Membrane association and kinase-like motifs of the RamC protein of Streptomyces coelicolor

The protein RamC is required for the production of the spore-forming cells called aerial hyphae by the filamentous bacterium Streptomyces coelicolor. We showed that RamC, which contains several weakly predicted membrane-spanning sequences, is located exclusively in the S. coelicolor membrane. By constructing site-directed mutants in the cloned ramC gene and complementing a ramC null mutant, we showed that protein kinase-like sequence motifs in the amino-terminal half of the protein are required for function in vivo. These data suggest that RamC is a membrane-associated receptor kinase.     

Dimerization of the RamC morphogenetic protein of Streptomyces coelicolor

RamC is required for the formation of spore-forming cells called aerial hyphae by the bacterium Streptomyces coelicolor. This protein is membrane associated and has an amino-terminal protein kinase-like domain, but little is known about its mechanism of action. In this study we found that the presence of multiple copies of a defective allele of ramC inhibits morphogenesis in S. coelicolor, consistent with either titration of a target or formation of inactive RamC multimers. We identified a domain in RamC that is C terminal to the putative kinase domain and forms a dimer with a K(d) of approximately 0.1 micro M. These data suggest that RamC acts as a dimer in vivo.     

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.     

SapB and the chaplins: connections between morphogenetic proteins in Streptomyces coelicolor

Morphogenesis in the streptomycetes features the differentiation of substrate-associated vegetative hyphae into upwardly growing aerial filaments. This transition requires the activity of bld genes and the secretion of biosurfactants that reduce the surface tension at the colony-air interface enabling the emergence of nascent aerial hyphae. Streptomyces coelicolor produces two classes of surface-active molecules, SapB and the chaplins. While both molecules are important for aerial development, nothing is known about the functional redundancy or interaction of these surfactants apart from the observation that aerial hyphae formation can proceed via one of two pathways: a SapB-dependent pathway when cells are grown on rich medium and a SapB-independent pathway on poorly utilized carbon sources such as mannitol. We used mutant analysis to show that while the chaplins are important, but not required, for development on rich medium, they are essential for differentiation on MS (soy flour mannitol) medium, and the corresponding developmental defects could be suppressed by the presence of SapB. Furthermore, the chaplins are produced by conditional bld mutants during aerial hyphae formation when grown on the permissive medium, MS, suggesting that the previously uncharacterized SapB-independent pathway is chaplin dependent. In contrast, a bld mutant blocked in aerial morphogenesis on all media makes neither SapB nor chaplins. Finally, we show that a constructed null mutant that lacks all chaplin and SapB biosynthetic genes fails to differentiate in any growth condition. We propose that the biosurfactant activities of both SapB and the chaplins are essential for normal aerial hyphae formation on rich medium, while chaplin biosynthesis and secretion alone drives aerial morphogenesis on MS medium.     

Extracellular complementation of a developmental mutation implicates a small sporulation protein in aerial mycelium formation by S. coelicolor

The filamentous bacterium S. coelicolor differentiates by forming aerial hyphae, which protrude into the air and metamorphose into chains of spores. Aerial hyphae formation is associated with the production of a small, abundant protein, SapB, which is present in a zone around colonies of differentiating bacteria. Production of SapB is impaired in bld mutants, which are blocked in aerial hyphae formation, but not in whi mutants in which spore formation is prevented. We report that aerial hyphae formation by a newly identified bld mutant is restored by juxtaposition of the mutant near colonies of SapB-producing bacteria or by the application of the purified protein near mutant colonies. These observations implicate SapB in aerial mycelium formation and suggest that SapB is a morphogenetic protein that enables hyphae on the surface of colonies to grow into the air.     

Cloning and analysis of a gene cluster from Streptomyces coelicolor that causes accelerated aerial mycelium formation in Streptomyces lividans.

