The cytoplasmic membrane protein TonB couples the proton electrochemical potential of the cytoplasmic membrane to transport events at the outer membrane of Gram-negative bacteria. The amino-terminal signal anchor of TonB and its interaction with the cytoplasmic membrane protein ExbB are essential to this process. The TonB signal anchor is predicted to form an α-helix, with a conserved face comprised of residues Ser16, His20, Leu27, and Ser31. Deletion of either Ser16 or His20 or of individual intervening but not flanking residues rendered TonB inactive and unable to assume a proton motive force-dependent conformation. In vivo formaldehyde cross-linking experiments revealed that the ability of this subset of mutants to form a characteristic heterodimer with ExbB was greatly diminished. Replacement of residues 17–19 by three consecutive alanines produced a wild type TonB allele, indicating that the intervening residues (Val, Cys, and Ile) contributed only to spacing. These data indicated that the spatial relationship of Ser16 to His20 was essential to function and suggested that the motif HXXXS defines the minimal requirement for the coupling of TonB to the cytoplasmic membrane electrochemical gradient. Deletion of Trp11 resulted in a TonB that remained active yet was unable to cross-link with ExbB. Because Trp11 was demonstrably not involved in the actual cross-linking, these results suggest that the TonB/ExbB interaction detected by cross-linking occurred at a step in the energy transduction cycle distinct from the coupling of TonB to the electrochemical gradient. The cytoplasmic membrane protein TonB couples the proton electrochemical potential of the cytoplasmic membrane to transport events at the outer membrane of Gram-negative bacteria. The amino-terminal signal anchor of TonB and its interaction with the cytoplasmic membrane protein ExbB are essential to this process. The TonB signal anchor is predicted to form an α-helix, with a conserved face comprised of residues Ser16, His20, Leu27, and Ser31. Deletion of either Ser16 or His20 or of individual intervening but not flanking residues rendered TonB inactive and unable to assume a proton motive force-dependent conformation. In vivo formaldehyde cross-linking experiments revealed that the ability of this subset of mutants to form a characteristic heterodimer with ExbB was greatly diminished. Replacement of residues 17–19 by three consecutive alanines produced a wild type TonB allele, indicating that the intervening residues (Val, Cys, and Ile) contributed only to spacing. These data indicated that the spatial relationship of Ser16 to His20 was essential to function and suggested that the motif HXXXS defines the minimal requirement for the coupling of TonB to the cytoplasmic membrane electrochemical gradient. Deletion of Trp11 resulted in a TonB that remained active yet was unable to cross-link with ExbB. Because Trp11 was demonstrably not involved in the actual cross-linking, these results suggest that the TonB/ExbB interaction detected by cross-linking occurred at a step in the energy transduction cycle distinct from the coupling of TonB to the electrochemical gradient. proton motive force carbonylcyanide m-chlorophenylhydrazone monoclonal antibody The outer membrane of Gram-negative bacteria serves as a diffusion barrier, that, by virtue of the lipopolysaccharide in the outer leaflet, presents a polar, negatively charged surface to the external surroundings, hindering the passage of detergents, hydrophobic antibiotics, and other toxic agents soluble in standard phospholipid bilayers. This barrier contains aqueous channels that are formed by general and substrate-specific porin proteins that enable diffusion of small, hydrophilic nutrients (<600 Da in Escherichia coli), across the outer membrane. Simple diffusion is not sufficient for the acquisition of either the nutrient cobalamin or Fe(III)-siderophore complexes. Instead, these nutrients are efficiently harvested at the outer membrane surface by high affinity receptors, which catalyze the active transport of bound ligand into the periplasmic space (1Bradbeer C. Biofactors. 1991; 3: 11-19PubMed Google Scholar, 2Postle K. J. Bioenerget. Biomembr. 