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

✍ Scribed by Mark Coy; Charles Doyle; Jaya Besser; J. B. Neilands

• Book ID: 104640599
• Publisher: Springer Netherlands
• Year: 1994
• Tongue: English
• Weight: 479 KB
• Volume: 7
• Category: Article
• ISSN: 1572-8773
• DOI: 10.1007/bf00144124

✦ Synopsis

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.