Generating Tetracycline-Inducible Auxotrophy in Escherichia coli and Salmonella enterica Serovar Typhimurium by Using an Insertion Element and a Hyperactive Transposase

We report the construction and application of a novel insertion element for transposase-mediated mutagenesis in gram-negative bacteria. Besides Km^r as a selectable marker, the insertion element InsTet^G−1 carries the anhydrotetracycline (atc)-regulated outward-directed P_A promoter so that atc-dependent conditional gene knockouts or knockdowns are generated. The complex formed between the purified hyperactive transposase and InsTet^G−1 was electroporated into Escherichia coli or Salmonella enterica serovar Typhimurium, and mutant pools were collected. We used E. coli strains with either TetR or the reverse variant revTetR^r2, while only TetR was employed in Salmonella. Screening of the InsTet^G−1 insertion mutant pools revealed 15 atc-regulatable auxotrophic mutants for E. coli and 4 atc-regulatable auxotrophic mutants for Salmonella. We have also screened one Salmonella mutant pool in murine macrophage-like J774-A.1 cells using ampicillin enrichment. Two mutants with the InsTet^G−1 insertion in the gene pyrE or argA survived this procedure, indicating a reduced intracellular growth rate in J774-A.1 cells. The nature of the mutants and the modes of their regulation are discussed.