Title: Cell Envelope Signaling in Escherichia coli
Abstract: The ferrichrome-iron receptor ofEscherichia coli is FhuA, an outer membrane protein that is dependent upon the energy-coupling protein TonB to enable active transport of specific hydroxamate siderophores, infection by certain phages, and cell killing by the protein antibiotics colicin M and microcin 25. In vivo cross-linking studies were performed to establish at the biochemical level the interaction between FhuA and TonB. In an E. coli strain in which both proteins were expressed from the chromosome, a high molecular mass complex was detected when the ferrichrome homologue ferricrocin was added immediately prior to addition of cross-linker. The complex included both proteins; it was absent from strains of E. coli that were devoid of either FhuA or TonB, and it was detected with anti-FhuA and anti-TonB monoclonal antibodies. These results indicate that,in vivo, the binding of ferricrocin to FhuA enhances complex formation between the receptor and TonB. An in vitro system was established with which to examine the FhuA-TonB interaction. Incubation of TonB with histidine-tagged FhuA followed by addition of Ni2+-nitrilotriacetate-agarose led to the specific recovery of both TonB and FhuA. Addition of ferricrocin or colicin M to FhuA in this system greatly increased the coupling between FhuA and TonB. Conversely, a monoclonal antibody that binds near the N terminus of FhuA reduced the retention of TonB by histidine-tagged FhuA. These studies demonstrate the significance of ligand binding at the external surface of the cell to mediate signal transduction across the outer membrane. The ferrichrome-iron receptor ofEscherichia coli is FhuA, an outer membrane protein that is dependent upon the energy-coupling protein TonB to enable active transport of specific hydroxamate siderophores, infection by certain phages, and cell killing by the protein antibiotics colicin M and microcin 25. In vivo cross-linking studies were performed to establish at the biochemical level the interaction between FhuA and TonB. In an E. coli strain in which both proteins were expressed from the chromosome, a high molecular mass complex was detected when the ferrichrome homologue ferricrocin was added immediately prior to addition of cross-linker. The complex included both proteins; it was absent from strains of E. coli that were devoid of either FhuA or TonB, and it was detected with anti-FhuA and anti-TonB monoclonal antibodies. These results indicate that,in vivo, the binding of ferricrocin to FhuA enhances complex formation between the receptor and TonB. An in vitro system was established with which to examine the FhuA-TonB interaction. Incubation of TonB with histidine-tagged FhuA followed by addition of Ni2+-nitrilotriacetate-agarose led to the specific recovery of both TonB and FhuA. Addition of ferricrocin or colicin M to FhuA in this system greatly increased the coupling between FhuA and TonB. Conversely, a monoclonal antibody that binds near the N terminus of FhuA reduced the retention of TonB by histidine-tagged FhuA. These studies demonstrate the significance of ligand binding at the external surface of the cell to mediate signal transduction across the outer membrane. High affinity iron uptake in Gram-negative bacteria such asEscherichia coli is a stepwise process that involves recognition of a ferric iron chelator (siderophore) by a receptor within the outer membrane, translocation of the siderophore-Fe(III) complex into the periplasm, and internalization of the iron by a cytoplasmic membrane permease in a periplasmic binding protein-dependent manner (reviewed in Refs. 1Postle K. Mol. Microbiol. 