title: A novel screening method for cell wall mutants in Aspergillus niger identifies UDP-galactopyranose mutase as an important protein in fungal cell wall biosynthesis.
abstract: To identify cell wall biosynthetic genes in filamentous fungi and thus potential targets for the discovery of new antifungals, we developed a novel screening method for cell wall mutants. It is based on our earlier observation that the Aspergillus niger agsA gene, which encodes a putative alpha-glucan synthase, is strongly induced in response to cell wall stress. By placing the agsA promoter region in front of a selectable marker, the acetamidase (amdS) gene of A. nidulans, we reasoned that cell wall mutants with a constitutively active cell wall stress response pathway could be identified by selecting mutants for growth on acetamide as the sole nitrogen source. For the genetic screen, a strain was constructed that contained two reporter genes controlled by the same promoter: the metabolic reporter gene PagsA-amdS and PagsA-H2B-GFP, which encodes a GFP-tagged nuclear protein. The primary screen yielded 161 mutants that were subjected to various cell wall-related secondary screens. Four calcofluor white-hypersensitive, osmotic-remediable thermosensitive mutants were selected for complementation analysis. Three mutants were complemented by the same gene, which encoded a protein with high sequence identity with eukaryotic UDP-galactopyranose mutases (UgmA). Our results indicate that galactofuranose formation is important for fungal cell wall biosynthesis and represents an attractive target for the development of antifungals.
authors.affiliation: Institute of Biology Leiden, Leiden University, Molecular Microbiology, Leiden, The Netherlands.
authors.forename: Robbert A
authors.initials: RA
authors.lastname: Damveld
authors.forename: Angelique
authors.initials: A
authors.lastname: Franken
authors.forename: Mark
authors.initials: M
authors.lastname: Arentshorst
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Punt
authors.forename: Frans M
authors.initials: FM
authors.lastname: Klis
authors.forename: Cees A M J J
authors.initials: CA
authors.lastname: van den Hondel
authors.forename: Arthur F J
authors.initials: AF
authors.lastname: Ram
mesh_terms: D001234:Aspergillus niger / Q000235:genetics*; D002473:Cell Wall / Q000201:enzymology / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics; D017353:Gene Deletion; D019751:Intramolecular Transferases / Q000235:genetics*; D016296:Mutagenesis; D009154:Mutation; D011995:Recombination, Genetic
chemical_list: D005656:Fungal Proteins; D019751:Intramolecular Transferases; C098051:UDP-galactopyranose mutase