The Chromosomally Encoded Cation Diffusion Facilitator Proteins DmeF and FieF from Wautersia metallidurans CH34 Are Transporters of Broad Metal Specificity

Abstract

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...

Siderophore-Based Iron Acquisition and Pathogen Control

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Identification and Characterization of a Major Zn(II) Resistance Determinant of Mycobacterium smegmatis

“…M. smegmatis mc 2 proteins are required for survival in an external contaminated environment. The presence of more than one member of a particular group may reflect differences in their substrate preference and efficiency (15).…”

“…The cation diffusion facilitators (CDFs) are integral membrane proteins that transport various divalent cations, including Zn(II) (19). Some of the CDF proteins have been shown to be specific for a single metal ion substrate whereas others possess broad substrate specificity (8,15).The genus Mycobacterium encompasses various environmental and pathogenic species. Zinc acts as an important component of many mycobacterial enzymes such as superoxide dismutase, alcohol dehydrogenase, carbonic anhydrase, etc.…”

“…The cation diffusion facilitators (CDFs) are integral membrane proteins that transport various divalent cations, including Zn(II) (19). Some of the CDF proteins have been shown to be specific for a single metal ion substrate whereas others possess broad substrate specificity (8,15).…”

“…Homologous CDFs MAP2784 and Rv2025c improved the growth of mutant K516 at 50 M zinc acetate but failed to provide zinc ion resistance at higher concentrations; nonspecific activity of the translational product of these genes towards different metal ions might be the reason for resistance at lower concentrations of the zinc ions. Many CDF proteins are known to transport more than one metal ion (2,15,26). The iron-transporting CDF protein (FieF) and the divalent cation-transporting CDF protein (DmeF) are also shown to transport zinc ions with lower specificity and provide some resistance towards zinc ions (9,15).…”