Characterization of C. jejuni Mutants

C. jejuni strain 81–176, a highly invasive isolate from a raw milk outbreak [31] (link), was used as the WT parental strain. All other strains are listed in Table 1. Targeted deletion mutants, such as ΔcprS, ΔflhA, and ΔflgR, have been described previously [19] (link), [32] (link). The ΔpflA mutant was isolated from a transposon mutant screen using the Mariner system developed for C. jejuni[33] (link). The double ΔcprS ΔflhA mutant was constructed by naturally transforming genomic DNA from ΔflhA into ΔcprS and selecting for Kan^R Cm^R colonies. C. jejuni strains were routinely cultured in Müller-Hinton (MH) broth (Oxoid, Hampshire, England) or on MH agar (1.7%) plates at 37°C under microaerobic conditions (6% O₂, 12% CO₂) in a Sanyo tri-gas incubator (plates and standing liquid cultures/biofilms) or generated using the Oxoid CampyGen system (shaking broth cultures). All media were supplemented with 10 µg mL⁻¹ vancomycin and 5 µg mL⁻¹ trimethoprim (Sigma, Oakville, ON). Where appropriate, antibiotics kanamycin (Kan), chloramphenicol (Cm), and streptomycin (Str) were added to a final concentration of 40 µg mL⁻¹, 15 µg mL⁻¹, and 100 µg mL⁻¹, respectively.