Surface-active proteins enable microbial aerial hyphae to grow into the air

Han A. B. Wösten1 and Joanne M. Willey2

Department of Microbiology, Groningen Biotechnology and Biomolecular Science Institute, Biological Centre, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands1
Department of Biology, Hofstra University, Hempstead, NY 11549, USA2

Author for correspondence: Han A. B. Wösten. Tel: +31 50 3632143. Fax: +31 50 3632154. e-mail: wostenha{at}biol.rug.nl

Keywords: streptomycetes, fungi, differentiation, aerial growth, surfactant, hydrophobin


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Although filamentous bacteria (i.e. the streptomycetes) and filamentous fungi belong to different kingdoms that diverged early in evolution, they adopted similar life styles. Both groups form aerial structures from which (a)sexual spores can develop. Some of the key processes involved in the formation of aerial hyphae by these microbes appear to be very similar. Both groups secrete highly surface-active molecules that lower the surface tension of their aqueous environment enabling hyphae to grow into the air. In the case of filamentous bacteria, small peptides (i.e. SapB and streptofactin) are secreted, while filamentous fungi use proteins known as hydrophobins to decrease the water surface tension. Although these fungal and bacterial molecules are not structurally related, they can, at least partially, functionally substitute for each other. Once escaped into the air, hyphae are covered with a hydrophobic film. In both filamentous fungi and filamentous bacteria this film is characterized by a mosaic of parallel rodlets. In fungi, this is the result of the self-assembly of hydrophobins but their bacterial equivalents have not yet been identified.


   Background
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Filamentous fungi and filamentous bacteria (i.e. actinomycetes, which include Streptomyces spp.) display a very similar lifestyle. In fact, it was only in the 1950s that it was established that these groups belong to different kingdoms (Goodfellow et al., 1983 ). Both filamentous fungi and filamentous bacteria occur in a wide variety of natural and man-made habitats. They are saprotrophic, and can be pathogens or symbionts of plants or animals. Representatives of both groups initially form a branched submerged mycelium consisting of filaments that are surrounded by rigid cell walls. These filaments grow at their apices and secrete large amounts of enzymes that degrade polymeric substrates (e.g. plant material) into small molecules that serve as nutrients. After a substrate feeding mycelium has been established, both filamentous fungi and filamentous bacteria may leave the hydrophilic environment to form aerial hyphae that may further differentiate to simple or elaborate spore-bearing aerial structures. Aerial hyphae of most species are hydrophobic and are characterized by rodlet-decorated surfaces. In this article we will describe observations that show that growth of aerial hyphae in fungi and bacteria involves surprisingly similar mechanisms. Although the molecules involved in aerial growth (i.e. SapB, streptofactin and hydrophobins) are not homologous, they can, at least partially, functionally substitute for each other. We have not extensively discussed the biochemical and biophysical properties of these surface-active molecules, although their characterization was instrumental in establishing their role in aerial growth. For this, we refer to recent reviews by Wösten et al. (1999a) and Wösten & de Vocht (2000) .


   Surface-active proteins involved in formation of aerial hyphae in filamentous fungi
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Hydrophobins have been identified as proteins involved in fungal emergent growth. They are small (~100 aa) secreted proteins characterized by similar hydropathy patterns and by the presence of eight conserved cysteine residues (Wessels, 1997 ). Class I hydrophobins have the remarkable capacity to self-assemble at any hydrophilic–hydrophobic interface into a 10 nm thick amphipathic protein film. The hydrophobic side of such films typically features a mosaic of parallel rodlets (Wösten et al., 1993 , 1994b , 1995 ). Self-assembly at the water–air interface is accompanied by a dramatic decrease in the water surface tension. For instance, self-assembled SC3 hydrophobin of the basidiomycete Schizophyllum commune reduces the water surface tension from 72 to as low as 24 mJ m-2, making it the most surface-active protein identified to date (Wösten et al., 1999b ). Hydrophobins fulfil a broad spectrum of functions in fungal development. They mediate escape of fungal hyphae from their hydrophilic environment, coat fungal surfaces exposed to the air with a hydrophobic membrane and mediate attachment of hyphae to hydrophobic surfaces (Wessels, 1997 ; Wösten & Wessels, 1997 ; Talbot, 1997 ; Wösten et al., 1999b ).

