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• W2105689172 abstract "The ferric siderophore transporters of the Gram-negative bacterial outer membrane manifest a unique architecture: Their N termini fold into a globular domain that lodges within, and physically obstructs, a transmembrane porin β-barrel formed by their C termini. We exchanged and deleted the N termini of two such siderophore receptors, FepA and FhuA, which recognize and transport ferric enterobactin and ferrichrome, respectively. The resultant chimeric proteins and empty β-barrels avidly bound appropriate ligands, including iron complexes, protein toxins, and viruses. Thus, the ability to recognize and discriminate these molecules fully originates in the transmembrane β-barrel domain. Both the hybrid and the deletion proteins also transported the ferric siderophore that they bound. The FepA constructs showed less transport activity than wild type receptor protein, but the FhuA constructs functioned with turnover numbers that were equivalent to wild type. The mutant proteins displayed the full range of transport functionalities, despite their aberrant or missing N termini, confirming (Braun, M., Killmann, H., and Braun, V. (1999) Mol. Microbiol. 33, 1037–1049) that the globular domain within the pore is dispensable to the siderophore internalization reaction, and when present, acts without specificity during solute uptake. These and other data suggest a transport process in which siderophore receptors undergo multiple conformational states that ultimately expel the N terminus from the channel concomitant with solute internalization. The ferric siderophore transporters of the Gram-negative bacterial outer membrane manifest a unique architecture: Their N termini fold into a globular domain that lodges within, and physically obstructs, a transmembrane porin β-barrel formed by their C termini. We exchanged and deleted the N termini of two such siderophore receptors, FepA and FhuA, which recognize and transport ferric enterobactin and ferrichrome, respectively. The resultant chimeric proteins and empty β-barrels avidly bound appropriate ligands, including iron complexes, protein toxins, and viruses. Thus, the ability to recognize and discriminate these molecules fully originates in the transmembrane β-barrel domain. Both the hybrid and the deletion proteins also transported the ferric siderophore that they bound. The FepA constructs showed less transport activity than wild type receptor protein, but the FhuA constructs functioned with turnover numbers that were equivalent to wild type. The mutant proteins displayed the full range of transport functionalities, despite their aberrant or missing N termini, confirming (Braun, M., Killmann, H., and Braun, V. (1999) Mol. Microbiol. 33, 1037–1049) that the globular domain within the pore is dispensable to the siderophore internalization reaction, and when present, acts without specificity during solute uptake. These and other data suggest a transport process in which siderophore receptors undergo multiple conformational states that ultimately expel the N terminus from the channel concomitant with solute internalization. Gram-negative bacteria recognize and transport ferric siderophores (1Neilands J.B. Annu. Rev. Microbiol. 