Fig 2.
SidJ deficiency increases the accumulation of FSC and its degradation products and decreases the transfer rate of iron to desferri-FC. (A) Representative reversed-phase HPLC analysis of the intracellular siderophore content of wt (shown in black) and ΔsidJ (shown in red) strains. A. fumigatus wt, ΔsidJ, and sidJc strains were grown for 22 h at 37°C in iron-limited liquid minimal medium (−Fe). Subsequently, mycelia were washed and transferred to fresh minimal medium containing 30 μM ferri-FSC and incubated for another 30 min or 120 min (sFSC 30 min and 120 min, respectively). The absorption at 210 nm is given in milliabsorption units (y axis), and the retention time is given in minutes (x axes). The intracellular siderophore content of the sidJc strain was wt-like and is therefore not shown. (B) Representative reversed-phase HPLC analysis of ferri-FSC incubated with cellular extracts of wt (shown in black) or ΔsidJ (shown in red) strains. Cellular extracts prepared from 30 mg iron-starved, freeze-dried mycelia were incubated with 1.0 mM ferri-FSC in 0.1 M Na phosphate buffer, pH 6.5, in a total volume of 0.7 ml at 37°C for 10 min or 60 min. (C) Mass spectrometry analysis of compound peaks 1 to 5 from panels A and B. The additional ionizing atoms are shown in bold, and the ionizing iron is connected to the molecules with two single bonds. The structures of these compounds are shown in Fig. S3 in the supplemental material.