Genetic insertion and exposure of a reporter epitope in the ferrichrome-iron receptor of Escherichia coli K-12

Moeck, Gregory S.; Bazzaz, Bibi Sedigheh Fazly; Gras, M. F.; Ravi, T. S.; Ratcliffe, Michael J. H.; Coulton, James W.

doi:10.1128/jb.176.14.4250-4259.1994

Cited by 43 publications

(48 citation statements)

References 37 publications

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“…Such duplication mutants display 64 to 100% of the wild-type level of vitamin B 12 transport activity (31). In FhuA, insertions of 4 to 16 heterologous residues in loops 4, 5, 7, or 10 result in derivatives that support growth on ferrichrome as the sole iron source and sensitivity to the FhuA ligands albomycin, colicin M, microcin J25, and the phages T1, T5, 80, and UC1 (30,36). The loops, therefore, display a complex behavior.…”

Section: Discussionmentioning

confidence: 99%

Defined Inactive FecA Derivatives Mutated in Functional Domains of the Outer Membrane Transport and Signaling Protein of Escherichia coli K-12

Sauter

Braun

2004

J Bacteriol

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The FecA outer membrane protein of Escherichia coli functions as a transporter of ferric citrate and as a signal receiver and signal transmitter for transcription initiation of the fec transport genes. Three FecA regions for which functional roles have been predicted from the crystal structures were mutagenized: (i) loops 7 and 8, which move upon binding of ferric citrate and close the entrance to the ferric citrate binding site; (ii) the dinuclear ferric citrate binding site; and (iii) the interface between the globular domain and the ␤-barrel. Deletion of loops 7 and 8 abolished FecA transport and induction activities. Deletion of loops 3 and 11 also inactivated FecA, whereas deletion of loops 9 and 10 largely retained FecA activities. The replacement of arginine residue R365 or R380 and glutamine Q570, which are predicted to serve as binding sites for the negatively charged dinuclear ferric citrate, with alanine resulted in inactive FecA, whereas the binding site mutant R438A retained approximately 50% of the FecA induction and transport activities. Residues R150, E541, and E587, conserved among energy-coupled outer membrane transporters, are predicted to form salt bridges between the globular domain and the ␤-barrel and to contribute to the fixation of the globular domain inside the ␤-barrel. Mutations E541A and E541R affected FecA induction and transport activity slightly, whereas mutations E587A and E587R more strongly reduced FecA activity. The double mutations R150A E541R and R150A E587R nearly abolished FecA activity. Apparently, the salt bridges are less important than the individual functions these residues seem to have for FecA activity. Comparison of the properties of the FecA, FhuA, FepA, and BtuB transporters indicates that although they have very similar crystal structures, the details of their functional mechanisms differ.

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Section: Discussionmentioning

confidence: 99%

Defined Inactive FecA Derivatives Mutated in Functional Domains of the Outer Membrane Transport and Signaling Protein of Escherichia coli K-12

Sauter

Braun

2004

J Bacteriol

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“…SDS-polyacrylamide gel electrophoresis (PAGE), immunoblotting, ELISA, and flow cytometry were performed as described previously (29). All ELISAs were performed in triplicate, with minimal variation in A 405 readings from experiment to experiment.…”

Section: Methodsmentioning

confidence: 99%

“…Hybridoma screening. Hybridoma supernatants were screened by either (i) ELISA against OM vesicles of SG303fhuA and SG303fhuA(pGC01) (29), (ii) flow cytometry against intact cells of strains CS2529fhuA and CS2529fhuA (pGC01) (29), or (iii) dot blotting against denatured Bac.FhuA(His) 6 . For dot blotting, inclusion bodies from B. subtilis IH6140(pKTH288) or IH6140(pGM01) were solubilized in urea and aspirated onto nitrocellulose, using a Milliblot-D apparatus (Millipore, Bedford, Mass.).…”

Section: Methodsmentioning

confidence: 99%

“…To examine directly the surface accessibility of FhuA sequences which were proposed to face the external milieu, we inserted a foreign antigenic determinant (the C3 epitope of poliovirus) at 16 different positions within FhuA, forming FhuA.C3 (29). Anti-C3 antibodies, when added to intact cells or to OM vesicles containing the FhuA.C3 proteins, bound the C3 epitope when it was inserted at positions 321, 405, and 417 of FhuA.…”

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confidence: 99%

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Topological analysis of the Escherichia coli ferrichrome-iron receptor by using monoclonal antibodies

