IDENTIFICATION OF A GENETIC-LOCUS IN PSEUDOMONAS-AUREOFACIENS INVOLVED IN FUNGAL INHIBITION

In iron-rich conditions, Pseudomonas aureofaciens PA147-2 produces an antibiotic-like compound that inhibits the growth of a plant fungal pa thogen, Aphanomyces euteiches. To contribute to the potential use of P A147-2 as a biocontrol organism, we report the identification of a gen etic locus important for antibiotic biosynthesis. Mutants defective fo r fungal inhibition (Af-) were generated by Tn5 mutagenesis. Southern hybridization of total DNAs from three Af- mutants indicated that loss of fungal inhibition was due to a single Tn5 insertion in each mutant . Restriction mapping of the mutation points showed that in two mutant s the Tn5 insertions were in the same 16.0-kb EcoRI fragment and were separated by 2.1 kb. A genomic library of PA147-2 was constructed and screened by using a region of DNA flanking the Tn5 insertion in one mu tant (PA109) as a probe to recover complementing cosmids. Three cosmid s containing a 16.0-kb EcoRI fragment complementary to the two mutants were recovered. Allele replacement by homologous recombination with p utative complementing cosmids restored one mutant to antifungal activi ty against A. euteiches. Southern analysis of the complemented mutants confirmed that allele replacement had occurred between cosmid DNA and Tn5. The wild-type 16.0-kb EcoRI fragment was cloned from the cosmid and complemented the two mutants to antifungal activity. An antifungal compound was isolated from PA147-2 grown on solid medium. Antifungal activity correlated to a peak on high-pressure liquid chromatography a nalysis. Under the same growth and extraction conditions, the antifung al activity seen in PA147-2 was absent in two Af- mutants. Furthermore , absence of an antifungal compound in each mutant correlated to the a bsence of the wild-type ''antifungal'' peak on high-pressure liquid ch romatography analysis.