1993; 25: 591-601PubMed Google Scholar, 3Braun V. FEMS Microbiol. Rev. 1995; 16: 295-307Crossref PubMed Scopus (283) Google Scholar). The release of cobalamin and Fe(III)-siderophores into the periplasmic space is energy-dependent, requiring an intact proton gradient at the cytoplasmic membrane (4Hancock R.E.W. Braun V. J. Bacteriol. 1976; 125: 409-415Crossref PubMed Google Scholar, 5Reynolds P.R. Mottur G.P. Bradbeer C. J. Biol. Chem. 1980; 255: 4313-4319Abstract Full Text PDF PubMed Google Scholar, 6Bradbeer C. J. Bacteriol. 1993; 175: 3146-3150Crossref PubMed Scopus (147) Google Scholar). The separation of transport events from their energy source suggests the need for an energy transducer; this need is met by the protein TonB (5Reynolds P.R. Mottur G.P. Bradbeer C. J. Biol. Chem. 1980; 255: 4313-4319Abstract Full Text PDF PubMed Google Scholar, 7Frost G.E. Rosenberg H. J. Bacteriol. 1975; 124: 704-712Crossref PubMed Google Scholar, 8Bassford P.J.J. Bradbeer C. Kadner R.J. Schnaitman C.A. J. Bacteriol. 1976; 128: 242-247Crossref PubMed Google Scholar, 9Hantke K. Braun V. FEBS Lett. 1975; 49: 301-305Crossref PubMed Scopus (103) Google Scholar). TonB is anchored to the cytoplasmic membrane presumably by a single transmembrane domain (amino acids 12–32), derived from an uncleaved signal sequence (10Postle K. Skare J.T. J. Biol. Chem. 1988; 263: 11000-11007Abstract Full Text PDF PubMed Google Scholar), with the bulk of the protein occupying the periplasmic space (11Hannavy 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, 12Roof S.K. Allard J.D. Bertrand K.P. Postle K. J. Bacteriol. 1991; 173: 5554-5557Crossref PubMed Google Scholar). TonB spans the periplasm and directly contacts outer membrane active transport proteins (13Skare J.T. Ahmer B.M.M. Seacord C.L. Darveau R.P. Postle K. J. Biol. Chem. 1993; 268: 16302-16308Abstract Full Text PDF PubMed Google Scholar, 14Moeck G.S. Postle K. Coulton J.W. J. Biol. Chem. 1997; 272: 28391-28397Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Roughly one-third to one-half of the total cellular TonB partitions exclusively with the outer membrane upon fractionation, suggesting that TonB functions as an intermembrane energy shuttle (15Letain T.E. Postle K. Mol. Microbiol. 1997; 24: 271-283Crossref PubMed Scopus (132) Google Scholar). The outer membrane components and specific features of TonB that support shuttling to the outer membrane are only defined in part, whereas the requisites for TonB interaction with the cytoplasmic membrane are clear: an intact, wild type signal anchor and the presence of two proteins, ExbB and ExbD (15Letain T.E. Postle K. Mol. Microbiol. 1997; 24: 271-283Crossref PubMed Scopus (132) Google Scholar, 16Larsen R.A. Thomas M.G. Postle K. Mol. Microbiol. 1999; 31: 1809-1824Crossref PubMed Scopus (154) Google Scholar). Both of these integral cytoplasmic membrane proteins physically interact with TonB, as evident by in vivo cross-linking (17Higgs P.I. Myers P.S. Postle K. J. Bacteriol. 1998; 180: 6031-6038Crossref PubMed Google Scholar), which, in the case of ExbB, appears to involve the TonB signal anchor (18Larsen R.A. Thomas M.T. Wood G.E. Postle K. Mol. Microbiol. 1994; 13: 627-640Crossref PubMed Scopus (85) Google Scholar). The TonB residues Ser16 and His20 occur on a conserved face of the predicted α-helical TonB transmembrane domain (19Koebnik R. Mol. Microbiol. 1993; 9: 219Crossref PubMed Scopus (29) Google Scholar). We previously noted that the deletion of a residue (Val17) occurring between the two conserved residues rendered TonB inactive (18Larsen R.A. Thomas M.T. Wood G.E. Postle K. Mol. Microbiol. 1994; 13: 627-640Crossref PubMed Scopus (85) Google Scholar), suggesting either that residue Val17 made a side chain-specific contribution to energy transduction or conversely that the spatial relationship between Ser16 and His20 was essential to function. To resolve this dichotomy, we constructed a series of single residue deletions that spanned the amino-terminal two-thirds of the TonB transmembrane domain. Characterization of the resultant deletion mutants and of several substitution mutants suggested by our initial findings indicated that the positional relationship of Ser16 to His20 was an essential feature for the apparent coupling of TonB to the proton motive force (pmf).1 All bacteria used in this study were derived from the E. coli K-12 strain W3110 (20Hill C.W. Harnish B.W. