A novel screening method for cell wall mutants in Aspergillus niger identifies UDP-galactopyranose mutase as an important protein in fungal cell wall biosynthesis.
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.
Damveld
Robbert A
RA
Institute of Biology Leiden, Leiden University, Molecular Microbiology, Leiden, The Netherlands.
Franken
Angelique
A
Arentshorst
Mark
M
Punt
Peter J
PJ
Klis
Frans M
FM
van den Hondel
Cees A M J J
CA
Ram
Arthur F J
AF
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
D005656:Fungal Proteins; D019751:Intramolecular Transferases; C098051:UDP-galactopyranose mutase