Identification and Characterization of a Novel ABC Iron Transport System, fit, in Escherichia coli

Effect of streptonigrin on growth of E. coli AA93 derivatives. Growth of strain AA93 with plasmids in LB (A), α-MEM (B), or LB with 200 μM dipyridyl (C) and growth of strain AA93/feoB with plasmids in LB (D) were measured. Open symbols, strains carrying pfit1; filled symbols, strains carrying pCR; squares, growth in medium without streptonigrin; triangles, growth in medium with 1 μg/ml streptonigrin; diamonds, growth in medium with 5 μg/ml. All experiments were performed at least three times to assess reproducibility. The figure presents the results of one typical experiment.

Measurement of the iron content of the E. coli AA93 derivatives by inductively coupled plasma atomic emission spectrometry. Cells were grown in α-MEM supplemented with 10 μM FeCl₃. The data were calculated from three independent replicates. Error bars indicate standard deviations. The asterisk indicates statistical significance using Student's t test (P < 0.05).

Expression of fit was induced by iron depletion. E. coli oy016 cells carrying pMP-fitB (A) or pMP-fitA (B) were first grown in LB medium for 3 h. Then the culture was split into two groups. Into one group, DIP was added at a final concentration of 200 μM, and into the other group no DIP was added. Cells were then allowed to grow, samples were taken at various time points, and β-galactosidase activities were measured as described elsewhere (21). Open bars, cells grown in LB; filled bars, cells grown in LB with DIP. Values are the means from three independent experiments. Error bars indicate standard deviations. Asterisks indicate statistical significance using Student's t test (P < 0.05).

Expression of fit was repressed by iron. E. coli oy016 cells carrying pMP-fitB (A) and pMP-fitA (B) were grown in MM9 medium with various metal ions. A 100 μM concentration each of Mn²⁺, Ca²⁺, Fe²⁺, Cu²⁺, Ni²⁺, and Rb⁺, 50 μM each of Zn²⁺ and Co²⁺, and 10 μM Cd²⁺ were used in the experiment. Cells were harvested at mid-log phase, and β-galactosidase activities were measured as described previously (21). Error bars indicate standard deviations (n = 3). Asterisks indicate statistical significance using Student's t test (P < 0.05).

Effects of pH and H₂O₂ on fit expression, based on β-galactosidase activities of oy016 cells carrying pMP-fitB (A and C) and pMP-fitA (B and D). (A and B) Cells were grown in LB at various pHs. (C and D) Cells were grown in LB, and when bacterial growth reached an A₆₀₀ of 0.6, various amounts of H₂O₂ were added to the medium. After 10 min, cells were collected and β-galactosidase activities were measured as described elsewhere (21). Values are the means from three independent experiments. Error bars indicate standard deviations (n = 3). Asterisks indicate statistical significance using Student's t test (P < 0.05).