1990; 4: 2019-2025Crossref PubMed Scopus (146) Google Scholar, 2Postle K. J. Bioenerg. Biomembr. 1993; 6: 591-601Google Scholar, 3Braun V. FEMS Microbiol. Rev. 1995; 16: 295-307Crossref PubMed Scopus (285) Google Scholar, 4Earhart C.F. Neidhart F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Brooks Low K. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. American Society for Microbiology, Washington, D. C.1996: 1075-1090Google Scholar). Dissection of the energy requirements for high affinity uptake of iron (5Hancock R.E.W. Braun V. J. Bacteriol. 1976; 125: 409-415Crossref PubMed Google Scholar) and of vitamin B12 (6Bradbeer C. Woodrow M.L. J. Bacteriol. 1976; 128: 99-104Crossref PubMed Google Scholar, 7Bassford Jr., P.J. Bradbeer C. Kadner R.J. Schnaitman C.A. J. Bacteriol. 1976; 128: 242-247Crossref PubMed Google Scholar, 8Reynolds P.R. Mottur G.P. Bradbeer C. J. Biol. Chem. 1980; 255: 4313-4319Abstract Full Text PDF PubMed Google Scholar, 9Bradbeer C. J. Bacteriol. 1993; 175: 3146-3150Crossref PubMed Scopus (147) Google Scholar), transport of which shares common elements with siderophore-Fe(III) transport mechanisms, identified the importance of the TonB and ExbB/D proteins of the cytoplasmic membrane to couple proton-motive force with active transport at the outer membrane (Ref.10Larsen R.A. Thomas M.G. Wood G.E. Postle K. Mol. Microbiol. 1994; 13: 627-640Crossref PubMed Scopus (85) Google Scholar; reviewed in Refs. 2Postle K. J. Bioenerg. Biomembr. 1993; 6: 591-601Google Scholar and 11Kadner R.J. Mol. Microbiol. 1990; 4: 2027-2033Crossref PubMed Scopus (181) Google Scholar). TonB homologues have been identified in many Gram-negative bacteria, including Salmonella enterica serovar Typhimurium (12Hannavy K. Barr G.C. Dorman C.J. Adamson J. Mazengera L.R. Gallagher M.P. Evans J.S. Levine B.A. Trayer I.P. Higgins C.F. J. Mol. Biol. 1990; 216: 897-910Crossref PubMed Scopus (100) Google Scholar), Yersinia enterocolitica (13Koebnik R. Bäumler A.J. Heesemann J. Braun V. Hantke K. Mol. Gen. Genet. 1993; 237: 152-160Crossref PubMed Scopus (38) Google Scholar), Haemophilus influenzae (14Jarosik G.P. Sanders J.D. Cope L.D. Muller-Eberhard U. Hansen E.J. Infect. Immun. 1994; 62: 2470-2477Crossref PubMed Google Scholar), andPseudomonas aeruginosa (15Poole K. Zhao Q. Neshat S. Heinrichs D.E. Dean C.R. Microbiology. 1996; 142: 1449-1458Crossref PubMed Scopus (59) Google Scholar). In addition, complexes between TonB or its homologues and other proteins (ExbB, ExbD, and as yet unidentified proteins) were detected with anti-E. coli TonB monoclonal antibodies (mAbs 1The abbreviations used are: mAb, monoclonal antibody; PVDF, polyvinylidene difluoride; NTA, nitrilotriacetate; LDAO, N,N-dimethyldodecylamine N-oxide; PAGE, polyacrylamide gel electrophoresis; TLN, Tris-HCl/LDAO/NaCl. ; Ref. 16Larsen R.A. Myers P.S. Skare J.T. Seachord C.L. Darveau R.P. Postle K. J. Bacteriol. 1996; 178: 1363-1373Crossref PubMed Google Scholar). These findings demonstrate that the TonB-dependent energy transduction system is shared among many Gram-negative bacteria and suggest that high affinity energy-dependent iron uptake in Gram-negative aerobes is accomplished using TonB and its accessory proteins (16Larsen R.A. Myers P.S. Skare J.T. Seachord C.L. Darveau R.P. Postle K. J. Bacteriol. 1996; 178: 1363-1373Crossref PubMed Google Scholar). Physical association between TonB and a TonB-dependent outer membrane receptor was first demonstrated biochemically byin vivo cross-linking experiments in which TonB was coupled to the E. coli enterobactin (also known as enterochelin) receptor, FepA (17Skare J.T. Ahmer B.M.M. Seachord C.L. Darveau R.P. Postle K. J. Biol. Chem. 1993; 268: 16302-16308Abstract Full Text PDF PubMed Google Scholar). This finding substantiated genetic analyses that suggested interactions between TonB and other outer membrane transporters, namely the ferrichrome-iron receptor FhuA (18Schöffler H. Braun V. Mol. Gen. Genet. 