Ferrichrome transport in Escherichia coli K-12: altered substrate specificity of mutated periplasmic FhuD and interaction of FhuD with the integral membrane protein FhuB

Rohrbach, Martin; Braun, Volkmar; Köster, Wolfgang

doi:10.1128/jb.177.24.7186-7193.1995

Cited by 101 publications

(86 citation statements)

References 55 publications

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“…Interactions between FhuD and FhuB have been demonstrated by cross-linking studies, protease protection assays (9), and enzyme-linked immunosorbent assay (10). Interaction of FhuB with FhuD is apparently independent of siderophore binding by FhuD (9). Taken together, these results suggest that FhuD functions as a carrier protein; ferrichrome released from the OM receptor is delivered by FhuD to the permease.…”

supporting

confidence: 50%

“…FhuD was reported to interact with regions of the CM-embedded permease FhuB. Interactions between FhuD and FhuB have been demonstrated by cross-linking studies, protease protection assays (9), and enzyme-linked immunosorbent assay (10). Interaction of FhuB with FhuD is apparently independent of siderophore binding by FhuD (9).…”

mentioning

confidence: 99%

“…Given the apparent weak affinity (1 M) of ferrichrome for FhuD (9), binding is unlikely to be a diffusion-governed process. Efficiency of siderophore capture would be enhanced by positioning a binding protein proximal to the lumen of the OM receptor.…”

mentioning

confidence: 99%

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Interactions between TonB from Escherichia coli and the Periplasmic Protein FhuD

Carter

Miousse

Gagnon

et al. 2006

Journal of Biological Chemistry

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supporting

confidence: 50%

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confidence: 99%

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Interactions between TonB from Escherichia coli and the Periplasmic Protein FhuD

Carter

Miousse

Gagnon

et al. 2006

Journal of Biological Chemistry

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“…Transport across the cytoplasmic membrane seems only to depend on the ABC transporter FhuCDB since fhuCDB fhuF ϩ mutants are completely unable to use ferrioxamine B as an iron source. The affinity of ferrioxamine to the binding protein FhuD is relatively low compared to the affinity of ferrichrome [31]. The combination of these uptake pathways across the outer membrane and the cytoplasmic membrane does not satisfy the iron needs of the cells since siderophore receptors unrelated to ferrioxamine B uptake in the outer membrane are strongly induced when E. coli cells are grown with ferrioxamine B [32].…”

Section: Fhuf Is Not a Transport Proteinmentioning

confidence: 99%

FhuF, an iron‐regulated protein of Escherichia coli with a new type of [2Fe‐2S] center

