Identification of a gene cluster encoding three 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 disease determinant of pathogenicity of Pseudomonas tolaasii on the cultivated 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 amount of tolaasin. In addition, TnphoA mutagenesis yielded a single tolaasin-negative mutant which was phoA active. Restriction enzyme mapping of mutant DNAs by Southern hybridization analysis revealed that the majority of Tn5 insertions were confined to a single genetic locus of approximately 65 kbp. Pulsed-field gel electrophoresis of representative Tn5 mutant DNAs showed that this region is at one end of a 640 kbp PacI chromosomal fragment and that the P. tolaasii genome is 6.7 Mbp. SDS-PAGE analysis of protein extracts from wild-type P. tolaasii demonstrated the presence of three high-molecular-weight proteins (designated TL1, TL2 and TL3). Alterations in the presence of these proteins, as well 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. Two other Tn5 mutations were also identified which resulted in a tolaasin-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 five genetic loci, including those encoding TL1, TL2 and TL3, are required for tolaasin synthesis.