Final Report Summary - STREP-CELL (Cellulose: a key component essential for development and attachment of Streptomyces coelicolor?)
Final report Strep-Cell (ERG-230944) Streptomycetes are soil-dwelling filamentous bacteria that produce about 70 % of all clinically used antibiotics. These bacteria grow as filaments (hyphae) that form a complex multicellular network called a mycelium. When nutrients become limiting, part of the vegetative mycelium is dismantled, concurring with antibiotic production. These secondary metabolites help to protect the pool of nutrients that is released during breakdown of the mycelium, and which is being used for a second phase of growth. During this phase, specialised hyphae are formed that grow away from the substrate into the air. This leads to the formation of spores that can survive the adverse environmental conditions. The power of actinomycetes to exploit nutrients in the soil is demonstrated by the enormous amount of secreted hydrolases, cellulases and chitinases, in total more than 100 proteins. Strikingly though, is the presence of a gene predicted to be involved in cellulose biosynthesis, rather than degradation. The putative cellulose synthase enzyme (SCO2836, hereinafter called cslA) is homologous to characterised cellulose synthases from other organisms, and has all the conserved catalytic residues required for cellulose synthesis. Prior to the start of the project, we discovered that this protein is important for the formation of aerial hyphae under a wide range of conditions. This suggested that CslA might have a structural role in the cell wall. However, soon after the start of this project a publication appeared describing a large part of the intended research (and partially confirming the original ideas). Therefore, the direction of the research changed, now focusing on the role of cslA in attachment, as well as on the identification and roles of (novel) cell surface proteins (see below).
The role of chaplins and cellulose in attachment.
The formation of aerial hyphae is accompanied by a change in the surface hydrophobicity of hyphae. Whereas vegetative hyphae are hydrophilic, aerial hyphae and spores are hydrophobic due to the presence of a surface layer called the rodlet layer (Wildermuth et al., 1971; Claessen et al., 2002). Our group has previously discovered the proteins that form this surface layer, which are called rodlins (Claessen et al., 2002) and chaplins (Claessen et al., 2003) 2004; (Elliot et al., 2003; Claessen et al., 2004). In this Marie Curie project, we discovered that the chaplin proteins are also important for attachment of hyphae to surfaces (de Jong et al., 2009b). Attachment may be important for the effective degradation of substrates, colonisation of specific niches such as the cuticle of leaf-cutting ants and the initiation of infection processes by pathogenic streptomycetes. During hyphal attachment, an intercellular network of fimbriae is formed composed of chaplins in association with a cellulose-like polymer produced by cslA (Xu et al., 2008; de Jong et al., 2009b). Interestingly, Various bacteria produce cellulose during formation of biofilms and adherence to plant tissues. For instance, the extracellular matrix produced by Salmonella enterica comprises, in addition to curli fimbriae, cellulose and one or more other polysaccharides (White et al., 2003). Importantly, we observed detachment of the Streptomyces fimbriae from the cell surface by enzymatic treatment with cellulase (which degrades cellulose), revealing a critical, and previously unknown role for this polymer in fimbrial anchoring (de Jong et al., 2009b). How cellulose mediates anchoring of the fimbrial structures, and whether this is important for fimbrial anchoring in pathogenic microbes, is still under current investigation. Results from such studies might be important to get a better understanding of the mechanisms that enable pathogenic organisms to interact with their host, and might lead in the long term to the development of an entire new class of drugs.
The role of NepA in control of spore germination
Previous work from our group strongly suggested the existence of a regulatory mechanism, which is activated as soon as aerial growth commences. This then leads to the expression of many aerial-hyphae specific genes (Claessen et al., 2004). We have 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 ?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 (Dalton et al., 2007). However, expression is also detected in developing spore chains, where NepA is secreted to end up as a highly insoluble protein in the cell wall (de Jong et al., 2009a). 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 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 unfavorable environmental conditions (de Jong et al., 2009a).
Structural characterisation of the chaplin cell surface proteins and possible uses thereof
The self-association of proteins into amyloid fibrils offers an alternative to the natively folded state of many polypeptides. Although commonly associated with disease, amyloid fibrils represent the natural functional state of some proteins, such as the chaplins from the soil-dwelling bacterium Streptomyces coelicolor, which coat the aerial mycelium and spores rendering them hydrophobic. We have undertaken a biophysical characterisation of the five short chaplin peptides ChpD-H to probe the mechanism by which these peptides self-assemble in solution to form fibrils. Each of the five chaplin peptides produced synthetically or isolated from the cell wall is individually surface-active and capable of forming fibrils under a range of solution conditions in vitro. These fibrils contain a highly similar cross-ß core structure and a secondary structure that resembles fibrils formed in vivo on the spore and mycelium surface. They can also restore the growth of aerial hyphae to a chaplin mutant strain. Such studies represent a significant development in the understanding of functional amyloid fibrils and provide a useful comparison of these structures with fibres associated with diseases, such as Alzheimer's. Our investigations are also of importance in the development of peptide-based materials for bio-/nanotechnology, in particular in applications where tuneable alterations in surface chemistry are required, such as biosensor coatings, because of the surfactant properties of the chaplin peptides.
