This chapter discusses the isolation of salmonella mutants defective for intracellular survival. The first step is to choose an appropriate random mutagenesis system. Historically, bacterial mutagenesis experiments were performed using chemical mutagens. Chemical mutagenesis might be useful if one were interested in identifying mutations that result in an enhanced level of intracellular survival compared with the wild-type, as mutants can be selected directly. Transposons are useful for a number of reasons; when the appropriate conditions are used, each mutant bears a single, independently derived insertion. Once the mutagenesis is completed, it is important to test whether a representative and random bank has been generated. If chemical mutagens are used, then reversion of the appropriate auxotrophic marker, perhaps from one of the Ames tester strains, should be used as a positive control. If mutagenesis was carried out using transposons, the frequency and distribution of auxotrophic mutations should be checked and compared with those found by other workers. When mutants that are apparently unable to survive inside cells have been identified, further precautions should be taken before they are characterized in detail. It is important to eliminate those mutants whose apparent inability to survive intracellularly is actually an artifact of the screening procedure. A number of tests may be carried out, depending on the particular screening system used.
ASJC Scopus subject areas
- Molecular Biology