Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB23EA, UK

Background and objectives
Pseudomonas tolaasii is the causal agent of brown blotch disease of the cultivated mushroom. Its key determinant of pathogenicity is synthesis of an extracellular toxin, tolaasin [1]. Tolaasin is a lipodepsipeptide (M, 1985) containing 18 amino-acid residues, 11 of which are D-isomers and/or require post-translational modification. Tolaasin has a broad spectrum of biological activity, forming voltage-gated ion channels, lysing erythrocytes via a colloidosmotic mechanism and as a biosurfactant. Tn5 mutagenesis of the wild-type pathogenic strain P. tolaasii 1116S and analysis of tolaasin-deficient mutants resulted in identification of the tolaasin synthetase gene cluster [1]. Since three high molecular proteins (435, 440 and 465 kDa) are encoded by this cluster, it was deduced that tolaasin is synthesised non-ribosomally by peptide synthetases. P. tolaasii also produces three fluorescent yellow-green pigments that have high iron-chelating activity, designated siderophores [2]. Pyoverdins, the siderophores isolated from the cultures of P. tolaasii, are linear peptides (Mr 1424, 1425 and 1453) containing 10 amino-acid residues, five of which are D-isomers. These siderophores may also play a role in pathogenicity of P. tolaasii on mushrooms. The objective of this study is to identify the biosynthetic mechanism of tolaasin and pyoverdin by P. tolaasii and the relationship between production of these molecules at the genetic and biochemical levels.

Materials and methods
In order to facilitate elucidation of the molecular basis of tolaasin biosynthesis, cloning and DNA sequencing of the tolaasin synthetase gene cluster was performed. The tolaasin synthetase gene cluster was isolated by cloning the DNA flanking tox::Tn5 insertions followed by constructing genomic libraries of the Tn5 tagged tolaasin-negative mutants. 31 cosmid clones were isolated from eight different Tn5 insertion sites. Physical mapping of these clones showed that they overlap and cover the entire 65-kb cluster. DNA sequencing of these clones was carried out in collaboration with the Human Genome Mapping Project Resource Centre in Cambridge by using the shot-gun strategy and standard DNA sequencing methods. Tn5 mutagenesis of 1116S and the non-pathogenic spontaneous mutant P. tolaasii 1116R also generated pyoverdin-deficient mutants. Pyoverdin synthesis by 1116S, 1116R and the Tn5 mutants was analysed by HPLC and using bioassays.

Results and conclusions
Analysis of the DNA sequence of the tolaasin synthetase gene cluster has shown that the tolaasin synthetases contain domains with high homology to those in the siderophore pyoverdin, surfactin and antibiotic phytinamycin synthetases. Progress in the sequence analysis of the tolaasin synthetase gene cluster will be reported. The profile of pyoverdin production by 1116R differs from that produced by 1116S, demonstrating that pyoverdin synthesis is regulated by the pheN regulatory pathway. In addition, out of 30 independent pyoverdin synthesis mutants, 27 were also shown to be tolaasin-deficient, suggesting a link between these pathways. Progress in the analysis of pyoverdin-deficient mutants of P. tolaasii will be described.

1. Rainey P, Brodey C, Johnstone K, 1993. Molecular Microbiology 8, 643-652.
2. Demange P, Bateman A, Mertz C et al., 1990. Biochemistry 29, 11041-11051.