We describe the cloning and analysis of two overlapping DNA fragments from Streptomyces coelicolor that cause aerial mycelium to appear more rapidly than usual when introduced into Streptomyces lividans on a low-copy-number plasmid vector. Colonies of S. lividans TK64 harboring either clone produce visible aerial mycelia after only 48 h of growth, rather than the usual 72 to 96 h. From deletion and sequence analysis, this rapid aerial mycelium (Ram) phenotype appears to be due to a cluster of three genes that we have designated ramA, ramB, and ramR. Both ramA and ramB potentially encode 65-kDa proteins with homology to ATP-dependent membrane-translocating proteins. A chromosomal ramB disruption mutant of S. lividans was found to be severely defective in aerial mycelium formation. ramR could encode a 21-kDa protein with significant homology to the UhpA subset of bacterial two-component response regulator proteins. The overall organization and potential proteins encoded by the cloned DNA suggest that this is the S. coelicolor homolog of the amf gene cluster that has been shown to be important for aerial mycelium formation in Streptomyces griseus. However, despite the fact that the two regions probably have identical functions, there is relatively poor homology between the two gene clusters at the DNA sequence level.     

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.     

Aerial development in Streptomyces coelicolor requires sortase activity

Streptomyces coelicolor is a multicellular bacterium whose life cycle encompasses three differentiated states: vegetative hyphae, aerial hyphae and spores. Among the factors required for aerial development are the 'chaplins', a family of eight secreted proteins that coat the surface of aerial hyphae. Three chaplins (the 'long' chaplins, ChpA, B and C) possess an LAXTG-containing C-terminal sorting signal and are predicted sortase substrates. The five remaining 'short' chaplins are presumed to be associated with the cell surface through interactions with the long chaplins. We show here that two sortase enzymes, SrtE1 and SrtE2, cleave LAXTG-containing peptides at two distinct positions in vitro, and are required for cell wall anchoring of ChpC in vivo. srtE1/E2 double mutants are delayed in aerial hyphae formation, do not sporulate and fail to display all short chaplins on their aerial surfaces. Surprisingly, these mutant characteristics were not shared by a long chaplin mutant, which exhibited only modest delays in aerial development, leading us to revise the current model of chaplin-mediated aerial development. The sortase mutant phenotype, instead, appears to stem from an inability to transcribe aerial hyphae-specific genes, whose products have diverse functions. This suggests that sortase activity triggers an important, and previously unknown, developmental checkpoint.     

Contribution of aerial hyphae of Aspergillus oryzae to respiration in a model solid-state fermentation system

Oxygen transfer is for two reasons a major concern in scale-up and process control in industrial application of aerobic fungal solid-state fermentation (SSF): 1) heat production is proportional to oxygen uptake and it is well known that heat removal is one of the main problems in scaled-up fermenters, and 2) oxygen supply to the mycelium on the surface of or inside the substrate particles may be hampered by diffusion limitation. This article gives the first experimental evidence that aerial hyphae are important for fungal respiration in SSF. In cultures of A. oryzae on a wheat-flour model substrate, aerial hyphae contributed up to 75% of the oxygen uptake rate by the fungus. This is due to the fact that A. oryzae forms very abundant aerial mycelium and diffusion of oxygen in the gas-filled pores of the aerial hyphae layer is rapid. It means that diffusion limitation in the densely packed mycelium layer that is formed closer to the substrate surface and that has liquid-filled pores is much less important for A. oryzae than was previously reported for R. oligosporus and C. minitans. It also means that the overall oxygen uptake rate for A. oryzae is much higher than the oxygen uptake rate that can be predicted in the densely packed mycelium layer for R. oligosporus and C. minitans. This would imply that cooling problems become more pronounced. Therefore, it is very important to clarify the physiological role of aerial hyphae in SSF.     

