The ZnuABC high-affinity zinc uptake system and its regulator Zur in Escherichia coli

Abstract

In Escherichia coli, lacZ operon fusions were isolated that were derepressed under iron-repletion and repressed under iron depletion. Two fusions were localized in genes that formed and operon whose gene products had characteristics of a binding protein-dependent transport system. The growth defect of these mutants on TY medium containing 5 mM EGTA was compensated for by the addition of Zn2+. In the presence of 0.5 mM EGTA, only the parental strain was able to take up 65Zn2+. This high-affinity transport was energized by ATP. The genes were named znuACB (for zinc uptake; former name yebLMI) and localized at 42 min on the genetic map of E. coli. At high Zn2+ concentrations, the znu mutants took up more 65Zn2+ than the parental strain. The high-affinity 65Zn2+ uptake was repressed by growth in the presence of 10 μZn2+. A znuA-IacZ operon fusion was repressed by 5 μMZn2+ and showed a more than 20-fold increase in β-galactosidase activity when Zn2+ was bound to 1.5 μM TPEN [tetrakis-(2-pyridylmethyl) ethylenediamine]. To identify the Zn2+-dependent regulator, constitutive mutants were isolated and tested for complementation by a gene bank of E. coli. A complementing gene, yjbk of the E. coli genome, was identified and named zur (for zinc uptake regulation). The Zur protein showed 27% sequence identify with the iron regulator Fur. High-affinity 65Zn2+ transport of the constitutive zur mutant was 10-fold higher than that of the uninduced parental strain. An in vivo titration assay suggested that Zur binds to the bidirectional promoter region of znuA and znuCB.