Chapter 5: Mutagenesis and Variant Selection in Salmonella

This chapter describes methods for mutagenesis of the Salmonella typhimurium genome and potential methods for screening a bank of mutants and genetic analysis of interesting variants. The chapter focuses on frequently used systems for transposon mutagenesis and variant selection. Variant selection describes in vitro cell culture assays designed to enrich a pool of mutants for those with defects in genes related to pathogenesis. Although the chapter focuses on methods developed in S. typhimurium, many of these are also applicable to other Salmonella serovars. Variants that have alterations in a defined region of the genome have been created in two independent ways: plasmid curing and construction of hybrids between different Salmonella strains. The breakthrough in creating large numbers of defined mutants that are easy to analyze came with the introduction of transposon mutagenesis. These mobile genetic elements insert more or less randomly in the genome, thus disrupting the function of the gene into which they have inserted. Transposons that are used for mutagenesis contain selectable markers. This important feature allows transfer of the mutation into a clean background, ensuring that an observed phenotype can be attributed to a single mutation.