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Linear DNA plasmids of Pichia inositovora are associated with a novel killer toxin activity
1Microbial Properties Research, U.S. Department of Agriculture, Peoria, IL 61604.
Abstract:
Pichia inositovora, strain NRRL Y-18709, which contains three linear double-stranded DNA plasmids, pPinl-1, pPinl-2 and pPinl-3, was cured of these plasmids both by growing the strain in the presence of 50 micrograms/ml bisbenzimide, and by exposure to ultraviolet light. Both cured and uncured strains were tested for growth on a variety of carbon sources. No differences in growth response were detected, indicating no discernible involvement of the linear plasmids in the catabolism of these compounds. Culture supernatants of Pichia inositovora were shown to contain a substance larger than 100 kDa that is toxic to Saccharomyces cerevisiae, strain GS 1688. Toxin activity was optimal in YEPD assay plates containing 50 mM citrate buffer with a pH between 3.4 and 4.2. Culture supernatants from P. inositovora were also weakly active against Cephaloascus albidus, strain NRRL Y-18710, and Citeromyces matritensis, strain NRRL Y-18711. Concentrated supernatants from cured P. inositovora strains did not exhibit these activities, consistent with the hypothesis that this toxic activity is linear plasmid-encoded. Unlike the well-known Kluyveromyces lactis system, or the newly identified P. acaciae system, P. inositovora strains cured of their linear plasmids do not become detectably sensitive to toxin produced by the wild-type strain suggesting a nonplasmid-encoded immunity function.
Insights
Pichia inositovora linear plasmids do not affect carbon source metabolism but encode a toxin toxic to other yeast species. Cured strains lack toxin production but retain immunity, suggesting a non-plasmid encoded immunity function.
Area of Science:
- Microbiology
- Molecular Biology
- Yeast Genetics
Background:
- Pichia inositovora possesses three linear double-stranded DNA plasmids (pPinl-1, pPinl-2, pPinl-3).
- Linear plasmids are known to influence yeast phenotypes, including toxin production and immunity.
Purpose of the Study:
- To investigate the role of linear plasmids in Pichia inositovora.
- To characterize the function of plasmid-encoded elements, specifically toxin production and carbon source metabolism.
Main Methods:
- Plasmid curing of Pichia inositovora using bisbenzimide and UV light.
- Growth assays on various carbon sources for cured and uncured strains.
- Toxin activity assays using culture supernatants against Saccharomyces cerevisiae and other yeast strains.
Main Results:
- No significant differences in carbon source utilization were observed between cured and uncured Pichia inositovora strains.
- Culture supernatants of wild-type Pichia inositovora exhibited toxicity towards Saccharomyces cerevisiae, with optimal activity at pH 3.4-4.2.
- Toxin activity was absent in supernatants from plasmid-cured strains, indicating plasmid-encoded toxin production.
- Pichia inositovora strains cured of plasmids did not show increased sensitivity to the wild-type toxin, suggesting a non-plasmid encoded immunity.
Conclusions:
- The linear plasmids of Pichia inositovora are responsible for the production of a secreted toxin but not for carbon source catabolism.
- The immunity of Pichia inositovora to its own toxin appears to be encoded by the host genome, not the plasmids.