IDENTIFICATION OF A GENE-CLUSTER ENCODING 3 HIGH-MOLECULAR-WEIGHT PROTEINS, WHICH IS REQUIRED FOR SYNTHESIS OF TOLAASIN BY THE MUSHROOM PATHOGEN PSEUDOMONAS-TOLAASII

The extracellular lipodepsipeptide toxin tolaasin is the primary disea se determinant of pathogenicity of Pseudomonas tolaasii on the cultiva ted mushroom, Agaricus bisporus. Transposon mutagenesis of P. tolaasii NCPPB 1116 with Tn5-generated 5000 chromosomal insertions of which 35 (0.7%) were tolaasin negative and 12 (0.25%) produced a reduced amoun t of tolaasin. In addition, TnphoA mutagenesis yielded a single tolaas in-negative mutant which was phoA active. Restriction enzyme mapping o f mutant DNAs by Southern hybridization analysis revealed that the maj ority of Tn5 insertions were confined to a single genetic locus of app roximately 65 kbp. Pulsed-field gel electrophoresis of representative Tn5 mutant DNAs showed that this region is at one end of a 640 kbp Pac l chromosomal fragment and that the P. tolaasii genome is 6.7 Mbp. SDS -PAGE analysis of protein extracts from wild-type P. tolaasii demonstr ated the presence of three high-molecular-weight proteins (designated TL1, TL2 and TL3). Alterations in the presence of these proteins, as w ell as apparently truncated forms of the 465 kDa (TL1), 440 kDa (TL2) and 435 kDa (TL3) proteins were observed in some mutants, enabling the direction and order of the transcriptional units to be determined. Tw o other Tn5 mutations were also identified which resulted in a tolaasi n-negative phenotype, but which did not affect the expression of TL1, TL2, or TL3. One of these mutants is linked to the TL-cluster, but the other is located outside this region. It is concluded that at least f ive genetic loci, including those encoding TL1, TL2 and TL3, are requi red for tolaasin synthesis.