Abstract
Pseudomonas tolaasii causes brown blotch disease on the cultivated mushrooms, such as Agaricus bisporus and Pleurotus ostreatu. It secretes a peptide toxin, tolaasin. The toxin consists of 18 amino acids and its molecular mass is 1,985 Da. The mechanism of membrane-pore formation of tolaasin molecule has not known in detail. However, tolaasin channels had been demonstrated in the artificial lipid bilayer. In order to characterize the pathogenic properties of toxin peptides, we isolated 23 strains of P. tolaasii from the infected mushrooms. To make sure that they are closely related but not the same bacteria, their 16S rRNA genes were sequenced and analyzed. They were divided into three subtypes, P1α, P1β, and P1γ, depending on the sequence of 16S rRNA genes, and 6, 16, and 1 strains were included in each subtypes, respectively. In the pitting test, all three subtypes were able to cause brown blotches on the surface of mushroom caps. Two subtypes, P1α and P1γ, showed hemolytic activities on erythrocytes; however, strains of P1β subtype did not show any hemolytic activity. Only three strains of P1α subtypes were able to form white lines with P. reactans, suggesting that the structures of their toxins are very similar to tolaasin. Since cytotoxic effects of toxins obtained from the same subtype strains were slightly different, further analyses were performed by measuring metabolic activities of these bacteria with API kit. In detail, there were 5 patterns in general microbial characters, 7 in biochemical tests, 4 in assimilation tests, and 5 in fermentation tests. Strains of P. tolaasii were divided into 6 groups by API test. Peptide toxins were obtained from each subtypes and purified by gel permeation chromatography, ion exchange chromatography, and HPLC. Their structures and functions are under investigation.
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