Characterization of the receptor protein for phage T5 and colicin M in the outer membrane of <i>E. coli</i> B

Braun, Volkmar; Wolff, Helga

doi:10.1016/0014-5793(73)80707-0

Cited by 69 publications

(21 citation statements)

References 16 publications

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“…Phage T5 binding is not affected by TonB-dependent conformational changes, which have to be assumed for binding of phages T1 and 80 since they bind irreversibly, accompanied by release of DNA from the phage head, only to energized TonB ϩ cells (17). Phage T5 binds to isolated FhuA and releases DNA (9,37,48), in contrast to phages T1 and 80, which do not bind to isolated FhuA. It is suggested that in corkless FhuA, the conformation of loop 4 is altered such that phage T5 cannot bind and/or that release of DNA from the phage head is not triggered.…”

Section: Discussionmentioning

confidence: 99%

In Vivo Reconstitution of the FhuA Transport Protein ofEscherichia coliK-12

et al. 2003

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The FhuA protein in the outer membrane of Escherichia coli actively transports ferrichrome and the antibiotics albomycin and rifamycin CGP 4832 and serves as a receptor for the phages T1, T5, and 80 and for colicin M and microcin J25. The crystal structure reveals a ␤-barrel with a globular domain, the cork, which closes the channel formed by the barrel. Genetic deletion of the cork resulted in a ␤-barrel that displays no FhuA activity. A functional FhuA was obtained by cosynthesis of separately encoded cork and the ␤-barrel domain, each endowed with a signal sequence, which showed that complementation occurs after secretion of the fragments across the cytoplasmic membrane. Inactive complete mutant FhuA and an FhuA fragment containing 357 N-proximal amino acid residues complemented the separately synthesized wild-type ␤-barrel to form an active FhuA. Previous claims that the ␤-barrel is functional as transporter and receptor resulted from complementation by inactive complete FhuA and the 357-residue fragment. No complementation was observed between the wild-type cork and complete but inactive FhuA carrying cork mutations that excluded the exchange of cork domains. The data indicate that active FhuA is reconstituted extracytoplasmically by insertion of separately synthesized cork or cork from complete FhuA into the ␤-barrel, and they suggest that in wild-type FhuA the ␤-barrel is formed prior to the insertion of the cork.The FhuA protein of Escherichia coli K-12 transports ferrichrome, the structurally related antibiotic albomycin, and the unrelated antibiotic rifamycin CGP 4832 across the outer membrane and serves as a receptor for colicin M, microcin J25, and the phages T1, T5, and 80 (7). Although extensive mutational analyses have been performed (6, 7, 33) and the crystal structure has been determined (15, 30), the molecular mechanism of FhuA transport is unclear.The protein consists of a ␤-barrel (residues 161 to 714) composed of 22 antiparallel ␤-strands that form a channel that is closed by a globular domain (residues 1 to 160), designated the cork or plug, that inserts from the periplasmic side into the ␤-barrel. Binding of ferrichrome elicits small, 1-to 2-Å movements of the cork domain relative to the ␤-barrel and a large 17-Å transition of E19 without opening the channel. Release of ferrichrome from the binding site formed by 10 amino acid residues located in the ␤-barrel and the cork, and opening of the channel, is thought to be triggered by interaction with the TonB protein, which requires the proton motive force of the cytoplasmic membrane to exert its action on FhuA. All FhuArelated activities, except infection by phage T5, depend on TonB.We previously reported that deletion of the cork (residues 5 to 160) results in a protein (FhuA⌬5-160) that still functions as an active TonB-dependent transporter and receptor (3,4,25). These experiments were performed with two fhuA mutant strains; mutant 41/2 contains a number of amino acid replacements and D348 is deleted (26), whereas PCR analysis indicated th...

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Section: Discussionmentioning

confidence: 99%

In Vivo Reconstitution of the FhuA Transport Protein ofEscherichia coliK-12

et al. 2003

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“…The first example of this multifunctionality was BtuB, the vitamin B12 receptor protein, that also recognizes E-group colicins (22) and the T5-like bacteriophage BF23 (8). TonA (later renamed FhuA) was another prototype of cell surface competition for reception of ferrichrome, colicin M, and the bacteriophages T1, T5, and 80 (13,82,93,101). Similarly, FepA (14), the OM receptor for FeEnt, also recognizes colicins B and D (100), but bacteriophage were not known to utilize FepA for penetration of the cell surface.…”

Section: Discussionmentioning

confidence: 99%

“…Ligandgated porins (LGP), which often function in the uptake of metals, show broad multifunctionality by also acting as receptors for bacteriophage, toxins, and antibiotics. One such LGP, FhuA, recognizes the hydroxamate siderophore ferrichrome; phages T1, T5, 80, and UC-1; colicin M; and the antibiotics albomycin and microcin 25 (11,13,43,51,82,93,101). Subsequent to binding, transport through the OM often requires another cell envelope protein, TonB (34,97,101), but different ligand molecules have different requirements for TonB.…”

