Streptonigrin sensitivity assay

For indirect quantification of the intracellular iron level, we performed a streptonigrin sensitivity assay as described previously [34]. Briefly, R. anatipestifer CH-1pLMF03, R. anatipestifer CH-1ΔfurpLMF03 and R. anatipestifer CH-1ΔfurpLMF03::fur were grown to OD₆₀₀ = 1.0 in GCB medium, GCB medium supplemented with 100 μM EDDHA, and GCB medium supplemented with 100 μM EDDHA and 200 μM Fe(NO₃)₃ at 37 °C in a shaking incubator. Cells were harvested by centrifugation at 6000 rpm for 10 min, and pellets were diluted with fresh PBS up to OD₆₀₀ = 0.5 and aliquoted at 1 mL/tube. Streptonigrin (Sigma-Aldrich, St. Louis, USA) was diluted to 1 µg/mL with sterile PBS, 0 µL, 50 µL, and 80 µL was added to each tube of bacterial solution, the final concentration of streptonigrin was 0 ng/mL, 50 ng/mL and 80 ng/mL, respectively. Then the samples were incubated in the static incubator at 37 °C for 30 min. After incubation, the bacterial solution was diluted and spread onto GCB plates for counting (T0, T50, and T80). After a 1-day incubation at 37 °C, the grown colonies were counted. The survival rate was calculated as (T50/T0) × 100% and (T80/T0) × 100%, and the experiments were performed in triplicate.