1989; 217: 378-383Crossref PubMed Scopus (147) Google Scholar, 19Günter K. Braun V. FEBS Lett. 1990; 274: 85-88Crossref PubMed Scopus (70) Google Scholar) and the vitamin B12 receptor BtuB (20Heller K. Kadner R.J. Günter K. Gene (Amst.). 1988; 64: 147-153Crossref PubMed Scopus (142) Google Scholar, 21Bell P.E. Nau C.D. Brown J.T. Konisky J. Kadner R.J. J. Bacteriol. 1990; 172: 3826-3829Crossref PubMed Google Scholar, 22Anton M. Heller K.J. Mol. Gen. Genet. 1993; 239: 371-377Crossref PubMed Scopus (11) Google Scholar). However, formation of the FepA-TonB complex appeared to be independent of the presence of ferric enterobactin, since the strains used carried mutations in the enterobactin biosynthetic genes and were negative on chrome azurol S plates used for detecting siderophore excretion (23Schwyn B. Neilands J.B. Anal. Biochem. 1987; 160: 47-56Crossref PubMed Scopus (4493) Google Scholar). Paradoxically, TonB did not appear to form cross-links with any of the other outer membrane receptors. BtuB and FhuA have subsequently been shown to compete for a limiting amount of TonB function; ferrichrome decreased the rate of vitamin B12 transport, and, conversely, vitamin B12 inhibited ferrichrome uptake if BtuB were overexpressed (24Kadner R.J. Heller K.J. J. Bacteriol. 1995; 177: 4829-4835Crossref PubMed Google Scholar). These results imply that ligand-bound outer membrane receptors have a means of signaling to TonB that they are occupied. Indeed, outer membrane receptors have been demonstrated to change conformations following addition of cognate ligands (25Liu J. Rutz J.M. Klebba P.E. Feix J.B. Biochemistry. 1994; 33: 13274-13283Crossref PubMed Scopus (46) Google Scholar, 26Moeck G.S. Tawa P. Xiang H. Turnbull J. Ismail A.A. Coulton J.W. Mol. Microbiol. 1996; 22: 459-471Crossref PubMed Scopus (62) Google Scholar, 27Jiang X. Payne M.A. Cao Z. Foster S.B. Feix J.B. Newton S.M.C. Klebba P.E. Science. 1997; 276: 1261-1264Crossref PubMed Scopus (91) Google Scholar). The binding of ferrichrome-iron to purified FhuA led to a conformational change in the receptor detectable both as a reduction in reactivity of certain anti-FhuA mAbs and as a gain in resistance to trypsinolysis at lysine 67 but not at lysine 5 of FhuA (26Moeck G.S. Tawa P. Xiang H. Turnbull J. Ismail A.A. Coulton J.W. Mol. Microbiol. 1996; 22: 459-471Crossref PubMed Scopus (62) Google Scholar). We proposed that such a conformational change in FhuA may act as a signal to TonB in vivo, indicating that the receptor is loaded with ligand. To understand the functional relevance of the ligand-induced conformational change in FhuA and to investigate whether receptors other than FepA can form complexes with TonB, we initiated studies of FhuA-TonB interactions. We now report that FhuA can be cross-linked to TonB and that the cross-linking between FhuA and TonB is significantly enhanced by the presence of the ligand ferricrocin. Additionally, we have established an in vitro system with which to evaluate the coupling between FhuA and TonB and the effect of normally membrane-impermeant macromolecules upon the interaction. Complementary results from in vivo and in vitro experiments confirm that ligand binding to FhuA enhanced its physical association with TonB. Goat anti-mouse immunoglobulin (Ig) G (heavy plus light chain)-horseradish peroxidase conjugate and goat anti-mouse IgG1-horseradish peroxidase conjugate antibodies were from Southern Biotechnology Associates (Birmingham, AL). The enhanced chemiluminescence Western kit was obtained from Amersham Corp. Immobilon-P polyvinylidene difluoride (PVDF) membrane was from Millipore Corp. Ni2+-nitrilotriacetate (NTA)-agarose resin was from QIAGEN, imidazole from ICN Biomedicals, Inc., and N, N-dimethyldodecylamine N-oxide (LDAO) detergent from Fluka. X-Omat AR-5 film was purchased from Eastman Kodak Co.; Reflection autoradiography film was from NEN Life Science Products. The E. coli K-12 strains used in this study were GM1 (araΔ(lac-pro) thi F′(lac-pro); Ref.28Sun T.P Webster R.E. J. Bacteriol. 