Mechanistic studies of metalloregulatory proteins controlling metal ion homeostasis in Escherichia coli.

摘要

Maintaining the delicate balance of metal ion concentrations inside the cytoplasm is a critical function in all cells since metals are both essential and potentially toxic. In bacteria, the MerR and Fur families of transcriptional regulators are responsible for controlling the expression of a diverse array of metal homeostasis genes. The total concentrations of zinc and iron in E. coli are shown to be in the 0.1 mM range and copper in the 10 μM range; however, by calibrating the Zn(II) sensors ZntR and Zur, we have shown that none of this is available as the free ion in the cytoplasm. ZntR is a MerR homologue that utilizes a DNA distortion mechanism to activate expression of ZntA, a Zn(II)-exporting ATPase. Zur, a Fur homologue, acts as a classical repressor by hindering RNA polymerase binding in the presence of Zn(II), thereby repressing expression of the znuABC Zn(II) uptake system. Surprisingly, the threshold free zinc concentrations that switch on transcriptional control by Zur and ZntR (2.0 × 10−16 M and 11.5 × 10−16 M, respectively) are six orders of magnitude lower than the concentration of one free zinc atom in the cell. This extraordinary Zn(II) responsiveness indicates that there is essentially no free zinc available in the cytoplasm of E. coli. Together with the spectroscopic characterization of the metal binding sites in ZntR, Zur, and ZntA also presented here, these experiments are providing a greater understanding of the inorganic chemistry of zinc in the cell.; In a manner similar to ZntR, the MerR homologue CueR is shown to use a DNA distortion mechanism to activate expression of copA, a Cu(I)-export protein homologous to ZntA. However, the metal inducibility of this system still remains to be resolved. Likewise, the Fur mechanism has been re-evaluated since the metal selectivity of this Fe(II) regulator has not been established in vitro. We present DNase I footprints for apo-Fur, Zn₁Fur, Zn₂Fur and Fe(II), Zn-Fur which reveal marked differences in the DNA binding characteristics of each form. These differences may help develop a new model for Fur regulation of iron homeostasis genes.