1995

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Ferrichrome-iron transport inThe uptake of ferric siderophores and vitamin B 12 across the outer membrane (OM) of gram-negative bacteria is driven by a poorly understood energy-coupled mechanism which involves the cytoplasmic membrane-associated proteins TonB, ExbB, and ExbD (reviewed in references 21, 26, 33). TonBdependent transport systems are characterized by OM receptors with high affinity and substrate specificity for their cognate siderophore or for vitamin B 12 . There are additional requirements for proteins within the periplasm and in the cytoplasmic membrane to complete the transport process of siderophore or vitamin B 12 . The Escherichia coli OM receptor for ferrichrome-iron is FhuA (M r , 78,992; 714 amino acids [11]), a protein which also acts as the receptor for phages T1, T5, UC-1, and 80, for colicin M, and for the peptide antibiotics albomycin and microcin 25. The periplasmic binding protein FhuD (5, 10) and the cytoplasmic membrane-associated proteins FhuB and FhuC are required for internalization of hydroxamate siderophores (reviewed in reference 3). FhuB and FhuC display characteristics of binding protein-dependent ATP-binding cassette transporters (18).A prerequisite to understanding the mechanism of active transport of ferrichrome-iron is to understand the molecular organization of FhuA within the OM. As with other OM proteins (OMPs), amphiphilic sequences of FhuA are thought to constitute membrane-spanning beta sheets, while intervening sequences (which often display a strongly hydrophilic character) probably form extramembranous loops. Experimental evidence generally supports these structural predictions and has provided some basis on which to model the topological organization of FhuA. Insertions of tetra-to hexadecapeptides at some sites within FhuA induced susceptibility to exogenously added proteases (28). Differential protease susceptibility of the insertion mutant FhuAs in whole cells as opposed to spheroplasts led to a prediction of the transmembrane arrangement of FhuA (28). Cells expressing FhuA⌬Asp-348 demonstrated reduced sensitivity to killing by phage T5 and complete resistance to T1, 80, and colicin M (23). These data supported the proposed cell surface exposure of a loop containing amino acids 316 to 356 of FhuA. Excision of amino acids 322 to 355 from FhuA transformed the ferrichrome-specific active transporter into a protein which displayed characteristics of a TonBindependent channel: cells expressing FhuA⌬322-355 became sensitive to bacitracin and sodium dodecyl sulfate (SDS) and formed stable conductance channels in black lipid membranes (22). It was proposed that the transmembrane strands of FhuA assume the conformation of a large beta barrel, with the predicted loop of amino acids 316 to 356 forming a ''gate'' that somehow regulates ferrichrome transport through the channel (4, 22). Recently, Killmann et al. (24) identified amino acids within the proposed gating loop of FhuA which contribute to the binding of phages T1, T5, and 80 and of colicin M. Preincubation o...

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“…Ampicillin was used at a concentration of 100 g/ml (Ap100). Strain AB2847⌬ara was created by P1 transduction of leu::Tn10 and ⌬ara714 from LMG194 (Invitrogen) into AB2847 (35 (37). The protein was purified as described in the literature (38) with the following changes: for binding experiments the purification was stopped before the detergent exchange from LDAO to DDAO.…”

Section: Construction Of Plasmids Encoding Tonbmentioning

confidence: 99%

Dimerization of TonB Is Not Essential for Its Binding to the Outer Membrane Siderophore Receptor FhuA of Escherichia coli

Koedding

Howard

Kaufmann

et al. 2004

Journal of Biological Chemistry

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FhuA belongs to a family of specific siderophore transport systems located in the outer membrane of Escherichia coli. The energy required for the transport process is provided by the proton motive force of the cytoplasmic membrane and is transmitted to FhuA by the protein TonB. Although the structure of full-length TonB is not known, the structure of the last 77 residues of a fragment composed of the 86 C-terminal amino acids was recently solved and shows an intertwined dimer (Chang, C., Mooser, A., Pluckthun, A., and Wlodawer, A. (2001) J. Biol. Chem. 276, 27535-27540). We analyzed the ability of truncated C-terminal TonB fragments of different lengths (77, 86, 96, 106, 116, and 126 amino acid residues, respectively) to bind to the receptor FhuA. Only the shortest TonB fragment, TonB-77, could not effectively interact with FhuA. We have also observed that the fragments TonB-77 and TonB-86 form homodimers in solution, whereas the longer fragments remain monomeric. TonB fragments that bind to FhuA in vitro also inhibit ferrichrome uptake via FhuA in vivo and protect cells against attack by bacteriophage ⌽80.

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Genetic insertion and exposure of a reporter epitope in the ferrichrome-iron receptor of Escherichia coli K-12

Cited by 43 publications

References 37 publications

Defined Inactive FecA Derivatives Mutated in Functional Domains of the Outer Membrane Transport and Signaling Protein of Escherichia coli K-12

Defined Inactive FecA Derivatives Mutated in Functional Domains of the Outer Membrane Transport and Signaling Protein of Escherichia coli K-12

Topological analysis of the Escherichia coli ferrichrome-iron receptor by using monoclonal antibodies

Dimerization of TonB Is Not Essential for Its Binding to the Outer Membrane Siderophore Receptor FhuA of Escherichia coli

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