Construction of EPEC null mutants and epitope-tagged strains encoding EscQ-3×FLAG.

Nonpolar EPEC mutants were generated using the Lambda Red recombinase system (λ-Red) (83). Briefly, a PCR fragment containing a kanamycin resistance cassette was amplified from the template plasmid pKD4 by using primers flanked by homologous sequences upstream and downstream of the gene to be deleted. The PCR product was electroporated into an EPEC strain carrying the pKD46 plasmid. Successful mutants were selected on LB-Km plates. EPEC strains expressing a C-terminally 3×FLAG-tagged EscQ protein in its chromosomal context were generated using a modification of the λ-Red recombinase system employing the pSUB11 plasmid as the template (84). For double mutant strains, the kanamycin cassette was excised from the corresponding single mutant strain by using the helper plasmid pFLP2, which expresses the FLP recombinase (85). The second allelic exchange was performed as described above. Mutant and tagged strains were confirmed by PCR. Importantly, the mutant strains used in this study were shown to be nonpolar, as the wild-type secretion phenotype was restored by introducing the plasmid(s) expressing the corresponding deleted gene(s).