1982; 36: 285-309Crossref PubMed Scopus (332) Google Scholar, 2Neilands J.B. J. Biol. Chem. 1995; 270: 26723-26726Abstract Full Text Full Text PDF PubMed Scopus (1196) Google Scholar) through proteins in their outer membrane (OM 1The abbreviations used are:OMouter membraneLGPligand-gated porinFeEntferric enterobactinFcferrichromemAbmonoclonal antibodyLDSlithium dodecyl sulfateMOPS4-morpholinepropanesulfonic acidPCRpolymerase chain reactionTBStris-buffered salineSulfoEGSethylene glycobis(sulfosuccimidylsuccinate)ELISAenzyme-linked immunosorbent assayPAGEpolyacrylamide gel electrophoresisESRelectron spin resonance). They secrete these chelators in the iron-deficient environments that they encounter in the wild and in the host. Because of its involvement in cellular processes, iron is essential to survival, and bacteria possess efficient systems to obtain it (3Braun V. FEMS Microbiol. Rev. 1995; 16: 295-307Crossref PubMed Scopus (283) Google Scholar, 4Klebba P.E. Newton S.M. Curr. Opin. Microbiol. 1998; 1: 238-247Crossref PubMed Scopus (81) Google Scholar, 5van der Helm D. Metal Ions Biol. Syst. 1998; 35: 355-401PubMed Google Scholar, 6Thulasiraman P. Newton S.M. Xu J. Raymond K.N. Mai C. Hall A. Montague M.A. Klebba P.E. J. Bacteriol. 1998; 180: 6689-6696Crossref PubMed Google Scholar). However, their competitors in the microbial world parasitize these iron uptake pathways. Bacteriocins and phages bind to siderophore receptors as an initial step in the penetration of the cell wall (7Guterman S.K. Biochem. Biophys. Res. Commun. 1971; 44: 1149-1155Crossref PubMed Scopus (12) Google Scholar, 8Di Masi D.R. White J.C. Schnaitman C.A. Bradbeer C. J. Bacteriol. 1973; 115: 506-513Crossref PubMed Google Scholar, 9Wayne R. Frick K. Neilands J.B. J. Bacteriol. 1976; 126: 7-12Crossref PubMed Google Scholar). Other microbes are not the only antagonists that target the iron portals of bacteria: synthetic antibiotics that couple antibacterial agents to organic iron complexes also enter through siderophore receptors (10McKee J.A. Sharma S.K. Miller M.J. Bioconjug. Chem. 1991; 2: 281-291Crossref PubMed Scopus (33) Google Scholar, 11Dolence E.K. Minnick A.A. Lin C.E. Miller M.J. Payne S.M. J. Med. Chem. 1991; 34: 968-978Crossref PubMed Scopus (31) Google Scholar, 12Miller M.J. McKee J.A. Minnick A.A. Dolence E.K. Biol. Met. 1991; 4: 62-69Crossref PubMed Scopus (23) Google Scholar, 13Dolence E.K. Minnick A.A. Miller M.J. J. Med. Chem. 1990; 33: 461-464Crossref PubMed Scopus (36) Google Scholar). Finally, iron acquisition is crucial to the pathogenesis of Gram-negative bacteria, includingSalmonella, Neisseria, Yersinia,Vibrio, and Hemophilus (14Fernandez-Beros M.E. Gonzalez C. McIntosh M.A. Cabello F.C. Infect. Immun. 1989; 57: 1271-1275Crossref PubMed Google Scholar, 15Furman M. Fica A. Saxena M. Di Fabio J.L. Cabello F.C. Infect. Immun. 1994; 62: 4091-4094Crossref PubMed Google Scholar, 16Cornelissen C.N. Sparling P.F. Mol. Microbiol. 1998; 27: 611-616Crossref PubMed Scopus (175) Google Scholar, 17Zhu W. Hunt D.J. Richardson A.R. Stojiljkovic I. J. Bacteriol. 2000; 182: 439-447Crossref PubMed Scopus (100) Google Scholar, 18Bearden S.W. Perry R.D. Mol. Microbiol. 1999; 32: 403-414Crossref PubMed Scopus (215) Google Scholar, 19Occhino D.A. Wyckoff E.E. Henderson D.P. Wrona T.J. Payne S.M. Mol. Microbiol. 1998; 29: 1493-1507Crossref PubMed Scopus (142) Google Scholar, 20Cope L.D. Hrkal Z. Hansen E.J. Infect. Immun. 2000; 68: 4092-4101Crossref PubMed Scopus (25) Google Scholar). In response, higher organisms defend themselves by the sequestration of iron in proteins like ferritin, transferrin, lactoferrin, and conalbumin (21Jurado R.L. Clin. Infect. Dis. 1997; 25: 888-895Crossref PubMed Scopus (323) Google Scholar, 22Cho S.S. Lucas J.J. Hyndman A.G. Brain Res. 1999; 816: 229-233Crossref PubMed Scopus (17) Google Scholar). The competition for iron in vivo is so fierce that one pathogenic species, Borrelia, evolved completely novel biochemical systems that do not require the metal (23Posey J.E. Gherardini F.C. Science. 