Molecular analysis of the Escherichia coli ferric enterobactin receptor FepA.
In Escherichia coli, the outer membrane protein FepA is a receptor for the siderophore complex ferric enterobactin and for colicins B and D. To identify protein domains important for FepA activity, the effects of deletion and linker insertion mutations on receptor structure and function were examined. In-frame internal deletion mutations removing sequences encoding up to 304 amino acid residues resulted in functionally defective FepA polypeptides, although most were translocated efficiently to the outer membrane. One exception, a derivative lacking 87 internal amino acid residues near the N terminus, showed an inability to transport ferric enterobactin but retained limited colicin receptor function. Analysis of cells carrying 3'-terminal fepA deletion mutations suggested that residues within the C terminus of FepA may be involved in secretion and proper translocation of the protein to the outer membrane. Introduction of the peptide Leu-Glu after FepA residues 55, 142, or 324 severely impaired receptor function for all three ligands, while the same insertion after residues 339 or 359 had virtually no detrimental effect on FepA function. Foreign peptides inserted after residues 204 or 635 restricted colicin B and D function only, leaving ferric enterobactin transport ability at near wild-type levels. The results presented in this study have identified key regions of FepA potentially involved in receptor function and demonstrate the presence of both shared and unique ligand-responsive domains.
Armstrong
S K
SK
Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia 65212.
Francis
C L
CL
McIntosh
M A
MA
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002872:Chromosome Deletion; D002874:Chromosome Mapping; D003087:Colicins / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D005798:Genes, Bacterial; D007501:Iron / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D002352:Carrier Proteins; D003087:Colicins; D015345:Oligonucleotide Probes; D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; C029489:enterobactin receptor; D007501:Iron