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7069-7072Crossref PubMed Scopus (174) Google Scholar). The majority of experiments used the ampicillin-resistanttonB deletion strain KP1344 and itsexbB::Tn10 derivative, KP1347 (16Larsen R.A. Thomas M.G. Postle K. Mol. Microbiol. 1999; 31: 1809-1824Crossref PubMed Scopus (154) Google Scholar). Transport assays used an aroB derivative of KP1344, KP1351 (16Larsen R.A. Thomas M.G. Postle K. Mol. Microbiol. 1999; 31: 1809-1824Crossref PubMed Scopus (154) Google Scholar). When necessitated by the use of ampicillin-selected plasmids, experiments were performed with the ampicillin-sensitivetonB deletion strains KP1229 (21Ahmer B.M.M. Thomas M.G. Larsen R.A. Postle K. J. Bacteriol. 1995; 177: 4742-4747Crossref PubMed Google Scholar) and KP1304 (constructed here by P1vir-mediated transduction (22Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 201-205Google Scholar) of anexbB::Tn10 originating in H1388 (23Hantke K. Zimmerman L. FEMS Microbiol. Lett. 1981; 12: 31-35Crossref Scopus (39) Google Scholar) into KP1229). A pACYC184 derivative (pKP325) carrying araC and thetonB gene under control of the arabinose promoter and an isogenic construct encoding TonBΔV17 (pKP362) have been described (24Levengood S.K. Beyer W.F.J. Webster Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar). In this a derivative carrying a was constructed by of a from a (13Skare J.T. Ahmer B.M.M. Seacord C.L. Darveau R.P. Postle K. J. Biol. Chem. 1993; 268: 16302-16308Abstract Full Text PDF PubMed Google Scholar) and to residues of the K. Braun V. J. Bacteriol. PubMed Google into a J. J. Bacteriol. 1995; 177: PubMed Scopus Google Scholar) that been at the and were for and were in to The resultant construct used the to an ExbB with an a and a residue between the and the residue of ExbB. The gene in the encoding was by the its and was performed in and by a from that described by 1994; 16: Google Scholar), a the is into a that a that the is in the In this the used for two the resultant with and to the at these Because the were to that characteristic detected by were to with the by in this are described in by by as and residue in the that consecutive residues occur in TonB at and and and Because deletion of either residue in a in the only a single deletion was made for with the resultant proteins as in the to and type by as K. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar) and K. Braun V. J. Bacteriol. PubMed Google Scholar) residue in the that consecutive residues occur in TonB at and and and Because deletion of either residue in a in the only a single deletion was made for with the resultant proteins as in the to in a and and and were made as described (22Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1972: 201-205Google Scholar). were with at in minimal with as and as indicated transport acids were by a defined and and were used at a of and for In initial cross-linking and strains were in to which resulted in TonB that of the for encoding deletions at were but the control not In experiments were with and with to to or that of were as previously described R.A. Myers P.S. Skare J.T. C.L. Darveau R.P. Postle K. J. Bacteriol. PubMed Google Scholar). The and were the of components were from was from formaldehyde was from and as were from was from were from were by was from All other were from either or and were from Molecular All other were from were in to of with a harvested in in of and as and of were to the surface in and for at were as the of the at which of the was were in and arabinose as to an A of and as previously described (18Larsen R.A. Thomas M.T. Wood G.E. Postle K. Mol. Microbiol. 1994; 13: 627-640Crossref PubMed Scopus (85) Google Scholar). The of the was as previously described (16Larsen R.A. Thomas M.G. Postle K. Mol. Microbiol. 1999; 31: 1809-1824Crossref PubMed Scopus (154) Google Scholar). an of was to to a of with with or for at for with to the and with and as described (16Larsen R.A. Thomas M.G. Postle K. Mol. Microbiol. 1999; 31: 1809-1824Crossref PubMed Scopus (154) Google Scholar). were in and arabinose as to an A of harvested in and in of by the of of formaldehyde were for at in of for at and at as were to on and the proteins were to were performed and as previously described R.A. Wood G.E. Postle K. Mol. Microbiol. 