Publications and other impact
The research carried out during this project has been described in four publications, including two in one of the leading microbiology journals Molecular Microbiology and a book chapter. Moreover, the work has been presented at various international research meetings. The reintegration grant has also contributed to the establishment of an independent research position for the fellow at Leiden University, where part of the research will be continued.
References
Claessen, D., Rink, R., de Jong, W., Siebring, J., de Vreugd, P., Boersma, F. G., Dijkhuisen, L., and W?sten, H. A. B. (2003) A novel class of secreted hydrophobic proteins is involved in aerial hyphae formation in Streptomyces coelicolor by forming amyloid-like fibrils. Genes Dev 17: 1714-1726.
Claessen, D., Stokroos, I., Deelstra, H. J., Penninga, N. A., Bormann, C., Salas, J. A., Dijkhuisen, L., and W?sten, H. A. B. (2004) The formation of the rodlet layer of streptomycetes is the result of the interplay between rodlins and chaplins. Mol Microbiol 53: 433-443.
Claessen, D., W?sten, H. A. B., van Keulen, G., Faber, O. G., Alves, A. M., Meijer, W. G., and Dijkhuisen, L. (2002) Two novel homologous proteins of Streptomyces coelicolor and Streptomyces lividans are involved in the formation of the rodlet layer and mediate attachment to a hydrophobic surface. Mol Microbiol 44: 1483-1492.
Dalton, K. A., Thibessard, A., Hunter, J. I., and Kelemen, G. H. (2007) A novel compartment, the'subapical stem'of the aerial hyphae, is the location of a sigN-dependent, developmentally distinct transcription in Streptomyces coelicolor. Mol Microbiol 64: 719-737.
de Jong, W., Manteca, A., Sanchez, J., Bucca, G., Smith, C. P., Dijkhuisen, L., Claessen, D., and W?sten, H. A. B. (2009a) NepA is a structural cell wall protein involved in maintenance of spore dormancy in Streptomyces coelicolor. Mol Microbiol 71: 1591-1603.
de Jong, W., W?sten, H. A. B., Dijkhuisen, L., and Claessen, D. (2009b) Attachment of Streptomyces coelicolor is mediated by amyloidal fimbriae that are anchored to the cell surface via cellulose. Mol Microbiol 73: 1128-1140.
Elliot, M. A., Karoonuthaisiri, N., Huang, J., Bibb, M. J., Cohen, S. N., Kao, C. M., and Buttner, M. J. (2003) The chaplins: a family of hydrophobic cell-surface proteins involved in aerial mycelium formation in Streptomyces coelicolor. Genes Dev 17: 1727-1740.
White, A. P., Gibson, D. L., Collinson, S. K., Banser, P. A., and Kay, W. W. (2003) Extracellular polysaccharides associated with thin aggregative fimbriae of Salmonella enterica serovar enteritidis. J Bacteriol 185: 5398-5407.
Wildermuth, H., Wehrli, E., and Horne, R. W. (1971) The surface structure of spores and aerial mycelium in Streptomyces coelicolor. J Ultrastruct Res 35: 168-180.
Xu, H., Chater, K. F., Deng, Z., and Tao, M. (2008) A cellulose synthase-like protein involved in hyphal tip growth and morphological differentiation in Streptomyces. J Bacteriol 190: 4971-4978.
The role of chaplins and cellulose in attachment.
The formation of aerial hyphae is accompanied by a change in the surface hydrophobicity of hyphae. Whereas vegetative hyphae are hydrophilic, aerial hyphae and spores are hydrophobic due to the presence of a surface layer called the rodlet layer (Wildermuth et al., 1971; Claessen et al., 2002). Our group has previously discovered the proteins that form this surface layer, which are called rodlins (Claessen et al., 2002) and chaplins (Claessen et al., 2003) 2004; (Elliot et al., 2003; Claessen et al., 2004). In this Marie Curie project, we discovered that the chaplin proteins are also important for attachment of hyphae to surfaces (de Jong et al., 2009b). Attachment may be important for the effective degradation of substrates, colonisation of specific niches such as the cuticle of leaf-cutting ants and the initiation of infection processes by pathogenic streptomycetes. During hyphal attachment, an intercellular network of fimbriae is formed composed of chaplins in association with a cellulose-like polymer produced by cslA (Xu et al., 2008; de Jong et al., 2009b). Interestingly, Various bacteria produce cellulose during formation of biofilms and adherence to plant tissues. For instance, the extracellular matrix produced by Salmonella enterica comprises, in addition to curli fimbriae, cellulose and one or more other polysaccharides (White et al., 2003). Importantly, we observed detachment of the Streptomyces fimbriae from the cell surface by enzymatic treatment with cellulase (which degrades cellulose), revealing a critical, and previously unknown role for this polymer in fimbrial anchoring (de Jong et al., 2009b). How cellulose mediates anchoring of the fimbrial structures, and whether this is important for fimbrial anchoring in pathogenic microbes, is still under current investigation. Results from such studies might be important to get a better understanding of the mechanisms that enable pathogenic organisms to interact with their host, and might lead in the long term to the development of an entire new class of drugs.
