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Yersinia pseudotuberculosis: Cultivation, Storage, and Methods for Introducing DNA
Robert K Davidson1, Kimberly M Davis1
1W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland.
Abstract:
Yersinia pseudotuberculosis has been studied for many decades, and research on this microbe has taught us a great deal about host-pathogen interactions, bacterial manipulation of host cells, virulence factors, and the evolution of pathogens. This microbe should not be cultivated at 37°C because this is a trigger that the bacterium uses to sense its presence within a mammalian host and results in expression of genes necessary to colonize a mammalian host. Prolonged growth at this temperature can result in accumulation of mutations that reduce the virulence of the strain, so all protocols need to be modified for growth at room temperature, or 26°C. This article describes protocols for cultivating this microbe and for its long-term storage and its genetic manipulation by transformation and conjugation. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Growth of Y. pseudotuberculosis from a stock Basic Protocol 2: Growth of Y. pseudotuberculosis in liquid medium from a single colony Basic Protocol 3: Freezing Y. pseudotuberculosis in glycerol for long-term storage Basic Protocol 4: Transformation of Y. pseudotuberculosis by electroporation Basic Protocol 5: Tri-parental mating/conjugation.
Insights
Cultivate Yersinia pseudotuberculosis at 26°C, not 37°C, to maintain virulence. This guide details methods for growing, storing, and genetically manipulating this important pathogen.
Area of Science:
- Microbiology
- Bacteriology
- Pathogen Research
Background:
- Yersinia pseudotuberculosis is a well-studied pathogen with significant implications for understanding host-pathogen interactions.
- Cultivation at 37°C, a mammalian host temperature, triggers virulence gene expression but can lead to reduced virulence due to accumulated mutations.
- Optimal growth conditions are crucial for maintaining the integrity and virulence of Y. pseudotuberculosis strains for research purposes.
Purpose of the Study:
- To provide standardized protocols for the cultivation of Yersinia pseudotuberculosis.
- To outline methods for the long-term storage of Y. pseudotuberculosis strains.
- To describe techniques for the genetic manipulation of Y. pseudotuberculosis, including transformation and conjugation.
Main Methods:
- Growth of Y. pseudotuberculosis from stock cultures and single colonies at a non-permissive temperature (26°C).
- Long-term storage of bacterial cultures using glycerol cryoprotection.
- Genetic manipulation via electroporation for transformation and tri-parental mating for conjugation.
Main Results:
- Established protocols ensure successful cultivation and maintenance of Y. pseudotuberculosis virulence.
- Demonstrated effective long-term storage of viable Y. pseudotuberculosis cultures.
- Provided reliable methods for genetic modification of the bacterium.
Conclusions:
- Cultivation at 26°C is essential for preserving the virulence of Yersinia pseudotuberculosis.
- The described protocols facilitate consistent research on Y. pseudotuberculosis, covering cultivation, storage, and genetic manipulation.
- These methods support ongoing studies into host-pathogen interactions and bacterial pathogenesis.
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