| Brown blotch disease in the commercial mushroom, Agaricus bisporus is caused by Pseudomonas tolaasii, which is found in two forms: the wild-type, which is pathogenic and the variant, which is non-pathogenic due to loss of production of the toxin, tolaasin. One such variant was found to have a mutation within the gacS gene, which encodes a sensor kinase protein. Using transposon and allele exchange mutagenesis studies of the GacS regulon were initiated to determine the regulatory network responsible for the expression of tolaasin biosynthesis genes of P. tolaasii. The specific aims of this research were: (1) to characterize the GacS/A signal transduction pathway in P. tolaasii ; (2) identify other regulators of tolaasin biosynthesis; and (3) identify signal molecules involved in host-pathogen communication during disease development.; We have isolated and identified GacA, the response regulator of the GacS sensor kinase in P. tolaasii. Both a gacA and gacS mutant were constructed which had similar phenotypes, confirming the role of GacA as the response regulator of GacS in P. tolaasii. The role of the alternative sigma factor, RpoS, in the regulation of tolaasin production was also elucidated in this study. A functional Gac system is required for the expression of rpoS as demonstrated by rpoS::lacZ expression studies. An rpoS mutant had increased levels of pathogenicity to mushrooms and produced higher levels of tolaasin in growing cultures. The sypB gene of P. tolaasii is required for tolaasin biosynthesis and full virulence during mushroom pathogenesis. A strain of P. tolaasii containing a sypB::lacZ fusion was used to detect transcription activation of the sypB gene by mushroom-derived metabolites. Among the mushroom compounds tested, trehalose, the main sugar in mushrooms, was shown to induce sypB giving rise to an increase in beta-galactosidase activity. To identify other genes involved in the regulation of tolaasin biosynthesis, a Tn5GGm library was generated in the sypB::lacZ strain and mutants screened for the lack of toxin production using a simple blue/white screening on X-gal plates. Using this screen the ABC-type and RND-like transporters involved in tolaasin secretion were identified. |