2000; 288: 1651-1653Crossref PubMed Scopus (392) Google Scholar). outer membrane ligand-gated porin ferric enterobactin ferrichrome monoclonal antibody lithium dodecyl sulfate 4-morpholinepropanesulfonic acid polymerase chain reaction tris-buffered saline ethylene glycobis(sulfosuccimidylsuccinate) enzyme-linked immunosorbent assay polyacrylamide gel electrophoresis electron spin resonance The OM proteins that initiate the uptake of iron, antibiotics, colicins, and phage function as ligand-gated porins (LGP (24Rutz J.M. Liu J. Lyons J.A. Goranson J. Armstrong S.K. McIntosh M.A. Feix J.B. Klebba P.E. Science. 1992; 258: 471-475Crossref PubMed Scopus (145) Google Scholar, 25Jiang X. Payne M.A. Cao Z. Foster S.B. Feix J.B. Newton S.M. Klebba P.E. Science. 1997; 276: 1261-1264Crossref PubMed Scopus (91) Google Scholar, 26Killmann H. Benz R. Braun V. EMBO J. 1993; 12: 3007-3016Crossref PubMed Scopus (116) Google Scholar)). They contain a transmembrane channel formed by amphiphilic β-strands that project to the cell surface as large loops (27Buchanan S.K. Smith B.S. Venkatramani L. Xia D. Esser L. Palnitkar M. Chakraborty R. van der Helm D. Deisenhofer J. Nat. Struct. Biol. 1999; 6: 56-63Crossref PubMed Scopus (485) Google Scholar, 28Locher K.P. Rees B. Koebnik R. Mitschler A. Moulinier L. Rosenbusch J.P. Moras D. Cell. 1998; 95: 771-778Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 29Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (661) Google Scholar). In this sense the proteins are fundamentally porins in nature (30Weiss M.S. Wacker T. Weckesser J. Welte W. Schulz G.E. FEBS Lett. 1990; 267: 268-272Crossref PubMed Scopus (170) Google Scholar, 31Cowan S.W. Schirmer T. Rummel G. Steiert M. Ghosh R. Pauptit R.A. Jansonius J.N. Rosenbusch J.P. Nature. 1992; 358: 727-733Crossref PubMed Scopus (1325) Google Scholar, 32Schirmer T. Keller T.A. Wang Y.F. Rosenbusch J.P. Science. 1995; 267: 512-514Crossref PubMed Scopus (531) Google Scholar). However, LGP differ from other porins, because they bind the molecules they transport with high affinity and because they contain a globular N terminus that resides within their transmembrane channel (Fig. 1). When ligands adsorb to siderophore receptors, they trigger unknown events that induce their own transport into the cell (33Liu J. Rutz J.M. Klebba P.E. Feix J.B. Biochemistry. 1994; 33: 13274-13283Crossref PubMed Scopus (46) Google Scholar, 34Moeck G.S. Tawa P. Xiang H. Ismail A.A. Turnbull J.L. Coulton J.W. Mol. Microbiol. 1996; 22: 459-471Crossref PubMed Scopus (62) Google Scholar, 35Letellier L. Locher K.P. Plancon L. Rosenbusch J.P. J. Biol. Chem. 1997; 272: 1448-1451Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In this sense the receptors are ligand-gated. Finally, LGP transport requires proton motive force (36Bradbeer C. J. Bacteriol. 1993; 175: 3146-3150Crossref PubMed Scopus (147) Google Scholar, 37Reynolds P.R. Mottur G.P. Bradbeer C. J. Biol. Chem. 1980; 255: 4313-4319Abstract Full Text PDF PubMed Google Scholar, 38Pugsley A.P. Reeves P. J. Bacteriol. 1976; 127: 218-228Crossref PubMed Google Scholar) and the participation of another cell envelope protein, TonB (39Wang C.C. Newton A. J. Biol. Chem. 1971; 246: 2147-2151Abstract Full Text PDF PubMed Google Scholar, 40Guterman S.K. Dann L. J. Bacteriol. 1973; 114: 1225-1230Crossref PubMed Google Scholar, 41Postle K. Good R.F. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5235-5239Crossref PubMed Scopus (90) Google Scholar), which presumably promotes the opening of the closed channels so ligands may enter. The crystal structures of the ferric enterobactin receptor, FepA (27Buchanan S.K. Smith B.S. Venkatramani L. Xia D. Esser L. Palnitkar M. Chakraborty R. van der Helm D. Deisenhofer J. Nat. Struct. Biol. 1999; 6: 56-63Crossref PubMed Scopus (485) Google Scholar), and the ferrichrome receptor, FhuA (28Locher K.P. Rees B. Koebnik R. Mitschler A. Moulinier L. Rosenbusch J.P. Moras D. Cell. 1998; 95: 771-778Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, 29Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (661) Google Scholar), defined the architecture of siderophore transporters but did not answer several pressing questions about their biochemical mechanisms. Their ligand recognition and transport processes, for example, are only vaguely defined. The OM receptors of Escherichia colidiscriminate numerous ferric siderophore complexes, which, despite their similar chelation geometry (hexacoordinate) and size (600–1000 Da), are distinct in composition and charge (1Neilands J.B. Annu. Rev. Microbiol. 1982; 36: 285-309Crossref PubMed Scopus (332) Google Scholar, 2Neilands J.B. J. Biol. Chem. 1995; 270: 26723-26726Abstract Full Text Full Text PDF PubMed Scopus (1196) Google Scholar, 42Raymond K.M. Pure Appl. Chem. 1994; 66: 773-781Crossref Scopus (53) Google Scholar). Each ferric siderophore enters through a different OM iron transporter. Ferric enterobactin (FeEnt), the native E. coli siderophore, penetrates through FepA (43McIntosh M.A. Earhart C.F. Biochem. Biophys. Res. Commun. 1976; 70: 315-322Crossref PubMed Scopus (30) Google Scholar), whereas ferrichrome (Fc), a fungal product that E. coli also utilizes, passes through FhuA (44Wayne R. Neilands J.B. J. Bacteriol. 1975; 121: 497-503Crossref PubMed Google Scholar). Does LGP specificity derive from residues in their surface loops or from amino acids in the N-domain, at the entrance to the membrane channel (Fig. 1)? Experiments on a FhuA mutant devoid of its N-terminal domain (45Braun M. Killmann H. Braun V. Mol. Microbiol. 1999; 33: 1037-1049Crossref PubMed Scopus (80) Google Scholar) were relevant to this issue, as well as that of ligand transport. In those studies the N-domain of FhuA was not necessary for Fc, colicin, and bacteriophage uptake. The experiments we report herein duplicated and confirmed those findings on FhuA and characterized comparable constructions from FepA, which gave almost identical results. Furthermore, when we genetically exchanged the N termini of FepA and FhuA, both hybrid proteins still bound and transported their appropriate ligands. Bacteria harboring plasmids (Table I) carrying the genes of interest were cultured in LB broth or MOPS minimal media (46Newton S.M. Igo J.D. Scott D.C. Klebba P.E. Mol. Microbiol. 1999; 32: 1153-1165Crossref PubMed Scopus (77) Google Scholar). It was impractical to create single-copy chromosomal derivatives of the numerous constructs we generated. Instead, we individually transferred all of the genes, including wild type fepA andfhuA, to the low-copy plasmid pHSG575, which exists inE. coli at a level of two to three copies/cell (47Hashimoto-Gotoh T. Franklin F.C. Nordheim A. Timmis K.N. Gene. 1981; 16: 227-235Crossref PubMed Scopus (137) Google Scholar).Table IStrains and plasmidsGenotypeReferenceStrainsBN1071F− thi,entA, pro, trp,rpsL(75Klebba P.E. McIntosh M.A. Neilands J.B. J. Bacteriol. 1982; 149: 880-888Crossref PubMed Google Scholar)KDF541BN1071 recA,fepA, fhuA, cir(24Rutz J.M. Liu J. Lyons J.A. Goranson J. Armstrong S.K. McIntosh M.A. Feix J.B. Klebba P.E. Science. 1992; 258: 471-475Crossref PubMed Scopus (145) Google Scholar)KDF571KDF541 tonB(24Rutz J.M. Liu J. Lyons J.A. Goranson J. Armstrong S.K. McIntosh M.A. Feix J.B. Klebba P.E. Science. 1992; 258: 471-475Crossref PubMed Scopus (145) Google Scholar)AN193F− pro leu, trp,thi, fhuA, lac, rpsL,entA(75Klebba P.E. McIntosh M.A. Neilands J.B. J. Bacteriol. 1982; 149: 880-888Crossref PubMed Google Scholar)PlasmidspHSG575(47Hashimoto-Gotoh T. Franklin F.C. Nordheim A. Timmis K.N. Gene. 1981; 16: 227-235Crossref PubMed Scopus (137) Google Scholar)PITS449fepA+ on pUC18(24Rutz J.M. Liu J. Lyons J.A. Goranson J. Armstrong S.K. McIntosh M.A. Feix J.B. Klebba P.E. Science. 