A flow equilibrium of zinc in cells of Cupriavidus metallidurans

Abstract

The hypothesis was tested that a kinetical flow equilibrium of uptake and efflux reactions is responsible for balancing the cellular zinc content. The experiments were done with the metal-resistant bacterium Cupriavidus metallidurans. In pulse-chase experiments, the cells were loaded with radioactive ⁶⁵Zn and chased with the 100-fold concentration of non-radioactive zinc chloride. In parallel, the cells were loaded with isotope-enriched stable ⁶⁷Zn and chased with non-enriched zinc to differentiate between zinc pools in the cell. The experiments demonstrated the existence of a kinetical flow equilibrium, resulting in a constant turnover of cell-bound zinc ions. The absence of the metal-binding cytoplasmic components, polyphosphate and glutathione, metal uptake, and metal efflux systems influenced the flow equilibrium. The experiments also revealed that not all zinc uptake and efflux systems are known in C. metallidurans. Cultivation of the cells under zinc-replete, zinc-, and zinc-magnesium-starvation conditions influenced zinc import and export rates. Here, magnesium starvation had a stronger influence compared to zinc starvation. Other metal cations, especially cobalt, affected the cellular zinc pools and zinc export during the chase reaction. In summary, the experiments with ⁶⁵Zn and ⁶⁷Zn demonstrated a constant turnover of cell-bound zinc. This indicated that simultaneously occurring import and export reactions in combination with cytoplasmic metal-binding components resulted in a kinetical flow equilibrium that was responsible for the adjustment of the cellular zinc content.

Importance: Understanding the biochemical action of a single enzyme or transport protein is the pre-requisite to obtain insight into its cellular function but this is only one half of the coin. The other side concerns the question of how central metabolic functions of a cell emerge from the interplay of different proteins and other macromolecules. This paper demonstrates that a flow equilibrium of zinc uptake and efflux reactions is at the core of cellular zinc homeostasis and identifies the most important contributors to this flow equilibrium: the uptake and efflux systems and metal-binding components of the cytoplasm.

Keywords: Cupriavidus metallidurans; zinc; zinc transport.

Fig 1

Schematic diagram of the methods used and the transport systems involved. Panel A. Growth curve of C. metallidurans. For the establishment of the method using stable ⁶⁷Zn, the metal was added at 100 Klett units, the cells were harvested and analyzed by inductively coupled plasma mass spectrometry (ICP-MS) at 150 Klett units. Panel B. Cells harvested at 150 Klett units were used for pulse-chase studies with radioactive ⁶⁵Zn (red) and in parallel with isotope-enriched ⁶⁷Zn (blue). The ellipsoids are the cells, the circles inside the zinc pools. Radioactive ⁶⁵Zn is the only zinc that can be measured with the scintillation counter. With stable isotope-enriched zinc and ICP-MS, a pool ZP1 (gray) with the natural isotope composition could be discriminated from a pool with a higher percentage of ⁶⁷Zn ZP2 (blue). Panel C. Transition metal cations and Mg(II) cross the outer membrane (OM) into the periplasm (gray) and further into the cytoplasm by uptake systems (on top). They can be bound inside the cells by glutathione, proteins, and polyphosphate (big circles). They may be exported from the cytoplasm to the periplasm by efflux systems (bottom row) and further on the outside by RND-driven transenvelope efflux systems (corners). Plasmid-encoded systems dashed, regulatory events concerning uptake systems (5) in blue.

Fig 2

Pulse-chase experiment with C. metallidurans strain AE104 and zinc. The cells were cultivated in TMM containing 200 nM Zn(II) (mZn-TMM, Panel A) or in a low metal medium without added zinc and 100 µM Mg(II) instead of 1 mM (lZn_lMg-TMM, Panel B). A preculture of the same medium was used. At turbidity of 150 Klett units, the cells were harvested by centrifugation, suspended in an equal volume of 10 mM TrisHCl (pH 7.0), and stored on ice until needed but no longer than a few hours. To 6 mL of this cell suspension, 2 g/L Na gluconate was added before the experiment. The uptake reaction (pulse) was started by the addition of 12 µL ⁶⁵Zn (500 µM, 12 µCi, 1 µM final concentration). The cells were incubated with shaking at 30°C. Samples of 500 µL were collected by filtration (0.2 µm pore size), washed twice with 5 mL ice-cold wash solution (50 mM Tris-HCl pH 7.0, 50 mM EDTA), and radioactivity was measured in a scintillation counter. After 20 min (bar), non-radioactive Zn(II) was added (chase) to a final concentration of 100 µM (closed circles), 1 mM (closed squares) or not (open circles) and sampling was continued. Three biological repeats and deviation bars are given.

Fig 3

Pulse-chase experiment with C. metallidurans AE104 uptake mutants ∆zupT, ∆7 and ∆9. Cells of strain ∆zupT (diamonds), ∆7 (∆zupT ∆corA1 ∆corA2 ∆corA3 ∆zntB ∆pitA ∆hoxN, squares), and ∆9 (∆7 ∆mgtA ∆mgtB::kan, triangles) were cultivated in TMM containing 200 nM zn(II) (mZn-TMM, Panel A) or no added Zn(II) and 0.1 mM Mg(II) (lZn_lMg, Panel B) as described in Fig. 2. Pulse at t = 0 with 1 µM ⁶⁵Zn(II), chase at t = 20 min with 100 µM non-radioactive Zn(II) (black symbols), or not chased (open symbols). Figure S5 provides an additional comparison with and without parent strain AE104.

Fig 4

Pulse-chase experiment with C. metallidurans AE104 efflux mutants ∆e2 and ∆e4. Cells of strain ∆e2 (∆zntA ∆cadA, squares) and ∆e4 (∆e2 ∆dmeF ∆fieF, triangles) were cultivated in TMM containing 200 nM Zn(II) (mZn-TMM, Panel A) or no added Zn(II) and 0.1 mM Mg(II) (lZn_lMg, Panel B) as described in Fig. 2. Pulse at t = 0 with 1 µM ⁶⁵Zn(II), chase at t = 20 min with 100 µM non-radioactive Zn(II) (black symbols), or not chased (open symbols). Figure S6 provides an additional comparison with parent strain AE104.

Fig 5

Pulse-chase experiment with C. metallidurans AE104 pool mutants ∆ppk::kan and ∆gshA::kan. Cells of strain AE104 (circles), ∆ppk::kan (squares), and ∆gshA::kan (triangles) were cultivated in TMM containing 200 nM zn(II) (mZn-TMM, Panels A and B) or no added Zn(II) and 0.1 mM Mg(II) (lZn_lMg, Panels C and D) as described in Fig. 2. Pulse at t = 0 with 1 µM ⁶⁵Zn(II), chase at t = 20 min with 100 µM non-radioactive Zn(II) (black symbols), or not chased (open symbols). Panels B and D contain the same data as Panels A and C except the AE104 values.