Monoclonal antibodies against the FhuA (TonA) protein of the Escherichia coli outer membrane

Dimoudis, Nikolaos

doi:10.1016/0378-1097(84)90054-5

Cited by 3 publications

(4 citation statements)

References 8 publications

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“…This finding was especially evident in afur background (15) with constitutive synthesis of iron-regulated proteins (not shown). By contrast to published antibody preparations (6,11), homogeneous FhuA protein (Fig. 3, lane 5) ies.…”

Section: Methodscontrasting

confidence: 56%

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

Hoffmann¹,

Fischer²,

Kraut³

et al. 1986

J Bacteriol

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A rapid and simple method for purification of the FhuA receptor protein from cell envelopes of a FhuA-overproducing strain of Escherichia coli K-12 was developed. The overproduction of FhuA was programmed by the thermoamplifiable plasmid pHK232, which carried thefljuACD genes of pLC19-19 of the Clarke and Carbon collection. At low temperature (27°C), pHK232 specified the overproduction of FhuA to levels comparable to those of major outer membrane proteins OmpF, OmpC, and OmpA. The amount of these proteins in the outer membrane was reduced along with overproduction of FhuA. Upon runaway replication of pHK232 at 37C, the precursor of the FhuA protein, proFhuA, was also accumulated in the cell envelope in amounts similar to FhuA. For extraction of the FhuA protein, crude cell envelopes were washed with 2% Triton X-100-6 M urea to remove less tightly bound proteins. Then FhuA but not proFhuA was solubilized by treating Triton X-100-urea-washed membranes with 1% octylglucoside-1 mM EDTA. This procedure yielded FhuA protein free from other membrane proteins. The amount of lipopolysaccharide and phospholipids was low and ranged from 5 to 15% and 10 to 25% of the weight of the FhuA protein, respectively. As shown by direct inactivation and by competition assays, the isolated FhuA protein retained receptor activity for ferrichrome, albomycin, colicin M, and phages T5 and Ti.The protein specified by the JhuA gene is a minor protein (approximately 103 copies per cell) of the outer membrane of Escherichia coli, with an apparent molecular weight of 78,000 (78K). It is a component of the transport system for the siderophores ferrichrome and albomycin, and it serves as a receptor for colicin M and for the phages T5, Ti and 4)80 (3,18). With the exception of the receptor function for phage T5, receptor activity of FhuA is linked to and may be modified by interaction with other proteins. The uptake of ferrichrome and albomycin depends on functions encoded by additional genes of the fhu operon (21), JfhuC, JhuD, and JhuB (13), and functions specified by the genes tonB (9) and exbB (19). Infection by phages Ti and 480 requiresfiiuA and tonB (14), while sensitivity to colicin M requiresfjzuA, tonB, and exbB (27). The molecular mechanisms underlying the different receptor functions of FhuA are unknown.

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Section: Methodscontrasting

confidence: 56%

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

Hoffmann¹,

Fischer²,

Kraut³

et al. 1986

J Bacteriol

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“…(24). Like Fhu3.1 and Fhu3.3, an anti-FhuA MAb which was described previously (MAb AJ216 [13]) inhibited the irreversible adsorption of T5 phage to FhuA and prevented cell killing by colicin M. This MAb was unable to inhibit ferrichrome-iron uptake. However, in that study, the MAb determinant on the primary sequence of FhuA was not identified.…”

Section: Discussionmentioning

confidence: 96%

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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“…Obviously, the binding sites for the various FhuA ligands overlap strongly, but mutations affecting only one or two ligands point to single residues important for binding of a single ligand (7,16,29,36 able information on the surface and periplasmic locations of regions, including data presented in this paper (pSKFT162-16, pSKF321-04, pSKF415-12, pSKF456-12, and pSKF531-12; Fig. 3), was incorporated into the FoxA model.…”

Section: Discussionmentioning

confidence: 99%

“…Ferrichrome and colicin M prevented T5 adsorption in deenergized cells and in tonB mutants (7,29). A monoclonal antibody inhibited adsorption of phages T5 and Ti and prevented killing of cells by colicin M, while phage +80 infection and ferrichrome uptake remained unaffected (16). Deletion of a single aspartate residue at position 348 abolished sensitivity to phages Ti and +80, as well as to albomycin, but these deletion mutants retained 1% of T5 sensitivity and 0.1% of colicin M sensitivity (36).…”

mentioning

confidence: 98%

Insertion derivatives containing segments of up to 16 amino acids identify surface- and periplasm-exposed regions of the FhuA outer membrane receptor of Escherichia coli K-12

1993

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The FhuA receptor in the outer membrane of Escherichwa coli K-12 is involved in the uptake of ferrichrome, colicin M, and the antibiotic albomycin and in infection by phages Ti, T5, and +80. Fragments of up to 16 amino acid residues were inserted into FhuA and used to determine FhuA active sites and FhuA topology in the outer membrane. For this purpose antibiotic resistance boxes flanked by symmetric polylinkers were inserted intohuiA and subsequently partially deleted. Additional in-frame insertions were generated by mutagenesis with transposon Tnl 725. The 68 FhuA protein derivatives examined contained segments of 4, 8, 12, 16, and 22 additional amino acid residues at 34 different locations from residues 5 to 646 of the mature protein. Most of the FhuA derivatives were found in normal amounts in the outer membrane fraction. Half of these were fully active toward all ligands, demonstrating proper insertion into the outer membrane. Seven of the 12-and 16-amino-acid-insertion derivatives (at residues 378, 402, 405, 415, 417, 456, and 646) were active toward all of the ligands and could be cleaved by subtilisin in whole cells, suggesting a surface location of the extra loops at sites which did not affect FhuA function. Two mutants were sensitive to subtilisin (insertions at residues 511 and 321) but displayed a strongly reduced sensitivity to colicin M and to phages +80 and Ti. Four of the insertion derivatives (at residues 162, 223, 369, and 531) were cleaved only in spheroplasts and probably form loops at the periplasmic side of the outer membrane. The number and size of the proteolytic fragments indicate cleavage at or close to the sites of insertion, which has been proved for five insertions by amino acid sequencing.Most mutants with functional defects were affected in their sensitivity to all ligands, yet frequently to different degrees. Some mutants showed a specifically altered sensitivity to a few ligands; for example, mutant 511-04 was partially resistant only to colicin M, mutant 241-04 was reduced in ferrichrome and albomycin uptake and showed a reduced colicin M sensitivity, and mutant 321-04 was fully resistant to phage Ti and partially resistant to phage +80. The altered residues define preferential binding sites for these ligands. Insertions of 4 to 16 residues at positions 69, 70, 402, 530, 564, and 572 resulted in strongly reduced amounts of FhuA in the outer membrane fraction, varying in function from fully active to inactive. These results provide the basis for a model of FhuA organization in the outer membrane.Uptake of substrates across the outer membrane of Escherichia coli occurs via diffusion, facilitated diffusion, and receptor-dependent transport. Hydrophilic substrates with molecular masses below 700 Da diffuse through the channels formed by the porins OmpF and OmpC. Examples for facilitated diffusion involving stereospecific recognition between substrate and porin are the maltodextrins which enter through the LamB pore (40), sucrose via the ScrY protein (52), nucleosides through the Ts...

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Monoclonal antibodies against the FhuA (TonA) protein of the Escherichia coli outer membrane

Cited by 3 publications

References 8 publications

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

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

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

Insertion derivatives containing segments of up to 16 amino acids identify surface- and periplasm-exposed regions of the FhuA outer membrane receptor of Escherichia coli K-12

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