Yersinia pseudotuberculosis: Cultivation, Storage, and Methods for Introducing DNA.

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.