Site-directed mutagenesis of the ferric uptake regulation gene of Escherichia coli

Abstract

The 12 histidine and four cysteine residues of the Fur repressor of Escherichia coli were changed, respectively, to leucine and serine by site-directed mutagenesis of the fur gene. The affects of these mutations were measured in vivo by ligation of the mutated genes to a wild-type fur promoter followed by measurement of the ability of these plasmids to regulate expression of a lacZ fusion in the aerobactin operon. In vitro affects were assayed by insertion of the mutated genes in the expression vector pMON2064 attended by isolation of the altered Fur proteins and appraisal of their capacity to bind to operator DNA. The results suggest that cysteine residues at positions 92 and 95 are important for the activity of the Fur protein.

Direct inhibition by nitric oxide of the transcriptional ferric uptake regulation protein via nitrosylation of the iron

“…This change in the metal environment could alter the conformation of the protein and, therefore, modify the ability of Fur to bind to DNA. On this purpose, site-directed mutagenesis experiments have shown that a single mutation of residue likely involved in Fe 2ϩ coordination yields an inactive Fur protein (45,46). Mutation especially of His-90, a highly conserved amino acid in Fur sequences, leads to an inactive protein still able to dimerize (45).…”

The ferric uptake regulator of Pseudomonas aeruginosa has no essential cysteine residues and does not contain a structural zinc ion

“…In this context, it is interesting that the Bacillus subtilis Fur apparently does not require Fe# + for DNA binding activity in vitro (Bsat & Helmann, 1999). Ligands to the structural Zn# + ion are believed to include Cys-92 and Cys-95, which have been shown to be essential for normal Fur activity in E. coli (Coy et al, 1994 ;Gonzalez de Peredo et al, 1999). Indeed, amongst a collection of Fur proteins substituted at all cysteine and histidine residues, those in which Cys-92 or Cys-95 were replaced by serine had much the most severe phenotypes (Coy et al, 1994).…”

Biochemical Characterization of the Structural Zn2+ Site in the Bacillus subtilis Peroxide Sensor PerR

“…The x-ray absorption spectroscopy of Escherichia coli Fur (Fur EC ) revealed Zn 2ϩ bound in a tetrahedral environment composed of two S and two N/O donor ligands (4). Subsequent studies using chemical modification and mass spectroscopy assigned Cys 92 and Cys 95 as Zn 2ϩ ligands (5), a conclusion supported by site-directed mutagenesis studies (6). To date, the only structure available for a protein from the Fur superfamily is Pseudomonas aeruginosa Fur (Fur PA ) that was crystallized in the presence of Zn 2ϩ (7).…”