Figure 5. Induction of cirA expression increases sensitivity to ColIb of E. coli.

(A) Ec^MG1655 and Ec^{MG1655 ΔcirA} were cultivated in M9 medium o.n., mixed with soft agar and indicated concentrations of FeCl₃ and overlaid on M9 agar plates. Paper discs with recombinant ColIb were placed on the agar plate and the diameter of the inhibition zone (halo) was measured after 24 h. The detection limit (dotted line) is the size of the paper-disc (6 mm). (B) cirA expression of E. coli grown in vitro in M9 medium supplemented with different concentrations of FeCl₃. Overnight cultures of Ec^MG1655 and Ec^{MG1655 ΔcirA} grown in M9 medium for 12 h were used for inoculation of 2 ml M9 medium supplemented with 1 µM, 10 µM, 0.1 mM or 1 mM FeCl₃ and subcultured for 7 h. From each subculture, 250 µl (for an OD₆₀₀ of 1) was taken, spun down at 4°C, 10 min, 10, 000 rpm. CirA was detected by immunoblot in bacterial lysates using affinity-purified rabbit-α-CirA-antiserum. E. coli DnaK was detected as loading control.