The efflux system CdfX exports zinc that cannot be transported by ZntA in Cupriavidus metallidurans

Cupriavidus metallidurans is able to survive exposure to high concentrations of transition metals, but is also able to grow under metal starvation conditions. A prerequisite of cellular zinc homeostasis is a flow equilibrium combining zinc uptake and efflux processes. The mutant strain ∆e4 of the parental plasmid-free strain AE104 with a deletion of all four chromosomally encoded genes of previously known efflux systems ZntA, CadA, DmeF, and FieF was still able to efflux zinc in a pulse-chase experiment, indicating the existence of a fifth efflux system. The gene cdfX, encoding a protein of the cation diffusion facilitator (CDF) family, is located in proximity to the cadA gene, encoding a P-type ATPase. Deletion of cdfX in the ∆e4 mutant resulted in a further decrease in zinc resistance. Pulse-chase experiments with radioactive ⁶⁵Zn(II) and stable-isotope-enriched ⁶⁷Zn(II) provided evidence that CdfX was responsible for the residual zinc efflux activity of the mutant strain ∆e4. Reporter gene fusions with cdfX-lacZ indicated that the MerR-type regulator ZntR, the main regulator of zntA expression, was responsible for zinc- and cadmium-dependent upregulation of cdfX expression, especially in mutant cells lacking one or both of the previously characterized efflux systems, ZntA and CadA. Expression of zntR also proved to be controlled by ZntR itself as well as by zinc and cadmium availability. These data indicate that the cdfX-cadA region provides C. metallidurans with a backup system for the zinc-cadmium-exporting P-type ATPase ZntA, with CdfX exporting zinc and CadA cadmium.IMPORTANCEBacteria have evolved the ability to supply the important trace element zinc to zinc-dependent proteins, despite external zinc concentrations varying over a wide range. Zinc homeostasis can be understood as adaptive layering of homeostatic systems, allowing coverage from extreme starvation to extreme resistance. Central to zinc homeostasis is a flow equilibrium of zinc comprising uptake and efflux reactions, which adjusts the cytoplasmic zinc content. This report describes what happens when an imbalance in zinc and cadmium concentrations impairs the central inner-membrane zinc efflux system for zinc by competitive inhibition for this exporter. The problem is solved by activation of Cd-exporting CadA or Zn-exporting CdfX as additional efflux systems.