Contributions of Five Secondary Metal Uptake Systems to Metal Homeostasis of Cupriavidus metallidurans CH34

Abstract

Cupriavidus metallidurans is adapted to high concentrations of transition metal cations and is a model system for studying metal homeostasis in difficult environments. The elemental composition of C. metallidurans cells cultivated under various conditions was determined, revealing the ability of the bacterium to shield homeostasis of one essential metal from the toxic action of another. The contribution of metal uptake systems to this ability was studied. C. metallidurans contains three CorA members of the metal inorganic transport (MIT) protein family of putative magnesium uptake systems, ZupT of the ZRT/IRT protein, or ZIP, family, and PitA, which imports metal phosphate complexes. Expression of the genes for all these transporters was regulated by zinc availability, as shown by reporter gene fusions. While expression of zupT was upregulated under conditions of zinc starvation, expression of the other genes was downregulated at high zinc concentrations. Only corA 1 expression was influenced by magnesium starvation. Deletion mutants were constructed to characterize the contribution of each system to transition metal import. This identified ZupT as the main zinc uptake system under conditions of low zinc availability, CorA 1 as the main secondary magnesium uptake system, and CorA 2 and CorA 3 as backup systems for metal cation import. PitA may function as a cation-phosphate uptake system, the main supplier of divalent metal cations and phosphate in phosphate-rich environments. Thus, metal homeostasis in C. metallidurans is achieved by highly redundant metal uptake systems, which have only minimal cation selectivity and are in combination with efflux systems that "worry later" about surplus cations.Sophisticated cellular biochemistry needs metals as cofactors. About 40% of all enzymes have them, ranking from Mg (16%) Ͼ Zn (9%) Ͼ Fe (8%) Ͼ Mn (6%) Ͼ Ca (2%) Ͼ Co and Cu (1%) down to K, Na, Ni, V, Mo, W, and only one example of Cd (59). It is an interesting question how the correct metal is allocated to the right protein, a challenge especially for the divalent metal cations Mg 2ϩ , Zn 2ϩ , Fe 2ϩ , Mn 2ϩ , Co 2ϩ , Ni 2ϩ , and Cu 2ϩ . These metals compete with each other for the metal binding sites in enzymes (16). Additionally, Fe 2ϩ/3ϩ and Cu ϩ/2ϩ promote dangerous reactive oxygen species in Fenton and Fenton-like reactions, as described by Haber and Weiss (13).Part of the solution to this problem might be to keep the metal cation bouquet in any cellular compartment in a way that minimizes competition for metal binding sites and the Fenton reaction. This leads to the question of how the cellular metal cation bouquet can be maintained in environments that may contain a single metal in a concentration range from pM to mM. The betaproteobacterium Cupriavidus metallidurans strain CH34 is able to keep its metal homeostasis under a variety of such adverse conditions (19,28,30). The organism can be found in many mesophilic metal-contaminated environments around the globe, such as zinc deserts of Belgium (8). Key to this a...

Influence of Copper Resistance Determinants on Gold Transformation by Cupriavidus metallidurans Strain CH34

“…metallidurans relies for its transition metal uptake on a battery of redundant and unspecific secondary metal uptake systems, namely, ZupT, PitA, CorA1, CorA2, CorA3, ZntB, and HoxN, which are transition metal transport systems with broad substrate specificity (44). The effect of deletion in zupT or all seven genes in the ⌬7 mutant strain of parent strain AE104 was investigated in dose-response curves, determined in 96-well plates (Fig.…”

FurC Regulates Expression of zupT for the Central Zinc Importer ZupT of Cupriavidus metallidurans

“…All genes for secondary zinc uptake systems were downregulated at high external zinc concentrations (1), in parallel with upregulation of the zntA and cadA genes for two zinc-and cadmiumexporting P IB2 -type ATPases (8). Additionally, zupT expression was upregulated by zinc starvation, e.g., in the presence of metal chelators, such as phosphate or EDTA.…”

“…This is achieved by highly redundant metal uptake systems, which have only minimal cation selectivity. These uptake systems are in combination with metal efflux systems, which remove surplus cations (1). The most sophisticated efflux systems and batteries of helper proteins are encoded by metal resistance determinants on the two native plasmids of wild-type strain CH34, pMOL28 and pMOL30, but a variety of resistance determinants also are located on the two chromosomes of this betaproteobacterium of the order Burkholderiales (2,3).…”

“…20), and, as uptake or efflux system, the MIT protein ZntB. C. metallidurans does not contain a primary ZnuABC-type ABC uptake system for this important metal (TC 3.A.3) but has two Ptype ATPases that may import Mg 2ϩ , Ca 2ϩ , and/or other divalent cations (1). None of the secondary transport systems alone was essential for zinc import, but ZupT was required under conditions of zinc starvation.…”

“…Additionally, zupT expression was upregulated by zinc starvation, e.g., in the presence of metal chelators, such as phosphate or EDTA. This effect could be antagonized best by addition of zinc ions (1), indicating that a zincdependent regulator should be in control of zupT expression.…”