1986; 165: 107-115Crossref PubMed Google Scholar), W3110 (F− IN(rrnD-rrnE)1; Ref.29Hill C.W. Harnish B.W. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7069-7072Crossref PubMed Scopus (175) Google Scholar), MC4100 (F− araD Δ(argF-lac)U169 rspL thi relA flbB deoC1 pstF rbsR; from T. J. Silhavy, Princeton University), SG303 (MC4100 aroB; Ref.30Carmel G. Hellstern D. Henning D. Coulton J.W. J. Bacteriol. 1990; 172: 1861-1869Crossref PubMed Google Scholar), KP1032 (W3110 tonB::kan; Ref. 10Larsen R.A. Thomas M.G. Wood G.E. Postle K. Mol. Microbiol. 1994; 13: 627-640Crossref PubMed Scopus (85) Google Scholar); KP1060 (GM1 Δ(ompT-fepA-entF); Ref. 17Skare J.T. Ahmer B.M.M. Seachord C.L. Darveau R.P. Postle K. J. Biol. Chem. 1993; 268: 16302-16308Abstract Full Text PDF PubMed Google Scholar), KP1120 (KP1060 Δ(trp-tonB-opp-ana)467; Ref. 31Jaskula J.C. Letain T.E. Roof S.K. Skare J.T. Postle K. J. Bacteriol. 1994; 176: 2326-2338Crossref PubMed Google Scholar), RK4691 (P1450 col M+; from R. J. Kadner, University of Virginia), and AW740 (hisG4 thr-1 fhuA31 tsx-78 ΔompF zcb::Tn10 ΔompC; Ref. 32Ingham C. Buechner M. Adler J. J. Bacteriol. 1990; 172: 3577-3583Crossref PubMed Google Scholar). Plasmid pGC01 (30Carmel G. Hellstern D. Henning D. Coulton J.W. J. Bacteriol. 1990; 172: 1861-1869Crossref PubMed Google Scholar) encodes the wild-type fhuA gene in pBR322; plasmid pHX405 encodes FhuA.H6 (26Moeck G.S. Tawa P. Xiang H. Turnbull J. Ismail A.A. Coulton J.W. Mol. Microbiol. 1996; 22: 459-471Crossref PubMed Scopus (62) Google Scholar), a derivative of FhuA containing a hexahistidine tag inserted after amino acid 405 of the deduced mature amino acid sequence of FhuA (33Coulton J.W. Mason P. Cameron D.R. Carmel G. Jean R. Rode H.N. J. Bacteriol. 1986; 165: 181-192Crossref PubMed Google Scholar). Plasmid pCG405 encodes FhuAΔ021–128.H6, a FhuA derivative that contains a hexahistidine tag inserted after amino acid 405 of the FhuAΔ021–128 protein (34Carmel G. Coulton J.W. J. Bacteriol. 1991; 173: 4394-4403Crossref PubMed Google Scholar). E. coli strain GSM01 was constructed by P1 transduction of thetonB::kan mutation from KP1032 into SG303fhuA, selecting for kanamycin resistance and screening for colicin B resistance. KP1060fhuA cells were isolated by selection of spontaneously occurring T5 phage-resistant colonies of KP1060 and screening for loss of sensitivity to the FhuA-specific phages UC-1 and φ80 and for resistance to colicin M and to the peptide antibiotic microcin 25. Lack of FhuA expression was confirmed by immunoblotting of outer membranes from KP1060fhuA with anti-FhuA mAbs (35Moeck G.S. Ratcliffe M.J.H. Coulton J.W. J. Bacteriol. 1995; 177: 6118-6125Crossref PubMed Google Scholar). Strain GSM02 was constructed by P1 transduction oftonBΔ66–100 (90% linked totrpB::Tn10) from KP1096 (36Larsen R.A. Wood G.E. Postle K. Mol. Microbiol. 1993; 10: 943-953Crossref PubMed Scopus (85) Google Scholar) into KP1060fhuA, selecting for tetracycline resistance and screening by immunoblot with anti-TonB mAb 4F1 (16Larsen R.A. Myers P.S. Skare J.T. Seachord C.L. Darveau R.P. Postle K. J. Bacteriol. 