Interplay of the Czc system and two P-type ATPases in conferring metal resistance to Ralstonia metallidurans

Abstract

Cadmium and zinc are removed from cells of Ralstonia metallidurans by the CzcCBA efflux pump and by two soft-metal-transporting P-type ATPases, CadA and ZntA. The czcCBA genes are located on plasmid pMOL30, and the cadA and zntA genes are on the bacterial chromosome. Expression of zntA from R. metallidurans in Escherichia coli predominantly mediated resistance to zinc, and expression of cadA predominantly mediated resistance to cadmium. Both transporters decreased the cellular content of zinc or cadmium in this host. In the plasmid-free R. metallidurans strain AE104, single gene deletions of cadA or zntA had only a moderate effect on cadmium and zinc resistance, but zinc resistance decreased 6-fold and cadmium resistance decreased 350-fold in double deletion strains. Neither single nor double gene deletions affected zinc resistance in the presence of czcCBA. In contrast, cadmium resistance of the cadA zntA double mutant could be elevated only partially by the presence of CzcCBA. lacZ reporter gene fusions indicated that expression of cadA was induced by cadmium but not by zinc in R. metallidurans strain AE104. In the absence of the zntA gene, expression of cadA occurred at lower cadmium concentrations and zinc now served as an inducer. In contrast, expression of zntA was induced by both zinc and cadmium, and the induction pattern did not change in the presence or absence of CadA. However, expression of both genes, zntA and cadA, was diminished in the presence of CzcCBA. This indicated that CzcCBA efficiently decreased cytoplasmic cadmium and zinc concentrations. It is discussed whether these data favor a model in which the cations are removed either from the cytoplasm or the periplasm by CzcCBA.

FIG. 1.

Prototypes of known efflux systems for zinc and cadmium in R. metallidurans. The Zn²⁺ and Cd²⁺ cations are exported from the cytoplasm (free or bound to thiols like glutathione) to the periplasm (grey area) by P-type ATPases (I) or by CDF (III) that are driven by the chemiosmotic gradient. The more complicated RND-driven efflux systems (II) are composed of an RND protein in the cytoplasmic membrane, an outer membrane factor, and a membrane fusion protein. Accordingly to the structures of the RND protein AcrB (21) and the outer membrane factor TolC (14), a trimeric state of all three subunits of the RND-driven efflux complex is assumed. CzcCBA is driven by the proton motive force (H⁺) (8, 23). The cationic substrates (white circles) may be directly exported out of the cell from the cytoplasm or periplasm (21).

FIG. 2.

Zinc and cadmium resistance of E. coli strains that express P-type ATPases from R. metallidurans. Dose-response curves are shown for E. coli strain GG48(ΔzntA ΔzitB) complemented in trans with genes encoding the R. metallidurans P-type ATPases CadA (g6751/691) (▴) and ZntA (g4648/649) (▪). Both genes were cloned into plasmid pASK3. The negative-control strain is GG48(pASK3) (○), and the positive-control wild-type strain is W3110(pASK3) (•). The means ± standard deviations (error bars) from three independent experiments are shown.

FIG. 3.

The presence of the CadA and ZntA P-type ATPases from R. metallidurans diminishes the accumulation of Cd²⁺ and Zn²⁺ in E. coli strain GG48 in assay buffer containing 10 μM ⁶⁵Zn²⁺ (A) or 10 μM ¹⁰⁹Cd²⁺ (B). E. coli strain GG48 (ΔzntA ΔzitB) was complemented in trans with the genes for the R. metallidurans P-type ATPases CadA (g6751/691) (▴) or ZntA (▪). The negative control (○) contained only the vector plasmid pASK3. The accumulation of Cd²⁺ and Zn²⁺ is shown in micromoles of the metal cation per gram (dry weight [d. w.]) of cells. The mean values of two independent experiments are shown.

FIG. 4.

Uptake of cadmium by R. metallidurans strains with mutations in the genes for CPx-type ATPases. Uptake of 1 μM ¹⁰⁹Cd²⁺ by cells of the megaplasmid-free R. metallidurans strain AE104 (•), its ΔzntA deletion derivative DN439 (▴), its ΔcadA deletion derivative DN438 (▪), and the ΔzntA cadA::pLO2 double mutant strain DN441 (○) is compared. Uptake is measured in nanomoles of Cd²⁺ per gram (dry weight [d.w.]) of cells.

FIG. 5.

Induction of a zntA-lacZ operon fusion by heavy metal cations in various R. metallidurans strains. The bacterial strains were DN444 (zntA-lacZ) (closed symbols) and DN445 (zntA-lacZ ΔcadA) (open symbols). Moreover, these strains were left alone (squares) or complemented in trans with czcCBADRS′ on plasmid pDNA385 (circles). The respective negative vector controls (plasmid pVDZ′2 only) gave results similar to the plasmid-free strains and are not shown. β-Galactosidase activity is measured in units per milligram (dry weight [d.w.]).

FIG. 6.

Induction of a cadA-lacZ operon fusion by heavy metal cations in various R. metallidurans strains. The bacterial strains were DN442 (cadA-lacZ) (closed symbols) and DN443 (cadA-lacZ ΔzntA) (open symbols). Moreover, these strains were left alone (squares) or complemented in trans with czcCBADRS′ on plasmid pDNA385 (circles). The respective negative vector controls (plasmid pVDZ′2 only) gave results similar to the plasmid-free strains and are not shown. β-Galactosidase activity is measured in units per milligram (dry weight [d.w.]).