1993; PubMed Scopus (85) Google Scholar). transport assays were from a study by and Braun Braun V. Mol. 1990; PubMed Scopus Google Scholar). The strain KP1351 carrying either a control or encoding either wild type TonB (pKP325) or was to of in with defined and were and to in by with for at of were and with to Transport was by the of of by of with at a in at for to with by at of were harvested at the indicated by which were three with of and was by and have to the transport to into E. coli A. PubMed Scopus Google Scholar, J. Bacteriol. 1975; PubMed Google Scholar), and their assays for TonB function. In this the of TonB proteins carrying single residue deletions in the transmembrane domain was by the was made to the proteins at and were in the deletion strain KP1344, with TonB by the presence of in the All assays were performed in Because this is a of is this of resulted in a of for wild type TonB (pKP325) that was from that with the TonB of W3110 case was evident at the data not In the of with wild type TonB was not In a study (16Larsen R.A. Thomas M.G. Postle K. Mol. Microbiol. 1999; 31: 1809-1824Crossref PubMed Scopus (154) Google Scholar), with of resulted in a of for wild type TonB, presumably a gene we not that the of TonB in this that by the by in were the of this with as described This the of single residue were Deletion of single residues from and of TonB on to single residue deletions in the of TonB with the of with was evident in two of three We have previously TonBΔV17 to inactive in this with of evident upon (16Larsen R.A. Thomas M.G. Postle K. Mol. Microbiol. 1999; 31: 1809-1824Crossref PubMed Scopus (154) Google Scholar). is that in the TonBΔV17 was to We previously that the of TonB with the presence or of (16Larsen R.A. Thomas M.G. Postle K. Mol. Microbiol. 1999; 31: 1809-1824Crossref PubMed Scopus (154) Google Scholar). of in the presence and of a or revealed that upon of the cytoplasmic membrane proton wild type TonB a distinct the amino-terminal residues an to by K. This ability to to is upon the presence of and is upon of either TonB residues Ser16 or His20 or the deletion of residue Val17 (16Larsen R.A. Thomas M.G. Postle K. Mol. Microbiol. 1999; 31: 1809-1824Crossref PubMed Scopus (154) Google Scholar). assays of were performed on the deletion series The results of the assays with proteins single residue deletions rendered to whereas proteins with deletions on either of this and In TonB derivative was to in indicating that not type TonB vivo with formaldehyde to form a of characteristic that a that with an apparent of a of that at an that at (13Skare J.T. Ahmer B.M.M. Seacord C.L. Darveau R.P. Postle K. J. Biol. Chem. 1993; 268: 16302-16308Abstract Full Text PDF PubMed Google Scholar), and a heterodimer with an apparent of (13Skare J.T. Ahmer B.M.M. Seacord C.L. Darveau R.P. Postle K. J. Biol. Chem. 1993; 268: 16302-16308Abstract Full Text PDF PubMed Google Scholar, P.I. Myers P.S. Postle K. J. Bacteriol. 1998; 180: 6031-6038Crossref PubMed Google Scholar). with TonBΔV17 indicated that the not form a with ExbB upon formaldehyde yet the ability to form the other characteristic (18Larsen R.A. Thomas M.T. Wood G.E. Postle K. Mol. Microbiol. 1994; 13: 627-640Crossref PubMed Scopus (85) Google Scholar). These experiments were here to the of single residue deletions in the cross-linking were in the signal of the several the of the signal of the for the and the of a by the in the for the Because the components of these are not as yet the of these were were the of the on the ability of TonB to form the heterodimer with ExbB. These results the results of the and with the ability of the mutants to form the characteristic with ExbB greatly or This was evident with the with the A. Larsen and K. suggest the to of the with ExbB to its apparent Because is the to of the with ExbB suggested the that the actual cross-linking occurred this To this two were the of TonB was by a residue with of ExbB was with a suggested that this was the residue with which TonB Trp11 The TonB of strains carrying or of these as by was from wild type not were and by in vivo formaldehyde cross-linking of TonB Trp11 with a residue not in the of cross-linking of TonB to ExbB. not the ability to cross-link to the of formed to with wild type TonB and vivo formaldehyde cross-linking of the strain KP1304 wild type TonB or with wild type ExbB, or the of the ExbB were and as with The of TonB and the TonB/ExbB heterodimer are indicated on the The deletion of either residue