The overall aim if this research proposal is to define the molecular mechanisms involved in iron-mediated regulation of gene expression in Escherichia coli, using the genes responsible for the biosynthesis (ent) and transport (fep, fes) of the catechol siderophore enterobactin as a model system. We have isolated and characterized six cistrons encoding enterobactin biosynthetic enzymes and seven genes specifying transport or iron release functions involving this siderophore, and we have defined two key genetic regions which control the iron-mediated expression of this genetic system. The objectives of the current study are: (i) to define the transcriptional boundaries of these iron- regulated genetic loci using nuclease protection and primer extension analyses: (ii) to determine (by site-directed mutagenesis of specially constructed fusion vectors) the precise nucleotides controlling this inducible expression; (iii) to examine what role secondary structures in the characterized leader transcripts upstream of several iron-inducible genes might play in post-transcriptional modulation of ent gene expression; (iv) to delineate protein-DNA interaction sites whereby the regulatory protein Fur mediates this control process, using in vitro and in vivo footprinting studies. This Expression as a complex metabolic pathway requiring regulatory coordination between biosynthetic and transport components. Aside from its basic scientific interest, this control is significant in that in all organisms studied, from bacteria to man, the metabolic iron state of the organism regulates the degree of nutritive iron absorption.