The role of hydrophobins in the formation of aerial hyphae is exemplified by the class I hydrophobin SC3 of Sch. commune. Like other filamentous fungi, Sch. commune colonizes moist substrates. After a feeding submerged mycelium has been established this fungus can form sterile aerial hyphae and fruiting bodies that are involved in sexual reproduction. In erecting these aerial structures, the fungus is confronted with the high surface tension of the water film surrounding the hyphae. To escape the barrier of the water surface tension (72 mJ m-2), Sch. commune secretes the SC3 hydrophobin which lowers the water surface tension enabling hyphae to breach the interface and to grow into the air (Fig. 1; Wösten et al., 1999b ). Young cultures not yet forming aerial structures do not express the SC3 gene and the water surface tension remains high. After 3 d growth, the first SC3 monomers are secreted into the culture medium and the surface tension drops to 45 mJ m-2, correlating with the emergence of the first aerial hyphae. Most aerial hyphae are formed after 4 d growth, which is accompanied by a high level of SC3 gene expression and a maximal drop in the medium surface tension to 27 mJ m-2. In an isogenic strain of Sch. commune in which the SC3 gene is disrupted (strain {Delta}SC3) (Wösten et al., 1994b ; van Wetter et al., 1996 ), surface tension decreases maximally to only 45 mJ m-2 and few aerial hyphae are observed (Wösten et al., 1999b ). However, the decrease in surface tension and the formation of aerial hyphae are restored by the addition of purified hydrophobin to the culture medium. The observation that in both the wild-type and the {Delta}SC3 strain aerial hyphae begin to emerge at a surface tension of 45 mJ m-2 indicates that this is a critical surface tension that allows hyphae to breach the interface (Wösten et al., 1999b ). At this surface tension, hyphae probably grow perpendicular to the interface, while hyphae that escape the interface at a lower surface tension may have met the surface at a lower contact angle.



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Fig. 1. Model for the formation of aerial hyphae in the filamentous fungus Sch. commune and the filamentous bacterium Str. coelicolor. After a submerged feeding mycelium has been formed, Sch. commune secretes SC3 into the medium, while Str. coelicolor produces SapB. These molecules lower the surface tension of the aqueous environment enabling hyphae to escape the substrate and to grow into the air. SC3 lowers the surface tension by assembling into an amphipathic membrane at the water–air interface. SC3 secreted by aerial hyphae of Sch. commune assembles at the interface between the hydrophilic cell wall and the hydrophobic air exposing its hydrophobic side, which is characterized by a mosaic of rodlets. The hydrophobic surface of aerial hyphae of Str. coelicolor is also typified by a rodlet layer. Although the molecules forming this layer have not yet been identified, evidence suggests it is not SapB.

 
Strain {Delta}SC3 does not form aerial hyphae in a liquid standing culture. However, when grown on solid medium the phenotype is less severe (van Wetter et al., 1996 ). This is probably due to a low level of the SC4 hydrophobin that is produced under these conditions. When this hydrophobin was expressed behind the SC3 promoter, formation of aerial hyphae in the {Delta}SC3 strain was restored (van Wetter et al., 2000 ). In contrast, when expression of SC4 was reduced by increasing the carbon dioxide concentration, formation of aerial hyphae was strongly reduced in the {Delta}SC3 strain.

