Insertional mutagenesis was applied for the first time to a fungal biocontrol agent, Pseudozyma flocculosa, in an attempt to obtain mutants with altered antagonistic properties. Transformants were obtained via DNA-mediated transformation. Molecular analyses of the transformants revealed that multiple copies of the plasmid were integrated in tandem at one to many chromosomal loci. The transformants were screened for their biocontrol properties using standard bioassays, and the 160 tested transformants were classified into four groups: group I mutants (22 transformants) showed a stronger antagonistic effect than the wild type (WT) while those of group II (107 transformants) had a comparable antagonistic effect; group III mutants (17 transformants) had a decreased antagonistic effect relative to WT and group IV mutants (14 transformants) had lost their biocontrol properties. Culture extracts of the mutants (group IV) and WT were analyzed and compared for the presence of active metabolites which were then separated by solid-phase extraction and purified using conventional methods. Nuclear magnetic resonance experiments and analytical studies on a metabolite specifically produced by the WT revealed the presence of 2-(2',4'-diacetoxy-5'-carboxy-pentanoyl) octadecyl cellobioside (flocculosin), a novel glycolipid with strong antifungal properties; the production of this compound would account for the biocontrol activity of P. flocculosa
NMR, structure, antifungal activity, cellobiose lipid, Pseudozyma flocculosa, insertional mutagenesis, DNA-mediated transformation, flocculosin, antifungal actiivity
NCBI PubMed ID: 12732526Publication DOI: 10.1128/aem.69.5.2595-2602.2003Journal NLM ID: 7605801Publisher: American Society for Microbiology
Correspondence: richard.belanger@plg.ulaval.ca
Institutions: Centre de Recherche en Horticulture, Université Laval, Québec, Canada, Pavillon Marchand, Université Laval, Québec, Canada, Département de Phytologie, Centre de Recherche sur la Function, la Structure et l’Ingénierie des Protéines, Université Laval, Québec, Canada, Département de Chimie, Université Laval, Québec, Canada, Département de Phytologie, Université Laval, Québec, Canada, Centre de Recherche sur la Function, la Structure et l’Ingénierie des Protéines, Département de Chimie, Université Laval, Québec, Canada
Methods: 13C NMR, 1H NMR, NMR-2D, FAB-MS, DNA techniques, 31P NMR, electrophoresis, HPLC, TOCSY, LC-ESI-MS, cell growth, mutagenesis, HMQC, COSY, antifungal activity assay, antifungal activity test, FABMS, LCMS-ESI
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