FIG 9.

Hemin binding assay of R. anatipestifer CH-1 and its derivative strains. Cells were incubated under iron starvation conditions, and a hemin-binding assay was performed. The mixture of 10 μg of hemin and 1 ml of bacteria (OD₆₀₀ = 1) was incubated for 2 h and 4 h, respectively. The binding reactions were terminated by centrifugation at 10,000 × g for 5 min. The amount of unbound hemin was determined by measuring the OD of hemin at 405 nm with a NanoDrop 2000 instrument (Thermo Scientific). The amount of bound hemin on the cells was calculated by subtraction. Each value indicates the mean of the results from three experiments, and the vertical bars denote the standard deviations. ****, P < 0.0001; ***, P < 0.001; **, P < 0.01; *, P < 0.05.