Comprehensive genomic analysis of cell wall genes in Aspergillus nidulans Piet W.J. de Groot a, * , Bernd W. Brandt b , Hiroyuki Horiuchi c , Arthur F.J. Ram d , Chris G. de Koster a , Frans M. Klis a a Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands b Centre for Integrative Bioinformatics (IBIVU), VU University Amsterdam, The Netherlands c Department of Biotechnology, The University of Tokyo, Japan d Molecular Microbiology, Institute of Biology, Leiden University, The Netherlands article info Article history: Received 19 May 2008 Accepted 31 July 2008 Keywords: 1,3-b-Glucan Chitin 1,3-a-Glucan 1,3/1,4-b-Glucan Mixed-linked glucanases GPI proteins Hydrophobins abstract Knowledge of the mechanisms underlying cell wall biosynthesis in Aspergillus spp. is of high relevance to medicine and food safety, and for biotechnological applications. The cell wall of Aspergillus nidulans is composed of galactomannoproteins, 1,3-a-glucan, b-glucans, and chitin. Here, we present a comprehen- sive inventory of the cell wall-related genes in A. nidulans. This includes glycan-synthetic and glycan-pro- cessing enzymes, spore wall maturation enzymes, GPI-anchor processing enzymes, GPI proteins and hydrophobins, and signaling proteins of the cell wall integrity pathway. All major known fungal cell wall-related genes are represented, except for Pir-CWPs. Importantly, we have identified a gene product that is possibly involved in the synthesis of 1,3/1,4-b-glucan, and we propose that four predicted GPI pro- teins, a mixed-linked b-glucanase and three amylase-like a-glucanases, may have transglucosidic activ- ities pertaining to the processing of 1,3/1,4-b-glucan and 1,3/1,4-a-glucan, respectively. We further present an updated survey of putative GPI proteins. Finally, we present mass spectrometric evidence sug- gesting the presence of at least twelve covalently-linked cell wall proteins in the hyphal wall of A. nidu- lans, including ten predicted GPI proteins, most of which belong to carbohydrate-active enzyme families that are also found in the walls of ascomycetous yeasts. Ó 2008 Elsevier Inc. All rights reserved. 1. Introduction The fungal wall accounts for a considerable percentage (20%) of the cellular biomass and thus represents a major metabolic invest- ment (for a review see Klis et al., 2007). The wall maintains the shape of the cell and is responsible for counteracting turgor pressure and for protecting the plasma membrane against mechanical damage. It is also involved in adhesion to biotic and abiotic surfaces and in biofilm formation, and depending on the species and its environ- mental niche it may have additional, more specialized functions (Yin et al., 2008). The walls of ascomycetous yeasts such as Saccharo- myces cerevisiae, Candida spp., and Schizosaccharomyces pombe have been studied in considerable detail and thus have served as a guide when analyzing the construction of the walls of filamentous fungi (De Groot et al., 2005; Klis et al., 2007). The walls of ascomycetous fungi are often bilayered with an internal layer containing the shape-conferring, stress-bearing polysaccharides and a fibrillar out- er layer consisting of (galacto)mannoproteins that extend away from the cell surface (Klis et al., 2007). In agreement with this, elec- tron micrographs of the hyphal walls of Aspergillus nidulans reveal a layered structure with an electron-transparent inner layer, probably representing the stress-bearing polysaccharides, and an electron- dense outer layer, probably largely consisting of glycoproteins (Jeong et al., 2004). Early analyses of the hyphal walls of A. nidulans show that glucose and (N-acetyl)glucosamine are the major sugars with galactose and mannose as minor components. The major polysaccharides are 1,3- a-glucan, 1,3-b-glucan, and chitin (Bull, 1970; Klis et al., 2007). Isolated walls also contain protein (this paper). In the related species Aspergillus niger, cell wall proteins in the hyphal walls were found to be linked through 1,6-b-glucan to 1,3-b-glucan in a similar way as described for the ascomycetous yeasts S. cerevisiae and Candida albicans (Brul et al., 1997; De Groot et al., 2005). Chemical analysis of fungal walls is often preceded by extraction with alkali, which removes cell wall proteins and 1,3-a-glucan, but leaves the b-glu- can–chitin network intact. The alkali-insoluble fraction of the hyphal walls of Aspergillus fumigatus accounts for about 40% of the original walls (Fontaine et al., 2000). It consists of a moderately branched 1,3-b-glucan whose non-reducing ends function as acceptor sites for chitin, as also shown for S. cerevisiae. Importantly, two other polymers, galactomannan and a linear 1,3/1,4-b-glucan, are also linked to the non-reducing ends of 1,3-b-glucan (Fontaine et al., 2000; Latgé, 2007). The enzymes involved in the synthesis of three major cell wall polysaccharides, 1,3-a-glucan, 1,3-b-glucan, and chitin, are known 1087-1845/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2008.07.022 * Corresponding author. Fax: +31 20 525 7056. E-mail address: P.W.J.deGroot@uva.nl (P.W.J. de Groot). Fungal Genetics and Biology 46 (2009) S72–S81 Contents lists available at ScienceDirect Fungal Genetics and Biology journal homepage: www.elsevier.com/locate/yfgbi