or or of the individual acids that between these resulted in the to support to assume a and greatly ability to form with ExbB in vivo these results to the that residues flanking residues made to energy not of the residues between and were of specific or as to the of between Ser16 and To this we these intervening residues and with three of the resultant to from wild type TonB on the of ability to assume a and in vivo formaldehyde cross-linking not To transport assays were The ability of to transport not from that of wild type TonB Transport was not evident for carrying a control or was in encoding wild type TonB by the of with the not of TonB and the in the indicated that the of were in either wild type TonB or not TonB functions as an energy the of the cytoplasmic membrane to active transport at the outer membrane of Gram-negative bacteria K. J. Bioenerget. Biomembr. 1993; 25: 591-601PubMed Google Scholar, C. Bradbeer C. Scholar, R.J. Mol. Microbiol. 1990; PubMed Scopus Google Scholar, G.S. Coulton J.W. Mol. Microbiol. 1998; PubMed Scopus Google Scholar). by (4Hancock R.E.W. Braun V. J. Bacteriol. 1976; 125: 409-415Crossref PubMed Google Scholar, 5Reynolds P.R. Mottur G.P. Bradbeer C. J. Biol. Chem. 1980; 255: 4313-4319Abstract Full Text PDF PubMed Google Scholar, 6Bradbeer C. J. Bacteriol. 1993; 175: 3146-3150Crossref PubMed Scopus (147) Google Scholar) in strains by C. J. Bacteriol. 1993; 175: 3146-3150Crossref PubMed Scopus (147) Google Scholar). these results suggest is but is the or the energy is of the energy suggest a of energy TonB a of that and release potential with to a energy to the energy source and to a energy to energy release to a outer membrane (16Larsen R.A. Thomas M.G. Postle K. Mol. Microbiol. 1999; 31: 1809-1824Crossref PubMed Scopus (154) Google Scholar). The by which TonB is to the of the cytoplasmic membrane is but is that the transmembrane signal anchor of TonB and its ability to interact with ExbB are and TonB to the energy source (10Postle K. Skare J.T. J. Biol. Chem. 1988; 263: 11000-11007Abstract Full Text PDF PubMed Google Scholar), the TonB signal anchor is essential for K. Higgins C.F. Mol. Microbiol. 1993; PubMed Scopus Google Scholar, T.E. S.K. Skare J.T. Postle K. J. Bacteriol. 1994; PubMed Google Scholar) and of the formaldehyde heterodimer T.E. S.K. Skare J.T. Postle K. J. Bacteriol. 1994; PubMed Google Scholar). we noted that TonB assume a conformation. This a signal anchor and with mutants as and unable to the an ExbB was (16Larsen R.A. Thomas M.G. Postle K. Mol. Microbiol. 1999; 31: 1809-1824Crossref PubMed Scopus (154) Google Scholar). for the of the TonB signal anchor from the ability of and to in for ExbB and ExbD V. J. Bacteriol. PubMed Google Scholar, J.T. Postle K. Mol. Microbiol. 1991; PubMed Scopus Google Scholar, V. C. Mol. Microbiol. 1993; PubMed Scopus Google Scholar). These protein are in sequence K. Braun V. J. Bacteriol. PubMed Google Scholar, Webster J. Bacteriol. PubMed Google Scholar) and K. Braun V. J. Bacteriol. PubMed Google Scholar, K. Braun V. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google Scholar, A. Webster R. J. Bacteriol. 1993; 175: PubMed Google Scholar, A. Beyer R. Webster J. Bacteriol. 1994; PubMed Google Scholar), with the evident in their transmembrane TonB and have a S.K. Allard J.D. Bertrand K.P. Postle K. J. Bacteriol. 1991; 173: 5554-5557Crossref PubMed Google Scholar, S.K. Beyer W.F.J. Webster Proc. Natl. Acad. Sci. U. S. A. 1991; PubMed Scopus Google Scholar), the not to the which are K. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S.K. Webster J. Bacteriol. PubMed Google Scholar), for their signal which a conserved of residues face of the transmembrane (19Koebnik R. Mol. Microbiol. 1993; 9: 219Crossref PubMed Scopus (29) Google Scholar). The conserved face to the signal of TonB and of the E. coli TonB residues Ser16, His20, Leu27, and in these of not to TonB S. Braun V. Mol. Microbiol. 1993; PubMed Scopus Google Scholar). In our either or have not been in high and for TonB (16Larsen R.A. Thomas M.G. Postle K. Mol. Microbiol. 1999; 31: 1809-1824Crossref PubMed Scopus (154) Google Scholar, R.A. Thomas M.T. Wood G.E. Postle K. Mol. Microbiol. 1994; 13: 627-640Crossref PubMed Scopus (85) Google Scholar, R.A. Postle K. J. Bacteriol. 1997; PubMed Google Scholar). of the residues in not function A. R. J. Bacteriol. 