Because the tips of aerial hyphae are isolated from the aqueous milieu of the feeding mycelium, SC3 secreted at the tips of such hyphae does not diffuse into the medium. Instead, SC3 monomers self-assemble at the interface between the hydrophilic cell wall and the hydrophobic air, forming an amphipathic film that coats the emergent aerial hyphae while at the same time conferring hydrophobicity to the hyphal surface (Fig. 1; Wösten et al., 1994a ). Self-assembled SC3 imparts a characteristic mosaic of parallel rodlets found on the surface of emergent hyphae (Wösten et al., 1993 ). However, only a fraction of the total hydrophobin produced is secreted by the hyphal tips of the emergent filaments. The majority of SC3 is secreted by submerged hyphae. It remains in the medium where it only facilitates hyphal erection into the air. To be coated with a hydrophobic film, emergent hyphae must themselves secrete SC3 (Fig. 1; Wösten et al., 1999b ).

If lowering the surface tension is required to allow hyphae to grow into the air, one would expect that other surface-active molecules would also be effective. Many surfactants, however, interfere with the plasma membrane and are therefore toxic (Lang & Wagner, 1993 ). Indeed, of all surfactants tested, only the fungal class I hydrophobins SC4 and ABH3, which are known to self-assemble, and the bacterial peptide streptofactin of Streptomyces tendae restore formation of aerial hyphae in the {Delta}SC3 strain (Wösten et al., 1999b ; Lugones et al., 1998 ). The capacity of fungal hydrophobins to lower the water surface tension is the result of a conformational change that occurs when they self-assemble at the water–air interface (van der Vegt et al., 1996 ; de Vocht et al., 1998 ). The resulting amphipathic film is not expected to diffuse through the cell wall and to interfere with the plasma membrane, which could explain their nontoxicity and their capacity to facilitate hyphal emergence. We expect that the model as presented for Sch. commune is a general mechanism for fungal aerial growth. Other fungi also secrete hydrophobins into the medium (see Wösten & Wessels, 1997 ), one of which, the ABH3 hydrophobin of Agaricus bisporus, restores lowering of the surface tension in a {Delta}SC3 strain (Lugones et al., 1998 ). A similar mechanism is thought to account for the capacity of bacterial amphipathic peptides to facilitate aerial growth (Tillotson et al., 1998 ; Richter et al., 1998 ).

Are hydrophobins the only class of structural proteins involved in formation of fungal aerial hyphae? Other proteins in the medium of a monokaryon of Sch. commune did not complement for the ability of SC3 to reduce the surface tension of the medium. Yet, it may very well be that other cell-wall proteins are indispensable for formation of aerial hyphae. In the maize pathogen Ustilago maydis a class of structural cell-wall proteins was identified that has a role in the formation of aerial hyphae (Wösten et al., 1996 ). Disruption of the Rep1 gene, which, by processing, produces 12 peptides of 35–53 aa called repellents, resulted in a strain that could not form aerial hyphae and had a wettable phenotype. This phenotype is very similar to that of a Sch. commune strain with a disrupted SC3 gene. Till now the function of repellents was not known.


   Structural peptides involved in formation of aerial hyphae by filamentous bacteria
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The role of amphipathic peptides in the emergence of aerial hyphae of filamentous bacteria has been best studied in Streptomyces coelicolor and Streptomyces tendae. In both species, ‘bald’ (bld) mutants have been isolated that are unable to form aerial hyphae under certain growth conditions. This was found to be correlated with the failure to secrete SapB and streptofactin, respectively (Willey et al., 1991 ; Richter et al., 1998 ). SapB consists of 18 amino acids (Willey et al., 1991 ), while streptofactin is an octapeptide (Richter et al., 1998 ). Both peptides bear a nonproteinaceous moiety and are believed to be synthesized non-ribosomally like the peptide antibiotics (Richter et al., 1998 ; Willey et al., 1993 ). Streptofactin and SapB are surface active. They reduce the water surface tension to as low as 39 mJ m-2 and 32 mJ m-2, respectively (Richter et al., 1998 ; Tillotson et al., 1998 ). Like SC3 of Sch. commune, both SapB and streptofactin are believed to facilitate the emergence of aerial hyphae by breaking the surface tension at the colony–air interface (Willey et al., 1991 ; Richter et al., 1998 ). The reduction in water surface tension by these peptides is at least enough to enable hyphae of Sch. commune to breach the water–air interface (see above).

