Surface topology of the Escherichia coli K-12 ferric enterobactin receptor

Abstract

Monoclonal antibodies (MAb) were raised to the Escherichia coli K-12 ferric enterobactin receptor, FepA, and used to identify regions of the polypeptide that are involved in interaction with its ligands ferric enterobactin and colicins B and D. A total of 11 distinct FepA epitopes were identified. The locations of these epitopes within the primary sequence of FepA were mapped by screening MAb against a library of FepA::PhoA fusion proteins, a FepA deletion mutant, and proteolytically modified FepA. These experiments localized the 11 epitopes to seven different regions within the FepA polypeptide, including residues 2 to 24, 27 to 37, 100 to 178, 204 to 227, 258 to 290, 290 to 339, and 382 to 400 of the mature protein. Cell surface-exposed epitopes of FepA were identified and discriminated by cytofluorimetry and by the ability of MAb that recognize them to block the interaction of FepA with its ligands. Seven surface epitopes were defined, including one each in regions 27 to 37, 204 to 227, and 258 to 290 and two each in regions 290 to 339 and 382 to 400. One of these, within region 290 to 339, was recognized by MAb in bacteria containing intact (rfa+) lipopolysaccharide (LPS); all other surface epitopes were susceptible to MAb binding only in a strain containing a truncated (rfaD) LPS core, suggesting that they are physically shielded by E. coli K-12 LPS core sugars. Antibody binding to FepA surface epitopes within region 290 to 339 or 382 to 400 inhibited killing by colicin B or D and the uptake of ferric enterobactin. In addition to the FepA-specific MAb, antibodies that recognized other outer membrane components, including Cir, OmpA, TonA, and LPS, were identified. Immunochemical and biochemical characterization of the surface structures of FepA and analysis of its hydrophobicity and amphilicity were used to generate a model of the ferric enterobactin receptor's transmembrane strands, surface peptides, and ligand-binding domains.

Two‐dimensional structure of the Opc invasin from Neisseria meningitidis

“…These criteria have been used to devise topological, two-dimensional models for porins (van der Ley et al, 1986;Tommassen, 1988), which were confirmed by the resolution of their crystal structure (Cowan et al, 1992). Similar rules seem to apply to other OMPs (Murphy et al, 1990;Koebnik and Braun, 1993;Brok et al, 1994;Dekker et al, 1995).…”

Biphasic Binding Kinetics between FepA and Its Ligands

“…This condition was met in Triton X-100, but not in DM (k 7 was Ͻ Ͻk 9 ). A possible explanation for this difference is that in DM the rapid release of FeEnt from FepA (k 9 ) results from an active solubilization of the siderophore off the surface of the receptor by the detergent. For release of ColB in the presence of FeEnt, k 10 reflects the ratelimiting step among k 8 , k 6 , k 1 , and k 3 .…”

“…In Vitro Ligand Binding in DM-The mutant protein FepAE280C (6, 10, 12, 31) binds and transports FeEnt, ColB, and ColD at wild type levels. Residue 280 lies in a proposed surface loop that participates in ligand binding (9,10). We derivatized FepAE280C with the sulfhydryl-specific reagent 5-iodoacetamidofluorescein.…”

“…The fifth proposed surface loop of FepA (PL5), bounded by residues 255-336, interacts with all three ligands during their passage through the OM (9,10). Deletions in this loop abolish FeEnt transport, render the bacteria ColB-and D-resistant (11), and convert FepA into a general porin through which sugars, antibiotics, and other small molecules may enter the cell (5).…”

PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin

“…This procedure is much more efficient than those previously used to disrupt Streptomyces genes (3). Our approach was based on the discovery that allelic exchanges on the Escherichia coli chromosome can be achieved by recombination with a PCR-generated selectable marker flanked at both ends by extensions of only a few tens of nucleotides homologous to the desired region of the chromosome, when the Red␣ (exo), Red␤ (bet), and Red␥ (gam) proteins of the phage are present in the targeted strain (4)(5)(6)(7)(8)(9). We have used -Red to promote recombination in E. coli between a PCR-amplified antibiotic resistance cassette selectable in E. coli and Streptomyces, and S. coelicolor DNA on a cosmid from the set used to sequence the S. coelicolor genome (10).…”