CzcD is a heavy metal ion transporter involved in regulation of heavy metal resistance in Ralstonia sp. strain CH34

Abstract

The Czc system of Ralstonia sp. strain CH34 mediates resistance to cobalt, zinc, and cadmium through ion efflux catalyzed by the CzcCB(2)A cation-proton antiporter. The CzcD protein is involved in the regulation of the Czc system. It is a membrane-bound protein with at least four transmembrane alpha-helices and is a member of a subfamily of the cation diffusion facilitator (CDF) protein family, which occurs in all three domains of life. The deletion of czcD in a Ralstonia sp. led to partially constitutive expression of the Czc system due to an increased transcription of the structural czcCBA genes, both in the absence and presence of inducers. The czcD deletion could be fully complemented in trans by CzcD and two other CDF proteins from Saccharomyces cerevisiae, ZRC1p and COT1p. All three proteins mediated a small but significant resistance to cobalt, zinc, and cadmium in Ralstonia, and this resistance was based on a reduced accumulation of the cations. Thus, CzcD appeared to repress the Czc system by an export of the inducing cations.

FIG. 1

Topological structure of CzcD. Parts of the czcD gene of Ralstonia were fused with the lacZ or phoA topological reporter gene. The czcD parts were from the 5′ end of the gene up to the points indicated by the positions of the bars. The relative activities of the reporter enzymes are indicated by overlapping white (LacZ) and black bars (PhoA), with error bars representing standard deviations. Above the hydrophobicity plot are the positions of putative metal-binding amino acid residues (in panel M, residues H [full-size bars] and C [half bars]), acidic amino acid residues (in panel A, residues D [two-thirds bars] and E [full-size bars]), and basic amino acid residues (in panel B, residues H [half bars], R [full-size bars], and K [two-thirds bars]).

FIG. 2

Effect of ΔczcD mutation on growth in presence of zinc. Cells of strain AE128(pMOL30) (● and ○), its ΔczcD mutant strain DN182(pMOL30-14) (■ and □), strain DN182 complemented in trans with pDNA176 containing czcD (▴ and ▵), pDNA178 containing ZRC1 (▾ and ▿), and pDNA177 containing COT1 (▸ and ▹) were cultivated for 48 h at 30°C in Tris-buffered mineral salts medium containing 2 g of sodium gluconate/liter as the carbon source. The cells were diluted in fresh medium containing 300 μM Zn²⁺ as the inducer (closed symbols) or no inducer (open symbols). Incubation was continued for 10 h, and then the cells were diluted to a cell density of up to 10 Klett units in fresh medium containing 2.5 mM Zn²⁺.

FIG. 3

CDF proteins mediate metal ion resistance. Ralstonia strain AE104 containing plasmids pVDZ′2 without an insert (○), pDNA176 with czcD (●), pDNA177 with COT1 (▴), or pDNA178 with ZRC1 (■) was cultivated in the presence of 100 μM Co²⁺ (A), 200 μM Zn²⁺ (B), or 100 μM Cd²⁺ (C), and the optical density over time was monitored. For each strain and metal ion, the results of two independent experiments are shown.

FIG. 4

Accumulation of heavy metal ions by Ralstonia strains expressing various CDF genes. Ralstonia strain AE104 containing plasmids pVDZ′2 without an insert (○), pDNA176 with czcD (●), pDNA177 with COT1 (▴), or pDNA178 with ZRC1 (■) was cultivated in Tris-buffered mineral salts medium containing 2 g of sodium gluconate/liter. The cells were harvested, washed, and suspended in 10 mM Tris-HCl buffer (pH 7.0) containing 2 g of sodium gluconate/liter. Radioactive metal isotopes at 1 or 100 μM were added, and incubation was continued with shaking at 30°C. Samples of 200 μl were removed, filtered (pore diameter, 0.45 μm), and washed twice on the filter with 2 ml of 10 mM Tris-HCl (pH 7.0) containing 10 mM MgCl₂. Radioactivity was determined with a scintillation counter (Beckman, Munich, Germany), and the dry weight (dw) was determined from the optical density with a calibration curve.

FIG. 5

Fast uptake of heavy metal ions by Ralstonia strains expressing various CDF genes. Ralstonia strain AE104 containing plasmids pVDZ′2 without an insert (○), pDNA176 with czcD (●), pDNA177 with COT1 (▴), or pDNA178 with ZRC1 (■) was cultivated in Tris-buffered mineral salts medium containing 2 g of sodium gluconate/liter. The cells were harvested, washed, and suspended in 10 mM Tris-HCl buffer (pH 7.0) containing 2 g of sodium gluconate/liter. The radioactive metal isotopes ⁶⁵Zn²⁺ (A), ⁵⁷Co²⁺ (B), and ¹⁰⁹Cd²⁺ (C) at 100 μM were added, and metal uptake was measured as described for Fig. 4. d.w., dry weight.