The functional homology of SapB and streptofactin is demonstrated by their ability to restore to bld mutants of either Str. coelicolor or Str. tendae the capacity to form aerial hyphae upon the exogenous addition of either peptide. Surprisingly, the structurally unrelated fungal SC3 hydrophobin also extracellularly complements bld mutants of both Streptomyces species (Tillotson et al., 1998 ), while the application of streptofactin to the {Delta}SC3 strain of Sch. commune restores its ability to form aerial hyphae (Wösten et al., 1999b ; see above). Whereas the vegetative (i.e. the substrate) hyphae formed by the bld mutants are hydrophilic, the aerial hyphae of extracellularly complemented bld mutants, like that of wild-type aerial hyphae and spores, are hydrophobic (Richter et al., 1998 ). More strikingly, unlike wild-type streptomycete aerial hyphae that curl and septate into spores, aerial hyphae formed by bld mutants after the addition of SapB, streptofactin or SC3 to the colony surface do not curl nor septate to form chains of spores (Tillotson et al., 1998 ). From this it is concluded that the addition of amphipathic peptides or proteins does not restore the capacity of the mutants to undergo complete morphological differentiation, rather it allows the release of vegetative hyphae from the colony surface. Their upward growth is responsible for the white, fuzzy appearance of the colony.

Similar to the situation in Sch. commune, not all surface-active molecules induce the formation of aerial hyphae (Richter et al., 1998 ). Viscosin of Pseudomonas fluorescens (Neu et al., 1990 ) and fengycin and surfactin of Bacillus subtilus (Vanittanakom et al., 1986 ; Kakinuma & Arima, 1969 ) are all shown to be ineffective. These data show that only specific surface-active molecules can restore aerial hyphae formation in filamentous micro-organisms and that the structurally unrelated molecules found in filamentous prokaryotic and eukaryotic microbes have been functionally converged.

It is not yet clear whether SapB and streptofactin have a role once the filaments of Str. coelicolor and Str. tendae have escaped the hydrophilic environment. It had been suggested that SapB might coat aerial structures of Str. coelicolor, making them hydrophobic (Willey et al., 1991 ). However, using immunogold labelling we were unable to localize this peptide either within the aerial hyphae or on their surface. A strong signal was found only in the culture medium (Fig. 2). A similar result was obtained in Streptomyces lividans, which is closely related to Str. coelicolor (data not shown). From these data we postulate that SapB reduces the surface tension of the medium but that other molecules coat aerial hyphae of Str. coelicolor to make them hydrophobic. Like the hydrophobin coating found on fungal aerial hyphae, the coating found on the aerial hyphae of streptomycetes is characterized by a rodlet layer (Wildermuth et al., 1972 ). Bradley & Ritzi (1968) suggested that the Streptomyces venezuelae rodlet mosaic is lipid-like, while Smucker & Pfister (1978) proposed that the rodlets of Str. coelicolor are composed of polymers of N-acetylated glucosamine. Yet, a significant amount of protein was shown to be present in the sample. However, the partial genome sequence of Str. coelicolor has not revealed protein sequences similar to those of the fungal hydrophobins.



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Fig. 2. Immunolocalization of SapB in a 5-d-old colony of Str. coelicolor grown on soy–mannitol medium at 30 °C. Aerial (a) and submerged hyphae (b) are shown. Fixation, embedding and immunolabelling of cultures were performed as described by Wösten et al. (1994a) except with the modification that Unicryl was substituted for K4M resin. Polyclonal antibodies raised against SapB (Willey et al., 1991 ) were purified with an acetone-powder of mycelium of a shaken culture of the bldA mutant of Str. coelicolor and diluted 1000 times. Similar results were obtained when freeze substitution instead of convential fixation was used or when Str. lividans instead of Str. coelicolor was labelled. No signals were observed in standing cultures of the bld261 mutant or in liquid shaken cultures of Str. coelicolor. Bar, 300 nm.