1996; 178: 1363-1373Crossref PubMed Google Scholar) for TonB protein of reduced molecular mass. Cultures were grown overnight in LB broth at 37 °C and then subcultured by 1:100 dilution into M9 glucose minimal medium supplemented with 0.2% casamino acids, tryptophan at 40 μg/ml, thiamine at 4 μg/ml, MgSO4 at 1 mm, and CaCl2 at 0.5 mm. FeCl3 was added to a final concentration of either 2 μm (for tonBstrains) or 20 μm (for aroB andentF strains). Cultures were grown with aeration at 37 °C to an A 550 of 0.5 (spectrophotometer path length, 1.5 cm). Ten nmol of the FhuA-specific hydroxamate siderophore ferricrocin-iron (hereafter referred to as ferricrocin) was added to 1 ml of cells at 0.5A 550 (ml) equivalents in 100 mmsodium phosphate, pH 6.8. Following an incubation of 5 min, formaldehyde was added to a final concentration of 1%. Cross-linking (17Skare J.T. Ahmer B.M.M. Seachord C.L. Darveau R.P. Postle K. J. Biol. Chem. 1993; 268: 16302-16308Abstract Full Text PDF PubMed Google Scholar) was allowed to proceed for 25 min. Lysine, cysteine, and tyrosine, and to a lesser extent tryptophan, histidine, aspartate, and arginine, are targets for cross-linking by formaldehyde (37Means G.E. Feeney R.E. Chemical Modification of Proteins. Holden-Day Publishers, San Francisco1971Google Scholar, 38Brinkley M. Bioconjugate Chem. 1992; 3: 2-13Crossref PubMed Scopus (502) Google Scholar). After formaldehyde cross-linking, cells were pelleted and solubilized in 4 × concentrated SDS-PAGE sample buffer with heating at 60 °C for 5 min. SDS-solubilized proteins (0.25 A 550 (ml) equivalents/lane) were resolved (200 mA·h/gel) on 8% polyacrylamide gels and transferred to PVDF membranes. Conditions for immunoblotting were those described (17Skare J.T. Ahmer B.M.M. Seachord C.L. Darveau R.P. Postle K. J. Biol. Chem. 1993; 268: 16302-16308Abstract Full Text PDF PubMed Google Scholar). Anti-FhuA mAbs Fhu8.3 and Fhu5.1 (35Moeck G.S. Ratcliffe M.J.H. Coulton J.W. J. Bacteriol. 1995; 177: 6118-6125Crossref PubMed Google Scholar) bind to determinants between residues 1–20 and 21–59, respectively, of the mature FhuA sequence (33Coulton J.W. Mason P. Cameron D.R. Carmel G. Jean R. Rode H.N. J. Bacteriol. 1986; 165: 181-192Crossref PubMed Google Scholar); anti-TonB mAbs 4H4 and 4F1 recognize epitopes corresponding to P77IPEPPKEAP and R120PASPFENT, respectively (16Larsen R.A. Myers P.S. Skare J.T. Seachord C.L. Darveau R.P. Postle K. J. Bacteriol. 1996; 178: 1363-1373Crossref PubMed Google Scholar), of the TonB sequence (39Postle K. Good R.F. Proc. Natl. Acad. Sci. U. S. A. 1983; 80: 5235-5239Crossref PubMed Scopus (90) Google Scholar). E. coliK-12 strains GSM02, KP1060fhuA, and KP1120fhuAwere grown to an A 600 of 1.0 in supplemented M9 glucose minimal medium. Cells were pelleted by centrifugation, resuspended in 1/30 volume of 100 mm Tris-HCl, pH 7.4, 1 mm EDTA, 0.5 mm phenylmethylsulfonyl fluoride, and lysed by two passes through a French cell press (Aminco) at 18,000 p.s.i., 4 °C. The cleared cell lysate was centrifuged at 15,000 × g for 15 min at 4 °C to pellet unbroken cells and much of the outer membrane fraction (40Nikaido H. Methods Enzymol. 1994; 235: 225-234Crossref PubMed Scopus (87) Google Scholar). The supernatant was centrifuged at 185,000 × g for 2 h at 4 °C. The pellet from ultracentrifugation, enriched for cytoplasmic membrane vesicles (40Nikaido H. Methods Enzymol. 1994; 235: 225-234Crossref PubMed Scopus (87) Google Scholar), was solubilized with stirring for 45 min at room temperature in 50 mm Tris-HCl, pH 7.8, 2% LDAO, and 0.5 mm phenylmethylsulfonyl fluoride to give a total soluble protein concentration of approximately 10 mg/ml. Extracts were stored at −20 °C. Immediately prior to column experiments the samples were thawed, diluted 10-fold with 50 mm Tris-HCl, pH 7.8, 0.1% LDAO, 100 mm NaCl (TLN) plus 5 mm imidazole, and filtered over 0.45-μm pore size cellulose acetate filters (Millipore Corp.). Hexahistidine-tagged FhuA (FhuA.H6) from the outer membrane of the E. coli K-12 strain AW740(pHX405) was purified to apparent homogeneity over Ni2+-NTA-agarose (26Moeck G.S. Tawa P. Xiang H. Turnbull J. Ismail A.A. Coulton J.W. Mol. Microbiol. 