Müller

Matzanke

Schünemann

et al. 1998

European Journal of Biochemistry

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We previously used fhuF as a sensitive reporter gene of the iron status of Escherichia coli. In this report, the fhuF gene was identified as open reading frame f262b at 99.2 min on the genome sequence map of E. coli K-12. The FhuF protein was labeled with a His-tag and then purified to electrophoretic homogeneity. Based on sulfur determinations and Mössbauer and EPR spectroscopy, FhuF was identified as a Keywords : iron-sulfur protein; ferrioxamine ; iron reductase; Mössbauer spectroscopy ; EPR.Under low-iron growth conditions, many bacteria secrete specific chelators called siderophores. Production of siderophores and transport systems for Fe 3ϩ -loaded siderophores is regulated in gram-negative bacteria by the repressor protein Fur with Fe 2ϩ as co-repressor [1]. In Escherichia coli, a fhuFϪlacZ operon fusion has been used as a reporter to study iron regulation [2] since this fusion reacts very sensitively to slight changes in the iron concentration of the medium. The only phenotype observed for fhuF mutations is a diminished ability of the cells to use ferrioxamine B as an iron source. This siderophore is a poor iron source for E. coli K-12 [3] because this strain lacks a specific receptor for ferrioxamine B in the outer membrane; this receptor is found in Yersinia [4] and other enterobacteria [5]. In an attempt to understand the function of FhuF, the protein was purified and characterized. Our results indicated that FhuF is an unusual [2Fe-2S] protein. EXPERIMENTAL PROCEDURESStrains and growth conditions. The E. coli strains, phages, and plasmids used in this study are described in Table 1 Mud1(Aplac) and λplacMu mutants were generated as described elsewhere [6,7]. P1 transductions were performed as described by Miller [8]. Bacteria were grown in TY medium containing (per liter) tryptone (8 g), yeast extract (5 g), and sodium chloride (5 g) or in M9 medium [8] or in minimal medium E [3]. Growth response to ferrioxamine B was tested on nutrient broth dipyridyl plates (8 g nutrient broth, 5 g NaCl, 15 g Difco agar/l and 0.2 mM 2,2′-dipyridyl), which were seeded with the appropriate strain in 2.5 ml water soft agar. Filter paper discs impregnated with 15 µl 2 mM ferrioxamine B were placed on the agar, and growth zones were measured after 18 h.Recombinant DNA techniques and cloning of fhuF. Standard procedures [9] or those recommended by the commercial supplier were followed for isolation of chromosomal and plasmid DNA, cleavage with restriction endonucleases, DNA modification, ligation, transformation, and agarose gel electrophoresis. DNA was sequenced by the dideoxy chain-termination method using the AutoRead sequencing kit and the A. L. F. sequencer (Pharmacia Biotech). A DNA Thermal Cycler (Perkin Elmer Cetus) was used for PCR.All E. coli chromosomal DNA positions and section numbers are those of the E. coli genome sequence [10,11].The fhuF gene region was cloned on a 3-kb EcoRV DNA fragment from λ phage 8D1 (672) of the Kohara collection [12] in the high-copy-number vector pSU18 and recloned into vector ...

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“…The excitation and emission slits were set at 1 and 10 nm, respectively, with excitation and emission wavelengths set at 283 and 348 nm, respectively. Experiments involving the E. coli His 6 -tagged FhuD were performed as previously described (13). The value for relative minimum fluorescence (F min ) was obtained using Equation 1.…”

Section: Fhud2 Function In S Aureusmentioning

confidence: 99%

The Role of FhuD2 in Iron(III)-Hydroxamate Transport in Staphylococcus aureus

Sebulsky¹,

Shilton

Speziali³

et al. 2003

Journal of Biological Chemistry

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The fhuD2 gene encodes a lipoprotein that has previously been shown to be important for the utilization of iron(III)-hydroxamates by Staphylococcus aureus. We have studied the function of the FhuD2 protein in greater detail, and demonstrate here that the protein binds several iron(III)-hydroxamates. Mutagenesis of FhuD2 identified several residues that were important for the ability of the protein to function in iron(III)-hydroxamate transport. Several residues, notably Tyr-191, Trp-197, and Glu-202, were found to be critical for ligand binding. Moreover, mutation of two highly conserved glutamate residues, Glu-97 and Glu-231, had no affect on ligand binding, but did impair iron(III)-hydroxamate transport. Interestingly, the transport defect was not equivalent for all iron(III)-hydroxamates. We modeled FhuD2 against the high resolution structures of Escherichia coli FhuD and BtuF, two structurally related proteins, and showed that the three proteins share a similar overall structure. FhuD2 Glu-97 and Glu-231 were positioned on the surface of the N and C domains, respectively. Characterization of E97A, E231A, or E97A/ E231A mutants suggests that these residues, along with the ligand itself, play a cumulative role in recognition by the ABC transporter FhuBGC 2 . In addition, small angle x-ray scattering was used to demonstrate that, in solution, FhuD2 does not undergo a detectable change in conformation upon binding iron(III)-hydroxamates. Therefore, the mechanism of binding and transport of ligands for binding proteins within this family is significantly different from that of other well studied binding protein families, such as that represented by maltosebinding protein.

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Ferrichrome transport in Escherichia coli K-12: altered substrate specificity of mutated periplasmic FhuD and interaction of FhuD with the integral membrane protein FhuB

Cited by 101 publications

References 55 publications

Interactions between TonB from Escherichia coli and the Periplasmic Protein FhuD

Interactions between TonB from Escherichia coli and the Periplasmic Protein FhuD

FhuF, an iron‐regulated protein of Escherichia coli with a new type of [2Fe‐2S] center

The Role of FhuD2 in Iron(III)-Hydroxamate Transport in Staphylococcus aureus

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