 

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Because SapB is diffusable, it is not surprising that Str. coelicolor bld mutants grown near a SapB-producing wild-type strain regain the capacity to erect aerial hyphae. Such hyphae are confined to the zone of SapB diffusion from the wild-type (Willey et al., 1991 ). However, an unexpected observation is that the formation of aerial hyphae can also be restored by growing certain pairs of bld mutants in close proximity to each other, even though all these mutants are blocked in SapB biosynthesis. Extracellular complementation is initially unidirectional; one mutant acting as a donor, the other as an acceptor (Willey et al., 1993 ). Such extracellular complementation assays reveal a hierarchical set of complementation groups in which each mutant can rescue morphogenesis in the mutant strain to the left: bldJ<bldK<bldA, bldH<bldG<bldC<bldD (Willey et al., 1993 ; Nodwell et al., 1996 ). Thus, bldJ (formerly referred to as bld261) can be rescued by all other bld mutants, while bldD can only be rescued by the wild-type strain. As expected from a complementation assay, once the acceptor is sufficiently complemented by the donor (i.e. sufficiently differentiated), it can ‘back-complement’ the donor strain, which ultimately leads to full differentiation of both bld mutants. The complementation tests suggest the existence of a total of at least five bld-dependent signalling molecules that are involved in morphological differentiation. Each signal would trigger the synthesis and release of the next signal, ultimately resulting in the bldD-dependent production of morphogenetic molecules like SapB (Willey et al., 1993 ; Nodwell et al., 1996 ). It was hypothesized that this cascade allows the bacterium to couple morphological differentiation with environmental cues like the nutritional status (Champness, 1988 ; Merrick, 1976 ; Pope et al., 1996 ) and cell density (Nodwell et al., 1996 ).

Although not all bld mutants fit neatly into the hierarchical cascade, the existence of a signalling cascade in Str. coelicolor is supported by genetic and biochemical data. The bldK locus encodes a putative oligopeptide permease of the ATP-binding cassette (ABC) membrane-spanning transporter family and bldK mutants are resistant to the toxic tripeptide bialaphos (Nodwell et al., 1996 ), indicating that the product of the bldK locus transports an extracellular peptide. It is hypothesized that the production of this peptide is under control of the bldJ gene. Partial purification of the BldK signalling molecule suggests that it is a 655 Da peptide (Nodwell & Losick, 1998 ). The model predicts that in the wild-type, import of this molecule by the BldK peptide permease induces production of a second extracellular signal that depends on the products of both bldA and bldH. This signal is secreted and triggers release of the next signal and so on until bldD is triggered to release its signal which either directly or indirectly results in SapB production (Nodwell et al., 1996 ). Whether SapB is produced by all growing submerged hyphae, like SC3 in Sch. commune (see below), is not known. Not all bld genes are directly involved in signalling molecule, transporter or receptor production. For instance, bldA encodes a leucyl-tRNA that is required for the translation of the rare UUA-containing mRNA (Lawlor et al., 1987 ).

Most recently, the production of an extracellular regulatory signal(s) from the Str. coelicolor bldF mutant strain 166 (Passantino et al., 1991 ) has been studied. The signal, which is heat stable, protease resistant and capable of passing through a dialysis tubing with a molecular mass cut-off of 3 kDa, is present on solid as well as in liquid complex medium (unlike other bld mutants). In contrast to the aerial hyphae formed upon the addition of amphipathic peptides, dilution of rich agar medium with strain 166-conditioned liquid medium restored the capacity of both bldK (strain NS40; Nodwell et al., 1996 ) and bldJ (strain HU261; Willey et al., 1993 ) mutants to undergo complete morphological differentiation (i.e. production of sporulating aerial hyphae) and to produce SapB as well as pigmented antibiotics (Fig. 3). Furthermore, the 166 spent medium also influenced the rate of differentiation in the wild-type, such that a 1:5 dilution (strain 166 spent medium:fresh culture medium) accelerated morphological differentiation in the wild-type strain Str. coelicolor J1501 by about 24 h. Presumably, wild-type Str. coelicolor is capable of producing the same extracellular signals as the bld mutants but signal synthesis is temporally regulated. The introduction of the signal(s) into the medium during vegetative growth would then be expected to initiate the reprogramming and acceleration of the developmental cycle. The capacity of strain 166 spent medium to accelerate morphogenesis is reminiscent of experiments in which it was shown that homoserine lactone-containing spent medium can elicit precocious expression of cell-density-dependent genes (for a review see Fuqua et al., 1996 ).