1996; 22: 459-471Crossref PubMed Scopus (62) Google Scholar). To construct the FhuA.H6-Ni2+-NTA-agarose column, a 2-mg aliquot of concentrated FhuA.H6 was diluted 1:10 in TLN and loaded onto a bed of 1 ml of Ni2+-NTA-agarose in an HR 5/5 column (Pharmacia Biotech Inc.). A flow rate of 0.5 ml/min was used for all column chromatography. The column was washed with 10 volumes of TLN plus 5 mm imidazole. A sample of TLN-solubilized proteins from the ultracentrifugation pellet of E. coli strain GSM02 containing TonBΔ66–100 was loaded onto the column. The resin was washed with 10 column volumes of TLN plus 5 mmimidazole and a linear gradient of imidazole to 500 mm was applied over 20 column volumes. A major peak was eluted at an imidazole concentration of approximately 80 mm. Samples (8 μl) of column fractions (1 ml) were mixed with an equal volume of 4 × concentrated SDS-PAGE sample buffer, boiled for 1 min, and then resolved on 9% polyacrylamide gels. Proteins were transferred to PVDF membranes and probed with anti-FhuA and anti-TonB mAbs. The primary antibodies were detected either with an anti-mouse κ chain-specific alkaline phosphatase-conjugated secondary antibody (187.1; Ref. 41Yelton D.E. Desaymard C. Scharff M.D. Hybridoma. 1981; 1: 5-11Crossref PubMed Scopus (192) Google Scholar) followed by visualization using 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium or with a goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody followed by visualization by enhanced chemiluminescence. In a parallel experiment, a duplicate 2-mg aliquot of concentrated FhuA.H6 was diluted 1:10 in TLN, mixed with a 20-fold molar excess of ferricrocin, and loaded onto a bed of 1 ml of Ni2+-NTA-agarose resin. Addition of solubilized proteins from E. coli strain GSM02 (containing TonBΔ66–100), column washing, and elution with imidazole was performed exactly as described above for the FhuA.H6 sample. To eliminate the possibility that the presence of TonBΔ66–100 in the eluate was due to some affinity of TonBΔ66–100 for the Ni2+-NTA-agarose resin, the experiment was repeated without application of FhuA.H6 to the resin. To investigate the influence of FhuA-specific ligands upon the protein-protein interaction, an in vitro experiment of smaller scale was devised. Aliquots (5 μg) of either FhuA.H6 or FhuAΔ021–128.H6 in TLN were incubated in solution with a 20-fold molar excess of ferricrocin, with 50 μl of undiluted RK4691 cell lysate containing colicin M (Ref. 34Carmel G. Coulton J.W. J. Bacteriol. 1991; 173: 4394-4403Crossref PubMed Google Scholar; 28-fold dilution of stock gave a clear zone of lysis on the indicator strain MC4100), with 50 μl of undiluted AY261 cell lysate containing microcin 25 (Ref. 42Salomón R.A. Farı́as R.N. J. Bacteriol. 1992; 174: 7428-7435Crossref PubMed Google Scholar; 28-fold dilution of stock gave a clear zone of lysis), or with 1011 plaque-forming units of the FhuA-specific phages T5 or φ80 for 10 min. All incubations were at 25 °C. For some experiments, hybridoma supernatants (specific Ig concentration, 5–10 μg/ml) containing anti-FhuA mAbs Fhu8.3 (G2a isotype), Fhu5.1 (G3 isotype), Fhu6.3 (M isotype), or Fhu8.1 (A isotype) were then added and incubated for another 10 min. These mAbs bind determinants located between amino acids 1–20, 21–59, 381–417, and 417–550 of FhuA, respectively (35Moeck G.S. Ratcliffe M.J.H. Coulton J.W. J. Bacteriol. 