1992; 258: 471-475Crossref PubMed Scopus (145) Google Scholar)pITS23fepA+ on pHSG5751-aAll constructions were analyzed as derivatives of the low copy plasmid PHSG575.This studypFepβfepAΔ17–150This studypFepβ2fepAΔ3–150This studypFepNFhuβfepA1–152::fhuA160–723This studypITS11fhuA+ on pHSG575This studypFhuβfhuAΔ5–160This studypFhuNFepβfhuA 1–155::fepA149–724This studypFhuNFepβ2fhuA 1–160::fepA153–724This study1-a All constructions were analyzed as derivatives of the low copy plasmid PHSG575. Open table in a new tab We used PCR to join the FepA N terminus (residues 1–152) to the FhuA C terminus (residues 160–723), and the FhuA N terminus (residues 1–155) to the FepA C terminus (residues 149–724). We first cloned the N- and C-terminal domains offepA and fhuA and then ligated them together. For instance, for FepNFhuβ, we PCR-amplified the DNA encoding FepN, incorporating PstI and BamHI sites in the forward and reverse primers, respectively, and inserted the product into the low copy plasmid pHSG575 (47Hashimoto-Gotoh T. Franklin F.C. Nordheim A. Timmis K.N. Gene. 1981; 16: 227-235Crossref PubMed Scopus (137) Google Scholar). We then PCR-amplified the DNA encoding Fhuβ, flanked by BamHI and SacI restriction sites, and ligated it to FepN DNA in pHSG575. Additionally, we employed oligonucleotide-directed mutagenesis (QuikChange, Stratagene, San Diego, CA) to delete residues 17–150 of fepA (creating Fepβ), residues 3–150 of fepA (Fepβ2), and residues 5–160 of fhuA (Fhuβ). Except when noted, fepA,fhuA, and their derivatives were all analyzed on pHSG575, which was used for binding, nutrition, and transport experiments. The adsorption of [59Fe]Ent and [59Fe]Fc was measured with metabolically inactive KDF541 (46Newton S.M. Igo J.D. Scott D.C. Klebba P.E. Mol. Microbiol. 1999; 32: 1153-1165Crossref PubMed Scopus (77) Google Scholar) expressing FepA or FhuA, respectively. [59Fe]Ent and [59Fe]Fc were prepared at a specific activity of ∼200 cpm/pm and chromatographically purified. Binding manipulations were performed at 0 °C. A mid-log bacterial culture was chilled on ice for 1 h, and an aliquot (containing ∼5 × 107 cells) was pipetted into a 50-ml test tube and incubated on ice. 25-ml volumes of ice-cold MOPS minimal media, containing varying concentrations of [59Fe]-siderophore, were poured into the tubes to achieve rapid and thorough mixing. After 1 min the binding reactions, which were performed in triplicate, were filtered through 0.45-μm nitrocellulose, the filters were washed with 10 ml of 0.9% LiCl and counted in a Packard Cobra gamma counter. The initial adsorption reaction reached equilibrium within 5 s at physiological temperatures and within 1 min on ice. The FepA-deficient strain KDF541 was simultaneously tested as a negative control, and any nonspecific adsorption of [59Fe]siderophores by this strain was subtracted from the experimental samples. Whenever necessary because of low cpm bound, the assay samples were counted for an extended period of time (up to 30 min) to decrease standard error. Binding data were analyzed using the Bound versus Total equation of Grafit 4.013 (Erithacus Ltd., Middlesex, UK). Ferric siderophore uptake was qualitatively evaluated by nutrition tests (46Newton S.M. Igo J.D. Scott D.C. Klebba P.E. Mol. Microbiol. 1999; 32: 1153-1165Crossref PubMed Scopus (77) Google Scholar), and the transport of [59Fe]Ent and [59Fe]Fc was quantitatively measured in live bacteria (46Newton S.M. Igo J.D. Scott D.C. Klebba P.E. Mol. Microbiol. 1999; 32: 1153-1165Crossref PubMed Scopus (77) Google Scholar). For ferric siderophore acquisition (and general and specific porin-mediated transport as well (48Nikaido H. Vaara M. Microbiol. Rev. 1985; 49: 1-32Crossref PubMed Google Scholar)), the outer membrane transport stage constitutes the rate-limiting step of the overall uptake process (4Klebba P.E. Newton S.M. Curr. Opin. Microbiol. 