RhlB sedimentation in the presence of 2 mM ATP. RhlB in the pellet, expressed as a percentage of total RhlB present in the polymerization reaction mixture, was plotted against calcium concentration. The insert shows an example of a Coomassie blue-stained gel of supernatant (S) and pellet (P) fractions from the sedimentation assay in the presence of ATP and Ca²⁺ (see results for ATP in Table ​Table11).The nonhydrolyzable ATP analogs ATPγS and AMP-PNP were approximately equivalent to ATP in promoting polymerization as monitored by the light scattering assay (Fig. ​(Fig.3B)3B) as well as in the electron microscopic studies. This suggests that nucleotide binding, but not hydrolysis, is required to promote RhlB polymerization. In this regard, RhlB resembles a number of other proteins, including F-actin, MreB, and MinD, where polymerization is induced by nucleotide binding (2, 5, 6, 18, 22). In these systems, subsequent ATP hydrolysis induces depolymerization, providing the basis for the dynamic behavior of the polymers within the cell. RhlB is an RNA-dependent ATPase (7), but it is not yet known whether ATP hydrolysis is associated with depolymerization in the RhlB system.Similar results were obtained when the extent of polymerization was monitored by a sedimentation assay, measuring the proportion of RhlB in the pellet fraction after centrifugation at 278,000 × g for 10 min (Table ​(Table1;1; Fig. ​Fig.3D).3D). Essentially all of the protein was sedimentable at pH 8 in the presence of Ca²⁺ and ATP. RhlB sedimentation returned to background levels when EGTA or EDTA was added to the reaction mixture (Table ​(Table1),1), confirming the Ca²⁺ requirement for RhlB polymerization in the electron microscopic and light scattering analyses. ATPγS was equivalent to ATP in the sedimentation assay, confirming the results described above. The relatively high background of RhlB sedimentation was not affected by prespinning the samples prior to addition of nucleotides and/or Ca²⁺.

TABLE 1.

Sedimentation assay for RhlB polymerization

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.     

Nonequilibrium Self-Assembly of a Filament Coupled to ATP/GTP Hydrolysis

We study the stochastic dynamics of growth and shrinkage of single actin filaments or microtubules taking into account insertion, removal, and ATP/GTP hydrolysis of subunits. The resulting phase diagram contains three different phases: two phases of unbounded growth: a rapidly growing phase and an intermediate phase, and one bounded growth phase. We analyze all these phases, with an emphasis on the bounded growth phase. We also discuss how hydrolysis affects force-velocity curves. The bounded growth phase shows features of dynamic instability, which we characterize in terms of the time needed for the ATP/GTP cap to disappear as well as the time needed for the filament to reach a length of zero (i.e., to collapse) for the first time. We obtain exact expressions for all these quantities, which we test using Monte Carlo simulations.     

Production of Glycolipid Biosurfactants,Mannosylerythritol Lipids,Using Sucrose by Fungal and Yeast Strains,and Their Interfacial Properties

Glycolipid biosurfactants, mannosylerythritol lipids (MELs), were produced from glucose and sucrose without vegetable oils. Pseudozyma antarctica JCM 10317, Ustilago maydis NBRC 5346, U. scitaminea NBRC 32730, and P. siamensis CBS 9960 produced mainly MEL-A, MEL-A, MEL-B, and MEL-C respectively. The sucrose-derived MELs showed excellent interfacial properties: low critical micelle concentration as well as that of oil-derived MELs.     

Ambient pH Is a Major Determinant in the Expression of Cuticle-Degrading Enzymes and Hydrophobin by Metarhizium anisopliae

Secretion of proteolytic and chitinolytic enzymes is a hallmark of infection processes of Metarhizium anisopliae in response to host (insect) cuticular signals. The regulation of these enzymes (subtilisin-like proteases [Pr1a and Pr1b], trypsin-like proteases [Pr2], metalloproteases, aspartyl proteases, aminopeptidase, and chitinases) and a hydrophobin was investigated by Northern analysis and/or enzyme assay. The production of each enzyme showed a differential expression pattern in response to ambient pH; enzymes were synthesized only at pHs at which they function effectively, irrespective of whether the medium contained an inductive cuticle substrate. Three aspartyl proteases (pH optimum, 3), and chitinase (pH optimum, 5) showed maximal accumulation at acidic pHs. The highest level of aminopeptidase (pH optimum, 7) was detected at pH 7. The highest levels of five metalloproteases (pH optima, ca. 7) were detected over the pH range 6 to 8. Two trypsins and several subtilisin-like Pr1 isoforms with pH optima of ca. 8 were produced only under alkaline conditions. Northern analysis of RNA species corresponding to seven cDNA sequences encoding proteases and chitinase confirmed that the ambient pH played a major role in gene expression of secreted proteins. Hydrophobin was expressed almost equally at pHs 5 and 8 but was not expressed at pH 3. During fungal penetration, the pH of infected cuticle rises from about 6.3 to 7.7. Consistent with pH regulation of enzyme production, serine and metalloproteases were produced in situ during infection, but no production of aspartyl proteases was found. We propose that the alkalinity of infected cuticle represents a physiological signal that triggers the production of virulence factors.     

Hydrophobic and acidic moments of a nucleoplasmin NP-core chaperone

A recent crystal structure, at atomic resolution, of the NO38-core chaperone has revealed a decamer comprised of a dimer of pentamers, with each pentamer consisting of closely coupled eight-stranded beta-barrel monomers. This N-terminal core domain of the chaperone shares the Nucleoplasmin family fold and is presumed to assist the binding of the core histones in their assembly into nucleosomes during DNA replication and repair. The present work provides a measure of the hydrophobic residue burial about the different interfaces and centers of the NO38-core multimeric structure. While the hydrophobic "pentameric ring," comprised of the hydrophobic cores of the monomers and prevalence of non-polar residues at their interfaces is observed, a hydrophobic bias with respect to the center of the pentamer is also found, and consequently also expected to contribute to the thermostability of the multimer. Structural and chromatographic analysis had shown the NO38-core chaperone to bind (H3-H4)2 histone tetramers as well as H2A-H2B dimers. The acidic dipole, which reflects the spatial disposition of the acidic residues of the core monomer points to the lateral region of the monomers comprising the oligomers, and thereby, shows it to be the region of charge that would optimally complement the basic charge of the histones in their electrostatic binding to the chaperone. It is also pointed out that the prevalence of basic residues on the short helices of the histone cores also provides regions of charge that would complement histone binding to the chaperone.