We previously used fhuF as a sensitive reporter gene of the iron status of Escherichia coli. In this report, the fhuF gene was identified as open reading frame f262b at 99.2 min on the genome sequence map of E. coli K-12. The FhuF protein was labeled with a His-tag and then purified to electrophoretic homogeneity. Based on sulfur determinations and Mössbauer and EPR spectroscopy, FhuF was identified as a Keywords : iron-sulfur protein; ferrioxamine ; iron reductase; Mössbauer spectroscopy ; EPR.Under low-iron growth conditions, many bacteria secrete specific chelators called siderophores. Production of siderophores and transport systems for Fe 3ϩ -loaded siderophores is regulated in gram-negative bacteria by the repressor protein Fur with Fe 2ϩ as co-repressor [1]. In Escherichia coli, a fhuFϪlacZ operon fusion has been used as a reporter to study iron regulation [2] since this fusion reacts very sensitively to slight changes in the iron concentration of the medium. The only phenotype observed for fhuF mutations is a diminished ability of the cells to use ferrioxamine B as an iron source. This siderophore is a poor iron source for E. coli K-12 [3] because this strain lacks a specific receptor for ferrioxamine B in the outer membrane; this receptor is found in Yersinia [4] and other enterobacteria [5]. In an attempt to understand the function of FhuF, the protein was purified and characterized. Our results indicated that FhuF is an unusual [2Fe-2S] protein.
EXPERIMENTAL PROCEDURESStrains and growth conditions. The E. coli strains, phages, and plasmids used in this study are described in Table 1 Mud1(Aplac) and λplacMu mutants were generated as described elsewhere [6,7]. P1 transductions were performed as described by Miller [8]. Bacteria were grown in TY medium containing (per liter) tryptone (8 g), yeast extract (5 g), and sodium chloride (5 g) or in M9 medium [8] or in minimal medium E [3]. Growth response to ferrioxamine B was tested on nutrient broth dipyridyl plates (8 g nutrient broth, 5 g NaCl, 15 g Difco agar/l and 0.2 mM 2,2′-dipyridyl), which were seeded with the appropriate strain in 2.5 ml water soft agar. Filter paper discs impregnated with 15 µl 2 mM ferrioxamine B were placed on the agar, and growth zones were measured after 18 h.Recombinant DNA techniques and cloning of fhuF. Standard procedures [9] or those recommended by the commercial supplier were followed for isolation of chromosomal and plasmid DNA, cleavage with restriction endonucleases, DNA modification, ligation, transformation, and agarose gel electrophoresis. DNA was sequenced by the dideoxy chain-termination method using the AutoRead sequencing kit and the A. L. F. sequencer (Pharmacia Biotech). A DNA Thermal Cycler (Perkin Elmer Cetus) was used for PCR.All E. coli chromosomal DNA positions and section numbers are those of the E. coli genome sequence [10,11].The fhuF gene region was cloned on a 3-kb EcoRV DNA fragment from λ phage 8D1 (672) of the Kohara collection [12] in the high-copy-number vector pSU18 and recloned into vector ...