The Escherichia coli yiiP gene encodes an iron transporter, ferrous iron efflux (FieF), which belongs to the cation diffusion facilitator family (CDF). Transcription of fieF correlated with iron concentration; however, expression appeared to be independent of the ferrous iron uptake regulator Fur. Absence of FieF led to decreased growth of E. coli cells in complex growth medium but only if fur was additionally deleted. The presence of EDTA was partially able to relieve this growth deficiency. Expression of fieF in trans rendered the double deletion strain more tolerant to iron. Furthermore, E. coli cells exhibited reduced accumulation of (55)Fe when FieF was expressed in trans. FieF catalyzed active efflux of Zn(II) in antiport with protons energized by NADH via the transmembrane pH gradient in everted membrane vesicles. Using the iron-sensitive fluorescent indicator PhenGreen-SK encapsulated in proteoliposomes, transmembrane fluxes of iron cations were measured with purified and reconstituted FieF by fluorescence quenching. This suggests that FieF is an iron and zinc efflux system, which would be the first example of iron detoxification by efflux in any organism.

Genomic sequencing of the ␤-proteobacterium Wautersia (previously Ralstonia) metallidurans CH34 revealed the presence of three genes encoding proteins of the cation diffusion facilitator (CDF) family. One, CzcD, was previously found to be part of the high-level metal resistance system Czc that mediates the efflux of Co(II), Zn(II), and Cd(II) ions catalyzed by the CzcCBA cation-proton antiporter. The second CDF protein, FieF, is probably mainly a ferrous iron detoxifying protein but also mediated some resistance against other divalent metal cations such as Zn(II), Co(II), Cd(II), and Ni(II) in W. metallidurans or Escherichia coli. The third CDF protein, DmeF, showed the same substrate spectrum as FieF, but with different preferences. DmeF plays the central role in cobalt homeostasis in W. metallidurans, and a disruption of dmeF rendered the high-level metal cation resistance systems Czc and Cnr ineffective against Co(II). This is evidence for the periplasmic detoxification of substrates by RND transporters of the heavy metal efflux family subgroup. (34,41,45). These transporters were initially identified as Co(II) or Zn(II) transporters (5, 36), but the family was subsequently shown to be able to transport Cd(II) (1, 18), Mn(II) (6), Ni(II) (46), or Fe(II) (12, 27) as well. For the zinc-and cadmium-transporting CDF protein CzcD from Bacillus subtilis, it was demonstrated that the mechanism of transport is electroneutral antiporting of the metal cation against H ϩ or K ϩ (18). Recently, kinetic parameters for transport for the Zn(II)-effluxing CDF ZitB from Escherichia coli were experimentally determined, suggesting that the ratio of Zn(II) to H ϩ is 1:1 and thus not electroneutral (3).The first characterized bacterial member of the CDF family, CzcD (36) from W. metallidurans, was shown to mediate the efflux of Cd(II), Zn(II), and Co(II) (1). The Czc system also comprises another tripartite efflux machinery, CzcCBA, that spans the cytoplasmic and outer membranes and mediates high-level resistance against Cd(II), Zn(II), and Co(II) by active efflux driven by the proton motive force (9, 35).Recently, the complete genome of W. metallidurans was made available. An analysis revealed the presence of two additional genes for CDF proteins in this organism. In contrast to czcD, these open reading frames are not located on one of the two indigenous megaplasmids (pMOL28 and pMOL30) but are carried by the bacterial chromosome. The first, named dmeF (divalent metal efflux), is located on contig 366 (gene number 7456), and the second, named fieF (ferrous iron efflux) after its orthologue from E. coli (12), is located on contig 369 (gene number 9480) (nomenclature is according to the web site http://www.genome.jgi-psf.org/draft_microbes/ralme/ralme.home .html).Using (28,35). This suggests that RND systems utilize Co(II) only from the periplasm and thus that RND-driven transport occurs from the periplasm rather than from the cytoplasm to the outside. MATERIALS AND METHODSBacterial strains and growth conditions. E. coli was gro...

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