Fur regulon in gram-negative bacteria. Identification and characterization of new iron-regulated Escherichia coli genes by a fur titration assay.
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.
Stojiljkovic
I
I
Lehrstuhl für Mikrobiologie/Membranphysiologie, Tübingen, Germany.
Bäumler
A J
AJ
Hantke
K
K
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003360:Cosmids; D004269:DNA, Bacterial; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005821:Genetic Techniques; D006090:Gram-Negative Bacteria / Q000235:genetics*; D007501:Iron / Q000378:metabolism*; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D015183:Restriction Mapping; D017186:Titrimetry
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012097:Repressor Proteins; C068477:ferric uptake regulating proteins, bacterial; D007501:Iron