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 andiron-storage/binding proteins was developed. The Fur titration assay (FURTA) enabled identification of cloned iron-regulated genes from different Gram-positiveand Gram-negative bacteria such as: Bacillus subtilis, Escherichia coli, Pantoea agglomerans, Pseudomanas putida, Salmonella typhimurium, Serratia marcescens and Yersinia enterocolitica. An ordered E. coli cosmidlibrary was screened for either new genes containing Fur-box nucleotide sequences or genes coding for iron-storage/binding proteins. Among 150 cosmids coveringapproximately 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-boxhalves was no longer bound by the Fur repressor complex in vivo. © 1994 Academic Press, Inc.