FIG. 3.

(A) λ-Red-mediated replacement of the tetRA element. Donor PCR products from coding sequences of fliA (σ²⁸) or flgM from A. aeolicus were flanked by 40-bp sequences of homology for recombination in the chromosome. The donor PCR products were electroporated into S. enterica serovar Typhimurium strains with tetRA elements inserted in the fliA or flgM genes. The tetRA element includes the coding sequences of tetR and tetA from transposon Tn10dTc and confers tetracycline resistance. The recombination was mediated by λ-Red (plasmid pKD46) (8), resulting in the loss of the tetRA element and replacement with the coding sequences of fliA or flgM from A. aeolicus by selection on tetracycline-sensitive medium (28). (B) Isolation of A. aeolicus fliA (σ²⁸) and flgM mutants using error-prone PCR and λ-Red-mediated replacement of the tetRA element. Replacement of the tetRA element were performed as described for panel A, except the donor PCR products of coding sequences from fliA (σ²⁸) or flgM were generated by error-prone PCR (as indicated by the sunburst symbol).