1998; 1: 238-247Crossref PubMed Scopus (81) Google Scholar, 6Thulasiraman P. Newton S.M. Xu J. Raymond K.N. Mai C. Hall A. Montague M.A. Klebba P.E. J. Bacteriol. 1998; 180: 6689-6696Crossref PubMed Google Scholar, 24Rutz J.M. Liu J. Lyons J.A. Goranson J. Armstrong S.K. McIntosh M.A. Feix J.B. Klebba P.E. Science. 1992; 258: 471-475Crossref PubMed Scopus (145) Google Scholar, 26Killmann H. Benz R. Braun V. EMBO J. 1993; 12: 3007-3016Crossref PubMed Scopus (116) Google Scholar, 46Newton S.M. Igo J.D. Scott D.C. Klebba P.E. Mol. Microbiol. 1999; 32: 1153-1165Crossref PubMed Scopus (77) Google Scholar, 49Cao Z. Qi Z. Sprencel C. Newton S.M. Klebba P.E. Mol. Microbiol. 2000; 37: 1306-1317Crossref PubMed Scopus (40) Google Scholar, 50Newton S.M. Allen J.S. Cao Z. Qi Z. Jiang X. Sprencel C. Igo J.D. Foster S.B. Payne M.A. Klebba P.E. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4560-4565Crossref PubMed Scopus (52) Google Scholar, 51Sprencel C. Cao Z. Qi Z. Scott D.C. Montague M.A. Ivanoff N. Xu J. Raymond K.M. Newton S.M. Klebba P.E. J. Bacteriol. 2000; 182: 5359-5364Crossref PubMed Scopus (62) Google Scholar). Periplasmic binding proteins (51Sprencel C. Cao Z. Qi Z. Scott D.C. Montague M.A. Ivanoff N. Xu J. Raymond K.M. Newton S.M. Klebba P.E. J. Bacteriol. 2000; 182: 5359-5364Crossref PubMed Scopus (62) Google Scholar) and the components of the inner membrane permease complexes are either constitutively expressed or de-repressed by iron starvation. Therefore, our transport data describe the outer membrane components of the process, FepA, FhuA, and their mutant derivatives. Transport manipulations were performed at 37 °C. A volume of mid-log bacterial culture (50–100 μl containing ∼5 × 107cells) was pipetted into a 50-ml test tube and incubated in a 37 °C water bath. Without delay, 25 ml of prewarmed MOPS minimal media, containing glucose (0.2%), appropriate nutritional supplements, and varying concentrations of [59Fe]siderophores, were poured into the tube to achieve rapid and thorough mixing. The transport reactions were quenched by the addition of a 1000-fold excess of nonradioactive ferric siderophores, immediately filtered through 0.45-μm nitrocellulose, and the filters were washed with 10 ml of 0.9% LiCl and counted in a Packard Cobra gamma counter. Kinetic parameters were determined from the initial rates of uptake, which were calculated at each substrate concentration from two independent measurements made in triplicate at 5 and 15 s: cpm bound to the cells at 5 s were subtracted from the cpm associated with the cells at 15 s (10-s uptakes). For some mutants with low uptake rates (FepNFhuβ, FhuNFepβ, Fepβ) we extended the uptake period to 60 min (6Thulasiraman P. Newton S.M. Xu J. Raymond K.N. Mai C. Hall A. Montague M.A. Klebba P.E. J. Bacteriol. 1998; 180: 6689-6696Crossref PubMed Google Scholar). In all transport experiments, the FepA-deficient strain KDF541 was simultaneously tested as a negative control, and any nonspecific adsorption of [59Fe]Ent by this strain was subtracted from the experimental samples. Transport results were analyzed according to the Michaelis-Menten equation, using Grafit 4.013. Serial dilutions of colicins B, D, or M, and bacteriophage T5 and φ80 were prepared in LB broth in microtiter plates, and 5-μl volumes of the dilutions were transferred to LB plates seeded with the strain of interest, using a Clonemaster (Immusine Corp., San Leandro, CA). The titer of the phage and colicins was expressed as the reciprocal of the highest dilution that cleared the bacterial lawn. Proteins were separated on SDS-PAGE gels (52Murphy C.K. Kalve V.I. Klebba P.E. J. Bacteriol. 