Fur regulon in gram-negative bacteria: Identification and characterization of new iron-regulated Escherichia coli genes by a Fur titration assay

Igor Stojiljkovic, Andreas J Baumler, Klaus Hantke

Research output: Contribution to journalArticle

294 Scopus citations

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, Pseudomanas 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.

Original languageEnglish (US)
Pages (from-to)531-545
Number of pages15
JournalJournal of Molecular Biology
Volume236
Issue number2
StatePublished - 1994
Externally publishedYes

Keywords

  • Escherichia coli
  • Fur repressor
  • Fur-box
  • Iron regulation
  • Iron-binding proteins

ASJC Scopus subject areas

  • Virology

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