1995; 177: 6118-6125Crossref PubMed Google Scholar). To these mixtures was added an aliquot (100 μl) of TLN-solubilized lysate (total protein concentration ∼1 mg/ml) from KP1060fhuA cells, prepared as described above. In some experiments, the TLN-solubilized lysate from KP1060fhuAcells was mixed with 1 μl of a 1 mm stock of either peptide 1 (corresponding to amino acids 1–18 of FhuA) or peptide 2 (corresponding to amino acids 636–651 of FhuA) for 10 min prior to the addition of the TonB-containing sample to FhuA.H6. Before use, the pH of the peptide solutions was adjusted to neutrality with 100 mm Tris-HCl, pH 7.4. After a 15-min incubation, 2.5 μl of TLN-equilibrated Ni2+-NTA-agarose resin was added and mixed for 10 min. The resin was pelleted by centrifugation and washed twice with TLN containing 5 mm imidazole, and bound proteins were eluted by addition of 30 μl of TLN containing 50 mm EDTA. Aliquots (5 μl) of the eluate were mixed with electrophoresis sample buffer, boiled for 1 min, and loaded onto 9% polyacrylamide gels. Resolved proteins were transferred to PVDF membranes and probed with anti-FhuA mAb Fhu8.4 and anti-TonB mAb 4H4, both of which are of the G1 isotype. Primary antibodies were detected by goat anti-mouse IgG1-horseradish peroxidase-conjugated antibodies and chemiluminescence. Relative amounts of FhuA.H6, FhuAΔ021–128.H6, and TonB were assessed by scanning (Hewlett Packard ScanJet IIcx flatbed scanner; 600 dots per inch resolution as a black and white photo; default settings of brightness and contrast) of the developed x-ray film and quantitation (ImageQuant software, Molecular Dynamics Inc., Sunnyvale, CA) of the bands by volume integration. To account for slight differences in loading of FhuA, TonB bands were normalized relative to each FhuA.H6band after background subtraction. At least three replicates of experiments with FhuA.H6 were performed. Previous results demonstrated the existence of a high molecular mass complex containing both FepA and TonB (17Skare J.T. Ahmer B.M.M. Seachord C.L. Darveau R.P. Postle K. J. Biol. Chem. 1993; 268: 16302-16308Abstract Full Text PDF PubMed Google Scholar). We posited that a complex between FhuA and TonB could be identified by using a similar in vivoformaldehyde cross-linking protocol. The anti-TonB mAb 4H4 detected prominent bands with apparent molecular masses of 57–59 kDa and ∼175 kDa (Fig. 1 A). Anti-TonB mAb-reactive bands with relative mobilities greater than the band at 69 kDa are not displayed because they have been characterized previously (10Larsen R.A. Thomas M.G. Wood G.E. Postle K. Mol. Microbiol. 1994; 13: 627-640Crossref PubMed Scopus (85) Google Scholar, 16Larsen R.A. Myers P.S. Skare J.T. Seachord C.L. Darveau R.P. Postle K. J. Bacteriol. 1996; 178: 1363-1373Crossref PubMed Google Scholar, 17Skare J.T. Ahmer B.M.M. Seachord C.L. Darveau R.P. Postle K. J. Biol. Chem. 1993; 268: 16302-16308Abstract Full Text PDF PubMed Google Scholar, 36Larsen R.A. Wood G.E. Postle K. Mol. Microbiol. 1993; 10: 943-953Crossref PubMed Scopus (85) Google Scholar). It should be noted that unequivocal assignments of molecular masses of bands based on their mobility in SDS-PAGE was not possible (as noted in Refs. 16Larsen R.A. Myers P.S. Skare J.T. Seachord C.L. Darveau R.P. Postle K. J. Bacteriol. 