1990; 172: 2736-2746Crossref PubMed Google Scholar), and either stained with Coomassie Blue or electrophoretically transferred to nitrocellulose paper. Western immunoblots were incubated with anti-FepA mAbs 26 or 45 (52), developed with 125I-protein A (49Cao Z. Qi Z. Sprencel C. Newton S.M. Klebba P.E. Mol. Microbiol. 2000; 37: 1306-1317Crossref PubMed Scopus (40) Google Scholar), quantitated by image analysis with a Packard Instant Imager, and visualized by exposure of x-ray films. The former antibody recognizes an epitope in the N-terminal globular domain of FepA (bounded by residues 27and 37 (52)); the latter binds in loop 4 of the C-terminal barrel domain, near residue 329 (49Cao Z. Qi Z. Sprencel C. Newton S.M. Klebba P.E. Mol. Microbiol. 2000; 37: 1306-1317Crossref PubMed Scopus (40) Google Scholar,52Murphy C.K. Kalve V.I. Klebba P.E. J. Bacteriol. 1990; 172: 2736-2746Crossref PubMed Google Scholar). For native gel electrophoresis, 50 μg of OM or 2 μg of purified protein was resuspended in LDS sample buffer and sonicated in an ice water bath for 5 min. After a 1-min centrifugation in a microcentrifuge, the samples were electrophoresed overnight at 5 mA and 4 °C on LDS-PAGE gels (53Liu J. Rutz J.M. Feix J.B. Klebba P.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10653-10657Crossref PubMed Scopus (67) Google Scholar). The expression of FepA and its mutants was quantitated by Western immunoblots of whole cell lysates with anti-FepA monoclonal antibodies 26 and 45 and125I-protein A. Protein concentrations were determined by image analysis, relative to standards. The localization of the chimeric proteins was determined by fractionation of inner and outer membranes on sucrose gradients (54Smit J. Kamio Y. Nikaido H. J. Bacteriol. 1975; 124: 942-958Crossref PubMed Google Scholar), followed by Western immunoblots of the fractions. Purified OM fragments or purified, denatured FepA, were suspended in 10 mm ammonium acetate, 10 mm ammonium carbonate, pH 8.3, at 10 and 0.5 μg/ml, respectively, dispensed into microtiter plates (Immulon), and incubated at 4 °C overnight. All further incubation steps were performed at 25 °C. In the morning, excess or unabsorbed antigen was removed by three washes with Tris-buffered saline containing 0.05% Tween 20, pH 7.4 (TBS-Tween), and the plates were blocked with 2% bovine serum albumin in TBS for 30 min. The plates were washed three times with TBS-Tween, and serial dilutions of anti-FepA mAbs in 50 μl of blocking buffer were added and incubated for 1 h. The blocking buffer was removed and 50 μl of a 1/100 dilution of goat-anti-mouse immunoglobulin-alkaline phosphatase (Sigma Chemical Co.) in blocking buffer was added to the plates. After incubation for 1 h at 25 °C, the plates were washed three times and developed with 50 μl of p-nitrophenyl phosphate (1 mg/ml, Sigma). After a 1-h incubation at 25 °C, 50 μl of 2n NaOH was added to stop the reaction and absorbance was measured at 405 nm with a microplate reader. Bacteria grown in MOPS minimal media or sucrose gradient-purified OM fractions were suspended at 109 cells/ml or 10 mg/ml, respectively, in 4 mmSulfoEGS, a water-soluble homobifunctional cross-linker (Mr 661 Da) that preferentially reacts with primary amines, for 2 h at 0 °C. The compound contains a 16-Å spacer arm, which is cleaved by reaction with hydroxylamine. After cross-linking, cells or OM proteins were solubilized in sample buffer, subjected to SDS-PAGE, and stained with Coomassie Blue. When indicated, 5 μm ferric enterobactin was added to the cells prior to cross-linking. Cross-linked bands were excised from the gels, cleaved with hydroxylamine, electroeluted, and re-electrophoresed: The identity of the cross-linked proteins was determined by sequence analysis of their N-terminal 15 residues (Protein and Nucleic Acid Sequence Facility, Medical College of Wisconsin). To understand the basis of ligand specificity in FepA and FhuA, we switched their N-terminal domains and biochemically characterized the resulting hybrid proteins. Although Braun et al. (45Braun M. Killmann H. Braun V. Mol. Microbiol. 