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Fig. 3. Extracellular complementation of bldJ and bldF. Str. coelicolor 166 (bldF) was plated on the left half of an R2Ye plate (red-pigmented cells), while strain Hu241 (bldJ) was plated on the right half. After 4 d growth at 30 °C, colonies of bldJ closest to those of bldF displayed abundant aerial hyphae formation.

 

   Regulation of aerial hyphae formation in Sch. commune
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The SC3 hydrophobin gene of Sch. commune is expressed only after a few days of submerged growth (see above). Expression of SC3 was studied in a culture using green fluorescent protein as a reporter. It was shown that most, if not all, submerged growing hyphae contribute to SC3 production in the medium and that this is not restricted to those hyphae that are destined to grow into the air (Lugones et al., 1999 ). It was proposed that transcription of SC3 begins only after a submerged mycelium has formed that can support growth of individual aerial hyphae and fruiting bodies by supplying nutrients (Wessels, 1992 ). How the mycelium senses that it has reached its ‘critical mass’ remains to be established.

The thn mutant of Sch. commune could be considered a phenotypic analogue of the bld mutants of Streptomyces. It does not produce aerial structures and does not express the SC3 gene (Wessels et al., 1991 ). Because a mutation in the THN gene has pleiotropic effects it is possible that it is a regulatory gene (Wessels et al., 1991 ). It was proposed (Wessels et al., 1995 ) that repression of THN gene expression in juvenile cultures prevents the premature transcription of genes involved in emergent growth. This would thus allow formation of an assimilative mycelium that could sustain the growth of emergent structures. Possibly, the THN gene is part of a signalling cascade analogous to that proposed in Str. coelicolor. Evidence exists that the filamentous fungus Histoplasma capsulatum can sense its own numbers (Strauss, 1999 ), while it was suggested that sensing cell density is involved in differentiation of aerial hyphae in Aspergillus nidulans (Lee & Adams, 1994 ). In fluG- deletion mutants, aerial hyphae do not differentiate to form asexual spores. The deletion could be rescued by growing the mutant next to a wild-type strain, as was done in the case of bld mutants of Str. coelicolor. It was suggested that FluG is involved in the production of an extracellular signalling molecule that above a certain concentration would trigger differentiation of aerial hyphae. The signalling cascades in filamentous fungi may not necessarily be as complex as that of Str. coelicolor. In the fungus Saccharomyces cerevisiae, a single kind of pheromone can induce an entire developmental process and this development is coupled to environmental signals such as the nutritional status (Herskowiz, 1989 ).


   Concluding remarks
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Fungi and bacteria appear to have independently evolved the ability to form aerial structures that allow efficient spore dispersal. Both groups of micro-organisms are confronted with the high water surface tension and the need for a submerged feeding mycelium. The former requires molecules that lower the surface tension, the latter monitoring of cell density. As long as the surface-activity is attained by self-assembly and is thereby not toxic to the cell, any such molecule should enable microbial hyphae to escape the aqueous environment to grow into the air. In contrast, monitoring cell density requires specific molecules.


   ACKNOWLEDGEMENTS
 
We are indebted to Klaas Sjollema for performing the immunolocalization of SapB and to Heine Deelstra for stimulating discussions.


   REFERENCES
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REFERENCES
 
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