Preparation of the FhuA (TonA) receptor protein from cell envelopes of an overproducing strain of Escherichia coli K-12

Hoffmann, H.; Fischer, Eckhard; Kraut, Heinrich; Braun, Volkmar

doi:10.1128/jb.166.2.404-411.1986

Cited by 32 publications

(24 citation statements)

References 45 publications

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“…However, this possibility is unlikely given that Fhu4.1 did not inhibit the killing of cells by colicin M or the growth promotion of cells by ferrichrome. These two processes are thought to occur through a conformational change in FhuA induced by energized TonB (16,19,26,33).…”

Section: Discussionmentioning

confidence: 99%

“…For immunoaffinity purification of FhuA, OM vesicles from AW740(pGC01) were solubilized in 50 mM Tris-HCl (pH 8.0)-6 M urea-2% Triton X-100 for 30 min at room temperature (19). The insoluble fraction was collected by centrifugation, solubilized as described above in TTE, and dialyzed against 10 mM Tris-HCl (pH 8.0)-0.1% Triton X-100 to remove EDTA.…”

Section: Methodsmentioning

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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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

1995

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“…To examine whether overproduced ExbB and ExbD (10,15). The wild-type strain AB2847(pFB103-1) transported ferrichrome at a constant rate, starting from a fourfold higher initial level (Fig.…”

Section: Methodsmentioning

confidence: 99%

Involvement of ExbB and TonB in transport across the outer membrane of Escherichia coli: phenotypic complementation of exb mutants by overexpressed tonB and physical stabilization of TonB by ExbB

1989

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The exb locus in Escherichia coli consists of two genes, termed exbB and exbD. Exb functions are related to TonB function in that most TonB-dependent processes are enhanced by Exb Transport of iron(III) siderophores and vitamin B12 across the outer membrane of Escherichia coli (2, 4, 17, 21) stands in contrast to the passive diffusion of most substrates through porins (18). The activity of the receptor proteins for iron(III) siderophore and vitamin B12 transport depends on the TonB protein of the cytoplasmic membrane, since these substrates stay at the receptor proteins in tonB mutants and in unenergized tonB+ cells (3; H. Schoffler and V. Braun, Mol. Gen. Genet., in press). The group B colicins also remain bound to the outer membrane receptors unless tonB+ cells are energized (3,20). Mutations in a single codon of the tonB gene suppressed the btuB451 point mutation in the structural gene of the vitamin B12 receptor (14), and mutations in theJhuA gene (Schoffler and Braun, in press), which encodes the receptor for ferrichrome, the antibiotic albomycin, colicin M, and the bacteriophages T5, Ti, and +80. Suppression of mutations in receptor genes by tonB mutations suggests a direct interaction between the TonB protein and the receptor proteins. The TonB function seems to be involved only in transport processes across the outer membrane.Exb designates a function of Escherichia coli similar to TonB with the difference that TonB-dependent processes absolutely require TonB whereas Exb-dependent functions require Exb to a different extent. There are TonB-dependent functions which do not require Exb, but all Exb-dependent functions rely on TonB. For example, tonB mutants are insensitive to colicins B and M and to the antibiotic albomycin and are unable to take up ferrichrome. exb mutants have the same properties except that they exhibit reduced sensitivity to colicin B. The exb locus has been mapped at min 65 of the E. coli linkage map (20). Recently, we cloned (9) and sequenced (8) the exb region and identified two genes, termed exbB and exbD, which are required for complement-* Corresponding author.ing exb mutants. The exbB and exbD genes encode proteins with molecular masses of 26.1 and 15.5 kilodaltons (kDa), respectively, which were both preferentially localized in the cytoplasmic membrane (8). Since the ExbB and ExbD proteins affect the same cellular activities as the TonB protein, and the requirement for ExbB and ExbD differs in various TonB-dependent functions, we studied whether overproduction of TonB compensates for the lack of ExbB and ExbD and vice versa. Indeed, the results presented in this paper show that the low sensitivity of exb mutants to colicins B, D, and M could be greatly increased by overeXpression of tonB. In addition, suppression of the btuB451 mutation by a mutation in tonB was enhanced in an exb+ background. The ExbB protein prevented degradation of the TonB protein. Radioactive ferrichrome taken up by exb mutants was released by a surplus of unlabeled ferrichrome, indicating that ferrichrome...

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“…To produce FepA, cells were grown in TY medium [8] with the above antibiotics at room temperature. Cultures, at their late log phase, were rapidly cooled by adding crushed ice and harvested by centrifugation.…”

Section: Introductionmentioning

confidence: 99%

Purification and crystallization of ferric enterobactin receptor protein, FepA, from the outer membranes of Escherichia coli UT5600/pBB2

Jalal

Helm

1989

FEBS Letters

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The ferric enterobactin receptor protein, FepA, was isolated and purified from the outer membranes of a genetically transformed strain of Escherichia coli (UT5600/pBB2) using anion-exchange chromatography, ehromatofoeusing and gel filtration. The purified protein was found to crystallize from 25 mM sodium phosphate buffer in the presence of 0.8~ fl-Doctylglucoside under a range of conditions. The protein formed mostly small rods and needle-shaped crystals in the hanging drop method.Ferric enterobactin protein; Purification; Crystallization; Outer membrane protein; (E. coli)

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Preparation of the FhuA (TonA) receptor protein from cell envelopes of an overproducing strain of Escherichia coli K-12

Cited by 32 publications

References 45 publications

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

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

Involvement of ExbB and TonB in transport across the outer membrane of Escherichia coli: phenotypic complementation of exb mutants by overexpressed tonB and physical stabilization of TonB by ExbB

Purification and crystallization of ferric enterobactin receptor protein, FepA, from the outer membranes of Escherichia coli UT5600/pBB2

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