1999; 33: 1037-1049Crossref PubMed Scopus (80) Google Scholar) reported the ability of an N-terminal deletion of FhuA to transport, they did not consider the affinity of the mutant for its ligands, nor the kinetic parameters of the uptake process. Our experiments duplicated their construction, created comparable deletion mutants of FepA, and created the two chimeric proteins that switched the N-domains of FepA and FhuA. In the latter case, the protein engineering conserved three structural features of FepA and FhuA: the globular N-domain, the β-barrel of the C-domain, and the β-turn that joins them (Fig.1). The resulting clones avoided the deletion or introduction of amino acids in the junction sequence: FepNFhuβ contained the N terminus and β-turn of FepA connected to the β-barrel of FhuA; FhuNFepβ contained the N terminus of FhuA linked to the β-turn and β-barrel of FepA. The hybrid proteins functioned with unexpected efficiency and selectivity, conferred by the β-barrels and their loops. The N-terminal domains did not affect the discrimination of ferric siderophores. FhuNFepβ bound FeEnt but not Fc, whereas FepNFhuβ bound Fc but not FeEnt. FepA adsorbs FeEnt with subnanomolar affinity, which persevered in the FhuNFepβ chimera: The Kdof its binding reaction with FeEnt was 0.2 nm (Fig.2, TableII). The reverse hybrid (FepNFhuβ) bound Fc with equivalent affinity to that of wild type FhuA: The Kd of its binding reaction with Fc was 0.6 nm (Fig. 2, TableII). So, despite their heterologous N termini, the chimeras maintained the selectivity of their β-barrels, with the affinity of wild type receptors.Table IIBiochemical properties of FepA and FepA mutantsStrain/plasmidFerric enterobactinProtein ligands2-aColicin and bacteriophage susceptibility was determined by measuring the killing of KDF541 expressing the wild type and mutant proteins by limiting dilutions of colicins B, D, and M, and phage T5 and φ80. The results are expressed as the titer of the killing agents on the different bacterial strains.MAbs2-dAnti-FepA mAbs 33 and 45, which recognize sites in loops 2 and 4, respectively, were used to measure the exposure of surface loops on the bacteria expressing the proteins of interest, relative to the FepA-deficient strain KDF541. The tabulated values are mean fluorescence intensity. The data are from a single experiment, but the experiment was repeated several times with little variation.Antibiotic2-eThe tabulated values are the diameter (millimeters) of the zone of clearing created by a 6-mm filter paper disk, embedded with an antibiotic (N, neomycin, 30 μg/ml; E, erythromycin, 15 μg/ml; B, bacitracin, 10 IU/ml) on a lawn of the bacteria.Exp.2-fThe expression of FepA and its chimeras (copies/cell) was measured by SDS-PAGE and immunoblots with anti-FepA mAbs and 125I-protein A (Fig. 4).Binding2-bKd (nm) and capacity (Cap) (pmol bound/109 cells) were determined from the concentration dependence of FeEnt binding, by analyzing the mean values from three independent experiments with Gra" @default.
• W2105689172 created "2016-06-24" @default.
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• W2105689172 date "2001-04-01" @default.
• W2105689172 modified "2023-09-27" @default.
• W2105689172 title "Exchangeability of N Termini in the Ligand-gated Porins ofEscherichia coli" @default.
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• W2105689172 doi "https://doi.org/10.1074/jbc.m011282200" @default.
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