Point mutations change specificity and kinetics of metal uptake by ZupT from Escherichia coli

Abstract

The ZIP (ZRT-, IRT-like Protein) protein ZupT from Escherichia coli is a transporter with a broad substrate range. Phenotypic and transport analysis showed that ZupT, in addition to Zn(II), Fe(II) and Co(II) uptake, is also involved in transport of Mn(II) and Cd(II). Competition experiments with other substrate cations suggested that ZupT has a slight preference for Zn(II) and kinetic parameters for Zn(II) in comparison to Co(II) and Mn(II) transport support this observation. Metal uptake into cells by ZupT was optimum at near neutral pH and inhibited by ionophores. Bicarbonate or other ions did not influence metal-uptake via ZupT. Amino acid residues of ZupT contributing to substrate specificity were identified by site directed mutagenesis. ZupT with a H89A exchange lost Co(II) and Fe(II) transport activity, while the S117V mutant no longer transported Mn(II). ZupT with E152D was impaired in overall metal uptake but completely lost its ability to transport the substrates Zn(II) and Mn(II). These experimental findings expand our knowledge on the substrate specificity of ZupT and provide further insight into the function of ZupT as a bacterial member of the vastly distributed and important ZIP family.

Survival of Escherichia coli Cells on Solid Copper Surfaces Is Increased by Glutathione

“…3 and 4) shows the higher toxicity of copper ions under anaerobic conditions in the presence of ascorbate. This may indicate that Cu I is more toxic than Cu II ; however, no uptake system was known for (28). On the other hand, upregulation of copA expression from background expression to about 350 copies per cell took only 2 min after addition of Cu II to the cells (32), and this regulatory event is performed by the MerR-type regulator CueR, which is usually constantly bound to its operator on the DNA (62).…”

“…Na I binds to copper-binding proteins (31) and vice versa; copper ions also inhibit Na I transport across membranes (63). So, copper ions could be able to enter the cytoplasm of E. coli by mimicking Na I or by ZupT as an import route for Cu II (28). As a practical consequence, sodium ions should not be present in solutions used to clean copper surfaces in hospitals.…”

“…If this is true, copper import into the cytoplasm should be a slow process in proteobacteria. The zinc importer ZupT is a low-rate transport system for Cu II (28) and should supply only minor amounts of copper to the cytoplasm. In contrast, import of the more toxic Cu I could be mediated unspecifically and rapidly by sodium or potassium transporters (29 (29,(34)(35)(36)(37)(38).…”

Coordinated transporter activity shapes high-affinity iron acquisition in cyanobacteria

“…An additional divalent metal transporter, ZIP (ZRT-, IRT-like protein), was also identified (Hopkinson and Barbeau, 2012). Both natural resistance-associated macrophage protein and ZIP have important roles in the acquisition of a wide range of divalent metals including Zn(II), Co(II), Fe(II), Mn(II) and Cd(II) (Nevo and Nelson, 2006;Taudte and Grass, 2010). These transporters were also identified in some freshwater and coastal cyanobacteria (Supplementary Figure S4).…”

Competition for zinc binding in the host-pathogen interaction

“…This metal permease has a broad metal specificity, but it displays a clear preference for zinc over other divalent metals (Grass et al, 2005). ZupT depends on the proton motive force to energize zinc import (Karlinsey et al, 2010; Taudte and Grass, 2010). …”