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Isolation of cell wall mutants in Aspergillus nidulans by screening for hypersensitivity to Calcofluor White

     Department of Biology, Rhodes College, Memphis, Tennessee 38112

Darlene M. Loprete

     Department of Chemistry, Rhodes College, Memphis, Tennessee 38112

Michelle Momany
Youngsil Ha

     Department of Plant Biology, The University of Georgia, Athens, Georgia 30602

Lisa M. Harsch
Jennifer A. Livesay
Amit Mirchandani
Jeremy J. Murdock
Michael J. Vaughan
Mridula B. Watt

     Departments of Biology and Chemistry, Rhodes College, Memphis, Tennessee 38112

As a first step toward identifying novel genes of wall metabolism in filamentous fungi, we have screened a collection of Aspergillus nidulans mutants for strains exhibiting hypersensitivity toward the chitin binding agent Calcofluor White (CFW). This strategy has been used previously to identify cell wall mutants in Saccharomyces cerevisiae. We have identified 10 mutants representing eight loci, designated calA through calH, for Calcofluor hypersensitivity. All cal mutants are impaired for sporulation at 30 C or 42 C or both, and in eight of the 10 mutations this sporulation defect shows at least partial osmotic remediability. All cal mutants show elevated sensitivity to one or more of the following agents: Caspofungin, Nikkomycin, Tunicamycin, Congo red and SDS, which are recognized wall-compromising agents or have been shown to be inhibitory to wall integrity mutants in yeast. Seven of the 10 cal mutants show swelling at elevated temperature, which in most cases is osmotically remediable. Spore swelling also can be induced at 30 C in all but one of the cal mutants by germination in the presence of one or more of the following: Caspofungin, Nikkomycin or Tunicamycin. Analysis of wall sugars showed no major changes in mutant strains. We also report that the chitin synthase inhibitor Nikkomycin induces excessive spore swelling during germination in all tested strains that have wild type cell wall metabolism (GR5, A4, A28 and AH12) at 42 C but not at 30 C. This effect mimics that of certain temperature-sensitive swollen cell (swo) mutations.

Key words: Calcofluor hypersensitivity, cell wall mutants, swollen cell phenotype

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