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A novel screening method for cell wall mutants in Aspergillus niger identifies UDP-galactopyranose mutase as an important protein in fungal cell wall biosynthesis

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Author: Damveld, R.A. · Franken, A. · Arentshorst, M. · Punt, P.J. · Klis, F.M. · Hondel, C.A.M.J.J. van den · Ram, A.F.J.
Type:article
Date:2008
Institution: TNO Kwaliteit van Leven
Source:Genetics, 2, 178, 873-881
Identifier: 240644
doi: doi:10.1534/genetics.107.073148
Keywords: Biology · Biotechnology · Acetamide · Alpha glucan synthase · Green fluorescent protein · Histone H2B · Mutase · Nuclear protein · Uridine diphosphate · Uridine diphosphate galactopyranose mutase · Article · Aspergillus niger · Biosynthesis · Cell wall · Controlled study · DNA sequence · Gene deletion · Gene expression · Mutagenesis · Nonhuman · PagsA gene · Priority journal · Reporter gene · Screening test · Temperature sensitive mutant · Aspergillus niger · Cell Wall · Fungal Proteins · Gene Deletion · Intramolecular Transferases · Mutagenesis · Mutation · Recombination, Genetic · Aspergillus niger · Eukaryota · Fungi

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 a-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. Copyright © 2008 by the Genetics Society of America.