NepA is a structural cell wall protein involved in maintenance of spore dormancy in Streptomyces coelicolor

Streptomycetes have a complex morphogenetic programme culminating in the formation of aerial hyphae that develop into chains of spores. After spore dispersal, environmental signals trigger dormant spores to germinate to establish a new colony. We here compared whole genome expression of a wild-type colony of Streptomyces coelicolor forming aerial hyphae and spores with that of the chp null mutant that forms few aerial structures. This revealed that expression of 244 genes was significantly altered, among which genes known to be involved in development. One of the genes that was no longer expressed in the Δ chpABCDEFGH mutant was nepA , which was previously shown to be expressed in a compartment connecting the substrate mycelium with the sporulating parts of the aerial mycelium. We here show that expression is also detected in developing spore chains, where NepA is secreted to end up as a highly insoluble protein in the cell wall. Germination of spores of a nepA deletion mutant was faster and more synchronous, resulting in colonies with an accelerated morphogenetic programme. Crucially, spores of the Δ nepA mutant also germinated in water, unlike those of the wild-type strain. Taken together, NepA is the first bacterial structural cell wall protein that is important for maintenance of spore dormancy under unfavourable environmental conditions.     

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.     

Regulation of Streptomyces development: reach for the sky!

Streptomyces coelicolor is characterized by a complex life cycle and serves as a model system for bacterial development. After a feeding substrate mycelium has been formed, this filamentous bacterium differentiates by forming aerial hyphae that septate into spores. The bld cascade regulates initiation of aerial growth, whereas the whi genes control spore formation. Recent findings indicate the existence of another regulatory pathway that operates after aerial hyphae have started to grow into the air, which we call the sky pathway. This pathway controls the expression of the chaplin and rodlin genes. These genes encode proteins that assemble into a rodlet layer that provides surface hydrophobicity to aerial hyphae and spores.     

A novel Streptomyces gene, samR, with different effects on differentiation of Streptomyces ansochromogenes and Streptomyces coelicolor

A 1.4-kb DNA fragment from Streptomyces ansochromogenes accelerated mycelium formation of S. ansochromogenes when present on a multicopy plasmid. The DNA fragment contains one complete open reading frame, designated samR, encoding a protein with 213 amino acids that contains a likely DNA-binding helix-turn-helix motif close to its N-terminus. The deduced SamR protein resembles the product of the hppR gene, which is involved in the regulation of catabolism of 3-(3-hydroxyphenyl) propionate in Rhodococcus globerulus. A samR disruption mutant was constructed that presented a bald phenotype and failed to form aerial hyphae and spores. We suggest that samR plays an important role in the emergence of aerial hyphae from substrate mycelium. An almost identical gene of Streptomyces coelicolor was also subjected to gene disruption. Surprisingly, the mutant was able to develop an aerial mycelium, but it remained white and deficient in sporulation instead of forming gray spores.     

Expression analysis of the spi gene in the pock-forming plasmid pSA1.1 from Streptomyces azureus and localization of its product during differentiation

The sporulation inhibitory gene spi in the pock-forming conjugative plasmid pSA1.1 of Streptomyces azureus was introduced into cells via a high or low copy number vector to examine the effect of gene dosage on the growth of Streptomyces lividans TK24 as a host. In transformants carrying a high spi copy number, nutrient mycelial growth was inhibited, as was morphological differentiation from substrate mycelium to aerial mycelium on solid media. The degree of inhibition depended on the spi gene dosage, but the presence of pSA1.1 imp genes, which encode negative repressor proteins for spi, relieved the inhibition. Confocal images of Spi tagged with enhanced green fluorescent protein in cells on solid media revealed that spi expression was initiated at the time of elongation of substrate mycelium, that its expression increased dramatically at septation in aerial hyphae, and that the expression was maximal during prespore formation. Expression of spi covered the whole of the hyphae, and the level of expression at the tip of the hyphae during prespore formation was about sixfold greater than during substrate mycelial growth and threefold greater than during aerial mycelial growth. Thus, localized expression of spi at particular times may inhibit sporulation until triggering imp expression to repress its inhibitory effects.