Fur Regulon in Gram-Negative Bacteria. Identification and Characterization of New Iron-Regulated Escherichia Coli Genes by a Fur Titration Assay

J Mol Biol. 1994 Feb 18;236(2):531-45. doi: 10.1006/jmbi.1994.1163.

Abstract

A highly sensitive genetic screen for the detection of cloned genes coding for iron-regulated and iron-storage/binding proteins was developed. The Fur titration assay (FURTA) enabled identification of cloned iron-regulated genes from different Gram-positive and Gram-negative bacteria such as: Bacillus subtilis, Escherichia coli, Pantoea agglomerans, Pseudomonas putida, Salmonella typhimurium, Serratia marcescens and Yersinia enterocolitica. An ordered E. coli cosmid library was screened for either new genes containing Fur-box nucleotide sequences or genes coding for iron-storage/binding proteins. Among 150 cosmids covering approximately 85% of the E. coli genome, 24 cosmids were identified as positive by FURTA. Nine of them contained new E. coli Fur-regulated genes and/or iron-storage/binding genes since they mapped at loci different to any of the known Fur-box containing genes. A new E. coli gene encoding a 8.7 kDa high-potential iron-sulfur-like protein was identified, cloned and sequenced. The Fur titration assay was also used to probe in vivo interaction between Fur repressor and different synthetic plasmid-located Fur-boxes. Non-optimal base-pairs in one half of the Fur-box nucleotide sequence led to a dramatic decrease of Fur repressor affinity. A synthetic Fur-box with changes in both Fur-box halves was no longer bound by the Fur repressor complex in vivo.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • Base Sequence
  • Cloning, Molecular
  • Cosmids
  • DNA, Bacterial
  • Escherichia coli / genetics*
  • Gene Expression Regulation, Bacterial*
  • Genetic Techniques
  • Gram-Negative Bacteria / genetics*
  • Iron / metabolism*
  • Molecular Sequence Data
  • Open Reading Frames
  • Promoter Regions, Genetic
  • Repressor Proteins / genetics*
  • Repressor Proteins / metabolism
  • Restriction Mapping
  • Titrimetry

Substances

  • Bacterial Proteins
  • DNA, Bacterial
  • Repressor Proteins
  • ferric uptake regulating proteins, bacterial
  • Iron

Associated data

  • GENBANK/X75948
  • GENBANK/X75949