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confidence: 99%

FepA- and TonB-Dependent Bacteriophage H8: Receptor Binding and Genomic Sequence

Rabsch

Wiley

et al. 2007

J Bacteriol

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H8 is derived from a collection of Salmonella enterica serotype Enteritidis bacteriophage. Its morphology and genomic structure closely resemble those of bacteriophage T5 in the family Siphoviridae. H8 infected S. enterica serotypes Enteritidis and Typhimurium and Escherichia coli by initial adsorption to the outer membrane protein FepA. Ferric enterobactin inhibited H8 binding to E. coli FepA (50% inhibition concentration, 98 nM), and other ferric catecholate receptors (Fiu, Cir, and IroN) did not participate in phage adsorption. H8 infection was TonB dependent, but exbB mutations in Salmonella or E. coli did not prevent infection; only exbB tolQ or exbB tolR double mutants were resistant to H8. Experiments with deletion and substitution mutants showed that the receptor-phage interaction first involves residues distributed over the protein's outer surface and then narrows to the same charged (R316) or aromatic (Y260) residues that participate in the binding and transport of ferric enterobactin and colicins B and D. These data rationalize the multifunctionality of FepA: toxic ligands like bacteriocins and phage penetrate the outer membrane by parasitizing residues in FepA that are adapted to the transport of the natural ligand, ferric enterobactin. DNA sequence determinations revealed the complete H8 genome of 104.4 kb. A total of 120 of its 143 predicted open reading frames (ORFS) were homologous to ORFS in T5, at a level of 84% identity and 89% similarity. As in T5, the H8 structural genes clustered on the chromosome according to their function in the phage life cycle. The T5 genome contains a large section of DNA that can be deleted and that is absent in H8: compared to T5, H8 contains a 9,000-bp deletion in the early region of its chromosome, and nine potentially unique gene products. Sequence analyses of the tail proteins of phages in the same family showed that relative to pb5 (Oad) of T5 and Hrs of BF23, the FepA-binding protein (Rbp) of H8 contains unique acidic and aromatic residues. These side chains may promote binding to basic and aromatic residues in FepA that normally function in the adsorption of ferric enterobactin. Furthermore, a predicted H8 tail protein showed extensive identity and similarity to pb2 of T5, suggesting that it also functions in pore formation through the cell envelope. The variable region of this protein contains a potential TonB box, intimating that it participates in the TonB-dependent stage of the phage infection process.Bacteriophage adsorb to components of the gram-negative bacterial outer membrane (OM) during the initial stages of their infectious processes (17,20,36,37,64,87,88,98). For example, phages Mu (84) and X174 (41) initially bind to lipopolysaccharide, whereas (95), T6 (86), and TLS (30) adsorb to the OM proteins LamB, Tsx, and TolC, respectively. T2 (53) and T4 (92, 102) utilize both lipopolysaccharide and surface proteins in their adsorption reactions. The surface receptor proteins are porins that nonspecifically (70, 71) or specifically (55, 56, 69) transport...

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“…Once in the periplasm, binding proteins adsorb ferric siderophores and deliver them to inner membrane (IM) permeases that actively transport either the metal complex or free iron into the cytoplasm. At the binding stage of the uptake process, ferric siderophores compete with noxious agents like bacteriophages (H8 [85a] and T5 and 80 [18,103]) and colicins (B, D [37], and M [18]) for entry into the cell. OM transport of metal complexes and susceptibility to phages and colicins require metabolic energy (13,25,85) and usually involve participation of an additional cell envelope protein, TonB.…”

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Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane

et al. 2008

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We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB ؉ bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepA⌬3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.Iron is one target of gram-negative bacterial cell envelope transport systems, and microbes elaborate high-affinity siderophores that complex extracellular iron (70). However, ferric siderophores, like ferric enterobactin (FeEnt), are too large (716 Da) to pass through general porins in the outer membrane (OM), necessitating a different type of transporter to acquire them. On the basis of their 22-stranded transmembrane ␤-barrels, OM metal transporters like FepA belong to the porin superfamily (89). Ligand binding to such receptors initiates the transport reaction through their transmembrane channels, which led to their designation as ligand-gated porins (LGP) (88), by analogy to the family of eukaryotic ligand-gated ion channels. It is noteworthy that LGP are mechanistically distinct from general, diffusive porins because they bind metal complexes with high affinity and actively transport them against a concentration gradient into the cell. Once in the periplasm, binding proteins adsorb ferric siderophores and del...

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Characterization of the receptor protein for phage T5 and colicin M in the outer membrane of E. coli B

Cited by 69 publications

References 16 publications

In Vivo Reconstitution of the FhuA Transport Protein ofEscherichia coliK-12

In Vivo Reconstitution of the FhuA Transport Protein ofEscherichia coliK-12

FepA- and TonB-Dependent Bacteriophage H8: Receptor Binding and Genomic Sequence

Insight from TonB Hybrid Proteins into the Mechanism of Iron Transport through the Outer Membrane

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