Molecular characterization of biosynthetic genes of an antifungal compound produced by Pseudomonas fluorescens MC07

Pseudomonas fluorescens MC07 is a growth-promoting rhizobacterium that suppresses mycelial growth in fungi such as Rhizoctonia solani, Pythium ultimum, Fusarium oxysporum, and Phytophthora capsici. To determine the role of the bacterium's antifungal activity in disease suppression, we screened 2,500 colonies generated by Tn5lacZ insertions, and isolated a mutant 157 that had lost antifungal activity. The EcoRI fragment carrying Tn5lacZ was cloned into pBluescript II SK(+) and used as a probe to isolate wild-type clones from a genomic library of the parent strain, MC07. Two overlapping cosmid clones, pEH4 and pEH5, that had hybridized with the mutant clone were isolated. pEH4 conferred antifungal activity to the heterologous host P.fluorescens strain 1855.344, whereas pEH5 did not. Through transposon mutagenesis of pEH4 and complementation analyses, we delineated the 14.7-kb DNA region that is responsible for the biosynthesis of an antifungal compound. DNA sequence analysis of the region identified 11 possible open reading frames (ORF), ORF1 through ORF11. A BLAST search of each putative protein implied that the proteins may be involved in an antifungal activity similar to polyketides.