1998; 180: PubMed Google Scholar). residues and not to essential for energy either Ser16 or His20 have a on TonB When at wild type (16Larsen R.A. Thomas M.G. Postle K. Mol. Microbiol. 1999; 31: 1809-1824Crossref PubMed Scopus (154) Google Scholar), and whereas for under of was greatly S. Braun V. Mol. Microbiol. 1993; PubMed Scopus Google Scholar). at the and residues of inactive A. R. J. Bacteriol. 1998; 180: PubMed Google Scholar). We previously a of function resulted from the deletion of a single residue (Val17) in the Ser16 and His20 (18Larsen R.A. Thomas M.T. Wood G.E. Postle K. Mol. Microbiol. 1994; 13: 627-640Crossref PubMed Scopus (85) Google Scholar). The of this was to that of the Ser16 and to the that ExbB for and were (16Larsen R.A. Thomas M.G. Postle K. Mol. Microbiol. 1999; 31: 1809-1824Crossref PubMed Scopus (154) Google Scholar, R.A. Thomas M.T. Wood G.E. Postle K. Mol. Microbiol. 1994; 13: 627-640Crossref PubMed Scopus (85) Google Scholar). When these and the results to or not transmembrane domain residues of were of to the coupling of TonB to the cytoplasmic membrane proton gradient. was a of single residue the of which are in deletion of either Ser16 or His20 resulted in a by to the results with these residues (16Larsen R.A. Thomas M.G. Postle K. Mol. Microbiol. 1999; 31: 1809-1824Crossref PubMed Scopus (154) Google Scholar). Deletion of single intervening residue produced a with the results (18Larsen R.A. Thomas M.T. Wood G.E. Postle K. Mol. Microbiol. 1994; 13: 627-640Crossref PubMed Scopus (85) Google Scholar). Deletion of single residues to the of His20 a wild type TonB, indicating that acids have in energy transduction and suggesting that the of Ser16 and His20 to the other conserved residues and was not essential to function. Deletion of single residues to the amino-terminal of Ser16 wild type TonB for cross-link with ExbB by and indicating that acids have in energy transduction and suggesting that the of Ser16 and His20 to the TonB cytoplasmic domain and the several residues of the signal anchor was not essential to function. These results suggested two either the of Ser16 and His20 to other was sufficient for coupling of TonB to the energy gradient or the side of or intervening residues contributed to the This was by the substitution of residues 17–19 by three consecutive The resultant was wild type by indicating that the intervening residues not side chain-specific essential for the of the that the positional relationship of Ser16 to His20 is to these results suggest that the motif residues and a face of the the minimal requirement for the coupling of TonB to the cytoplasmic membrane proton electrochemical gradient. noted that the initial that Ser16 and His20 were of from their in and the that these residues the face of an (19Koebnik R. Mol. Microbiol. 1993; 9: 219Crossref PubMed Scopus (29) Google Scholar). In the of a the data the to for a The of to form with ExbB, wild is initial that the for this to the was the of cross-link was not we are with the that this of cross-linking of an TonB the actual cross-linking on TonB is to a on ExbB. This suggests that the TonB/ExbB interaction detected by cross-linking is not essential to energy is that cross-linking an interaction that defines the by which TonB is to a form to the or of cross-linking to ExbB by TonB mutants unable to support energy transduction the that are in a of the cycle that to the by cross-linking to ExbB. the contribution of the TonB signal anchor to the of the shuttling cycle are The study the of the TonB signal anchor in the step of the cycle and defined appears to the minimal requirement of TonB for coupling to the cytoplasmic membrane proton gradient. is evident that this is by ExbB and the by which this ExbB and ExbD at (17Higgs P.I. Myers P.S. Postle K. J. Bacteriol. 1998; 180: 6031-6038Crossref PubMed Google Scholar). Because ExbB and ExbD interact in V. S. C. K. H. J. Bacteriol. PubMed Google Scholar), occur as a with the that a proton are for TonB to to the presence or of (16Larsen R.A. Thomas M.G. Postle K. Mol. Microbiol. 1999; 31: 1809-1824Crossref PubMed Scopus (154) Google Scholar). ExbB and ExbD essential for the by which TonB A step involve or not ExbB directly with the TonB motif which features of ExbB in an We Coulton for the and for of We and for
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