The role of NepA in control of spore germination
Previous work from our group strongly suggested the existence of a regulatory mechanism, which is activated as soon as aerial growth commences. This then leads to the expression of many aerial-hyphae specific genes (Claessen et al., 2004). We have 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 ?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 (Dalton et al., 2007). However, expression is also detected in developing spore chains, where NepA is secreted to end up as a highly insoluble protein in the cell wall (de Jong et al., 2009a). 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 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 unfavorable environmental conditions (de Jong et al., 2009a).
Structural characterisation of the chaplin cell surface proteins and possible uses thereof
The self-association of proteins into amyloid fibrils offers an alternative to the natively folded state of many polypeptides. Although commonly associated with disease, amyloid fibrils represent the natural functional state of some proteins, such as the chaplins from the soil-dwelling bacterium Streptomyces coelicolor, which coat the aerial mycelium and spores rendering them hydrophobic. We have undertaken a biophysical characterisation of the five short chaplin peptides ChpD-H to probe the mechanism by which these peptides self-assemble in solution to form fibrils. Each of the five chaplin peptides produced synthetically or isolated from the cell wall is individually surface-active and capable of forming fibrils under a range of solution conditions in vitro. These fibrils contain a highly similar cross-ß core structure and a secondary structure that resembles fibrils formed in vivo on the spore and mycelium surface. They can also restore the growth of aerial hyphae to a chaplin mutant strain. Such studies represent a significant development in the understanding of functional amyloid fibrils and provide a useful comparison of these structures with fibres associated with diseases, such as Alzheimer's. Our investigations are also of importance in the development of peptide-based materials for bio-/nanotechnology, in particular in applications where tuneable alterations in surface chemistry are required, such as biosensor coatings, because of the surfactant properties of the chaplin peptides.
Publications and other impact
The research carried out during this project has been described in four publications, including two in one of the leading microbiology journals Molecular Microbiology and a book chapter. Moreover, the work has been presented at various international research meetings. The reintegration grant has also contributed to the establishment of an independent research position for the fellow at Leiden University, where part of the research will be continued.
References
Claessen, D., Rink, R., de Jong, W., Siebring, J., de Vreugd, P., Boersma, F. G., Dijkhuisen, L., and W?sten, H. A. B. (2003) A novel class of secreted hydrophobic proteins is involved in aerial hyphae formation in Streptomyces coelicolor by forming amyloid-like fibrils. Genes Dev 17: 1714-1726.
Claessen, D., Stokroos, I., Deelstra, H. J., Penninga, N. A., Bormann, C., Salas, J. A., Dijkhuisen, L., and W?sten, H. A. B. (2004) The formation of the rodlet layer of streptomycetes is the result of the interplay between rodlins and chaplins. Mol Microbiol 53: 433-443.
Claessen, D., W?sten, H. A. B., van Keulen, G., Faber, O. G., Alves, A. M., Meijer, W. G., and Dijkhuisen, L. (2002) Two novel homologous proteins of Streptomyces coelicolor and Streptomyces lividans are involved in the formation of the rodlet layer and mediate attachment to a hydrophobic surface. Mol Microbiol 44: 1483-1492.
Dalton, K. A., Thibessard, A., Hunter, J. I., and Kelemen, G. H. (2007) A novel compartment, the'subapical stem'of the aerial hyphae, is the location of a sigN-dependent, developmentally distinct transcription in Streptomyces coelicolor. Mol Microbiol 64: 719-737.
de Jong, W., Manteca, A., Sanchez, J., Bucca, G., Smith, C. P., Dijkhuisen, L., Claessen, D., and W?sten, H. A. B. (2009a) NepA is a structural cell wall protein involved in maintenance of spore dormancy in Streptomyces coelicolor. Mol Microbiol 71: 1591-1603.
de Jong, W., W?sten, H. A. B., Dijkhuisen, L., and Claessen, D. (2009b) Attachment of Streptomyces coelicolor is mediated by amyloidal fimbriae that are anchored to the cell surface via cellulose. Mol Microbiol 73: 1128-1140.
Elliot, M. A., Karoonuthaisiri, N., Huang, J., Bibb, M. J., Cohen, S. N., Kao, C. M., and Buttner, M. J. (2003) The chaplins: a family of hydrophobic cell-surface proteins involved in aerial mycelium formation in Streptomyces coelicolor. Genes Dev 17: 1727-1740.
White, A. P., Gibson, D. L., Collinson, S. K., Banser, P. A., and Kay, W. W. (2003) Extracellular polysaccharides associated with thin aggregative fimbriae of Salmonella enterica serovar enteritidis. J Bacteriol 185: 5398-5407.
Wildermuth, H., Wehrli, E., and Horne, R. W. (1971) The surface structure of spores and aerial mycelium in Streptomyces coelicolor. J Ultrastruct Res 35: 168-180.
Xu, H., Chater, K. F., Deng, Z., and Tao, M. (2008) A cellulose synthase-like protein involved in hyphal tip growth and morphological differentiation in Streptomyces. J Bacteriol 190: 4971-4978.