Promoter and operator determinants for fur-mediated iron regulation in the bidirectional fepA-fes control region of the Escherichia coli enterobactin gene system.
The fepA-entD and fes-entF operons in the enterobactin synthesis and transport system are divergently transcribed from overlapping promoters, and both are inhibited by the Fur repressor protein under iron-replete conditions. A plasmid harboring divergent fepA'-phoA and fes-entF'-'lacZ fusions, both under the control of this bidirectional regulatory region, was constructed for the purpose of monitoring changes in expression of the two operons simultaneously. Deletion analysis, site-directed mutagenesis, and primer extension were employed to define both a single promoter governing the expression of fes-entF and two tandemly arranged promoters giving rise to the opposing fepA-entD transcript. A single Fur-binding site that coordinately regulates the expression of all transcripts emanating from this control region was identified by in vitro protection from DNase I nicking. The substitution of one base pair in the Fur recognition sequence relieved Fur repression but did not change the in vitro affinity of Fur for its binding site. Additional mutations in a limited region outside of the promoter determinants for either transcript inhibited expression of both fes and fepA. These observations suggest a mechanism of Fur-mediated regulation in this compact control region that may involve other regulatory components.
Hunt
M D
MD
Department of Molecular Microbiology and Immunology, University of Missouri, School of Medicine, Columbia 65212.
Pettis
G S
GS
McIntosh
M A
MA
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D002265:Carboxylic Ester Hydrolases / Q000235:genetics; D002352:Carrier Proteins / Q000235:genetics; D004252:DNA Mutational Analysis; D004758:Enterobactin / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors; D007501:Iron / Q000494:pharmacology*; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic / Q000235:genetics; D009876:Operon / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012097:Repressor Proteins / Q000378:metabolism; D017384:Sequence Deletion
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C029489:enterobactin receptor; C068477:ferric uptake regulating proteins, bacterial; D004758:Enterobactin; D007501:Iron; D002265:Carboxylic Ester Hydrolases; C075139:ferric enterobactin esterase, E coli