1996; 178: 1363-1373Crossref PubMed Google Scholar and 17Skare J.T. Ahmer B.M.M. Seachord C.L. Darveau R.P. Postle K. J. Biol. Chem. 1993; 268: 16302-16308Abstract Full Text PDF PubMed Google Scholar). Since formaldehyde cross-links are heat-labile, protein samples were not completely denatured by boiling prior to application to the polyacrylamide gel. The prominent band of ∼175 kDa was present in cells in which FhuA was expressed at chromosomal levels (E. coli strain SG303) but absent from SG303fhuA cells. This band was detected when FhuA was expressed from the multicopy plasmid pGC01 in SG303fhuA cells but was lost in the isogenictonB::kan strain GSM01(pGC01). This band therefore represented a candidate FhuA-TonB complex. To establish the specificity of the candidate FhuA-TonB complex, the effect of adding the FhuA-specific siderophore ferricrocin immediately prior to formaldehyde cross-linking was examined. Ferricrocin induced the appearance of the band migrating to ∼175 kDa in strain SG303 and increased the abundance of the complex in SG303fhuA(pGC01). Addition of ferricrocin did not significantly change the SDS-soluble protein profile of either SG303fhuA or GSM01(pGC01). To verify that the ∼175-kDa complex contained FhuA, an identical panel of cross-linked proteins was probed by immunoblotting with the anti-FhuA mAb Fhu8.3 (Fig. 1 B). In the SDS-soluble cross-linked proteins from strain SG303, the most prominent band corresponded to monomeric wild-type FhuA of 80 kDa. Less intensely mAb-reactive bands of ∼90, 100, and 110 kDa were also identified. Upon longer exposures of the x-ray film (Fig. 1 C), a band migrating with an apparent molecular mass of ∼175 kDa was detected in the SDS-soluble extract from SG303 cells that had been preincubated with ferricrocin. As was apparent upon probing with the anti-TonB mAb, no band migrating to ∼175 kDa was present when FhuA was expressed from the chromosome in the absence of exogenously added ferricrocin. The formation of this ∼175-kDa complex was dependent upon both FhuA and TonB since in strains SG303fhuA and GSM01(pGC01) the complex was absent. Furthermore, the complex that was detected by the anti-FhuA mAb displayed an identical pattern of abundance as influenced by addition of ferricrocin. These data therefore demonstrated that a complex between FhuA and TonB was formed in vivo and that its amount increased by preincubation with ferricrocin. When FhuA was expressed from the high copy number plasmid pGC01, a family of FhuA-containing, formaldehyde-cross-linked complexes appeared above the FhuA monomer in the range of 90–130 kDa (Fig.1 B). These bands had mobilities identical to the less prominent bands that were seen above the FhuA monomer when FhuA was expressed from the chromosome in strain SG303 (particularly in longer exposures; see Fig. 1 C) and were independent of the presence of TonB since their profile was unchanged between SG303fhuA(pGC01) and GSM01(pGC01). At present, we have not established the identity of these FhuA-containing complexes. A similar set of anti-FhuA mAb-reactive bands was disclosed after in vivo cross-linking with E. coli strain SG303fhuA(pCG405) (data not shown). In these experiments, the FhuA-containing complexes migrated slower than the FhuAΔ021–128.H6 monomer, which had a relative mobility of ∼72 kDa. This observation indicates that amino acids 21–128 of FhuA are apparently not required for the formation of these higher molecular mass complexes. We developed anin vitro system with which to examine the interaction between FhuA and TonB and that would allow for further assessment of its specificity. Purified FhuA.H6 protein was applied to a Ni2+-NTA-agarose column. Detergent-solubilized samples containing TonB were introduced into the column, the resin was washed and bound proteins were eluted with imidazole. Initial experiments showed that trace amounts of wild-type TonB were retained by the Ni2+-NTA-agarose resin even in the absence of FhuA.H6. Two modifications were employed to circumvent this limitation. We postulated that the proline-rich region of TonB might be responsible for the interaction betwe