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 beta-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.

FIG. 1.

Zinc resistance of E. coli strains that express dmeF or fieF from W. metallidurans. Cultures grown overnight in LB medium were diluted 1:400 in fresh medium with the inducer anhydrotetracycline and increasing ZnCl₂ concentrations. The cultures were cultivated for 6 h at 37°C, and the optical densities at 600 nm were determined. The strains tested included GG48 (zntA::kan ΔzitB::cat) (pASK-IBA3) (○) as a negative control, GG48(pZITB)(pASK-IBA3::zitB) (•) as a positive control, ECA166(pASK-IBA3::dmeF) (▴), and ECA168(pASK-IBA3::fieF) (▪). The data shown are the averages and standard deviations for three independent experiments.

FIG. 2.

Iron uptake into E. coli by FieF or DmeF from W. metallidurans. Cultures that were grown overnight in minimal medium were inoculated at 30 Klett units into fresh medium at 37°C and grown up to an optical density of 60 Klett units. Uptake was started by the addition of a reaction mix of ⁵⁵Fe (1 μCi), FeSO₄ (5 μM final concentration), and 1 mM ascorbate. At defined time points, cellular iron accumulation was determined by the filtration method. The data shown are for E. coli strains ECA187 (ΔfieF) (pASK-IBA3) (○), ECA187(pECD884)(pASK-IBA3::fieF [from E. coli]) (•), ECA187(pECD873)(pASK-IBA3::dmeF) (▴), and ECA187(pECD875)(pASK-IBA3::fieF [from W. metallidurans]) (▪). The data shown are the averages and standard deviations for three independent experiments. (Inset) Western blot of StreptagII-labeled CDF proteins from Fig. 1 and 2 in isolated E. coli membranes (100 μg of total protein for each lane): 1, vector control; 2, DmeF from W. metallidurans; 3, FieF from W. metallidurans; 4, FieF from E. coli; 5, ZitB from E. coli.

FIG. 3.

Influence of disruptions of dmeF or fieF on metal resistance of W. metallidurans strain AE104. Cultures grown in minimal medium for 48 h were diluted 1:100 in fresh medium and cultivated with shaking for 24 h at 30°C. The cultures were inoculated 1:50 into fresh minimal medium with several metal salt concentrations and cultivated for 18 h, after which the optical densities at 600 nm were determined. The metals tested included Co(II) (A), Ni(II) (B), Zn(II) (C), and Cd(II) (D), and the strains were AE104 (plasmid free) (•), AE104 dmeF::pLO2 (○), and AE104 fieF::pLO2 (□). The experiments were performed in triplicate, and averages and standard deviations were calculated.

FIG. 4.

Nickel uptake into a dmeF or fieF mutant of W. metallidurans strain AE104. Cultures grown in minimal medium for 48 h were inoculated at 30 Klett units into fresh medium at 30°C and grown up to an optical density of 60 Klett units. Uptake was started by the addition of a reaction mix of ⁶³Ni (1 μCi) and NiCl₂ (5 μM final concentration), and incubation was continued with shaking at 30°C. At defined time points, nickel samples were withdrawn and the cellular accumulation of nickel was determined for W. metallidurans strains AE104 (•), AE104 dmeF::pLO2 (○), and AE104 fieF::pLO2 (□). The data shown are the averages and standard deviations for three independent experiments.

FIG. 5.

Influence of disruption of dmeF or fieF on Co(II) resistance of W. metallidurans wild-type strain CH34(pMOL30, pMOL28). Cultures were grown as described in the legend for Fig. 3. The W. metallidurans strains were CH34(pMOL30, pMOL28) (•), CH34 dmeF::pLO2(pMOL30, pMOL28) (○), and CH34 fieF::pLO2(pMOL30, pMOL28) (□).

FIG. 6.

CzcD mediates low-level Co(II) resistance in the absence of DmeF in W. metallidurans strain AE128(pMOL30). Cultures were grown as described in the legend for Fig. 3. The W. metallidurans strains were AE128(pMOL30) (•), DN182(pMOL30-14 ΔczcD) (), AE128 dmeF::pLO2(pMOL30) (), and AE128 dmeF::pLO2(pMOL30-14 ΔczcD) (○).