A study of antifungal activity by a potential biological control strain, Pseudomonas aureofaciens strain PA147-2

In iron-rich conditions, Pseudomonas aureofaciens PA147-2 produces an antibiotic-like compound that inhibits the growth of a number of plant fungal pathogens. To contribute to the potential use of PA147-2 as a biocontrol organism, I report the identification of a genetic locus important for antibiotic biosynthesis. Mutants defective for fungal inhibition (Af-), were generated by Tn5 mutagenesis. Southern hybridisation of genomic DNA from the Af- mutants indicated that in each case loss of fungal inhibition was due to a single Tn5 insertion. Restriction mapping of cloned DNA showed that in two mutants, PA1 and PA109, the Tn5 insertions were in the same 16kb EeoRI fragment, separated by 2.1kb. Allele replacement, by homologous recombination with cosmids from a genomic library of PA147-2, restored one mutant (PA109) to antifungal activity. The 16kb EeoRI wildtype fragment complemented PA109 and PA1 in trans to antifungal activity. Saturation mutagenesis using a miniTn10 transposon identified a region of at least 13kb that is required for fungal inhibition in culture. Under growth room conditions, PA147-2 protected asparagus seedlings from Phytophthora megasperma root rot while the Af- mutant, PA109, did not suppress the pathogen. An inhibitory compound isolated from PA147-2 and analysed by HPLC, was absent in three Af- mutants. The region flanking Tn5 insertion in PA109 was partially sequenced. Thia region in mutant PA109 has significant homology to several two-component regulators and appears to be necessary for wildtype antifungal activity on PBPDA and in planta. The homologous wildtype region was sequenced and a single open reading frame encoding a putative regulatory protein was identified as a "hybrid kinase", a member of the family of two-component signal proteins in bacteria. The regulatory gene is located within the putative cluster that is involved in antibiotic-mediated biological control of fungal pathogens