Originally published as Genetics Published Articles Ahead of Print on February 3, 2008.

Genetics, Vol. 178, 873-881, February 2008, Copyright © 2008
doi:10.1534/genetics.107.073148

A Novel Screening Method for Cell Wall Mutants in Aspergillus niger Identifies UDP-Galactopyranose Mutase as an Important Protein in Fungal Cell Wall Biosynthesis

Robbert A. Damveld*,1, Angelique Franken*, Mark Arentshorst*, Peter J. Punt{dagger}, Frans M. Klis{ddagger}, Cees A. M. J. J. van den Hondel* and Arthur F. J. Ram*,2

* Institute of Biology Leiden, Leiden University, Molecular Microbiology, 2333 AL, Leiden, The Netherlands, {dagger} TNO Quality of Life, Department of Microbiology, 3704 HE, Zeist, The Netherlands and {ddagger} Swammerdam Institute for Life Sciences, University of Amsterdam, BioCentrum Amsterdam, 1018 WV, Amsterdam, The Netherlands

2 Corresponding author: Institute of Biology, Clusius Laboratory, Molecular Microbiology, Leiden University, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands.
E-mail: a.f.j.ram{at}biology.leidenuniv.nl

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.