Elsevier

Methods in Enzymology

Volume 236, 1994, Pages 531-545
Methods in Enzymology

Isolation of hyperinvasive mutants of Salmonella

https://doi.org/10.1016/0076-6879(94)36041-3Get rights and content

Publisher Summary

This chapterdescribes an alternative approach to identify genes involved in invasion. These methods allow positive selection of mutations that affect invasion and are applicable to microorganisms that require many genes for invasion. The goal is to isolate bacterial mutants, called hyperinvasive mutants that have increased ability to enter epithelial cells. This procedure could be used for the study of a variety of invasive pathogens, but the specific methods described focus on the isolation of hyperinvasive Salmonella mutants. The basic steps for isolation of hyperinvasive mutants are (l) to prepare a pool of bacterial mutants, (2) to grow the pool of mutants under repressing growth conditions, (3) to incubate the pool of mutants with mammalian cells to allow the hyperinvasive mutants to enter the cells, (4) to treat the infected mammalian cells with gentamicin to kill the noninvasive, extracellular bacteria preferentially, and (5) to recover the viable intracellular hyperinvasive bacteria that have survived the gentamicin treatment. There are several aspects of the isolation procedure that should be optimized: the mutagenesis technique, the repressing growth conditions, the number of enrichment cycles for selection of hyperinvasive mutants, and the identification of independent mutations.

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