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

Abstract

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...

The Generalized TransducingSalmonellaBacteriophage ES18: Complete Genome Sequence and DNA Packaging Strategy

“…This agrees with the fact that ES18 is known to infect both O-antigen-producing (smooth) and O-antigen-defective (rough) strains of Salmonella enterica (48). It adsorbs to the host outer membrane FhuA protein, a TonB-dependent ferrichrome transporter that is also the receptor in E. coli for phages 80, T1, and T5 (6,47). However, the ES18 J-like protein is only rather distantly related to receptor binding proteins in these three phages (80 [G. Plunkett, personal communication], T1 [73], and T5 [accession no.…”

Characterization of the two-protein complex in Escherichia coli responsible for lipopolysaccharide assembly at the outer membrane

“…Alternatively, and perhaps more likely, LptE may form a plug that sits at the base of, or is somewhat buried within, the lumen of the β-barrel formed by the C-terminal domain of LptD. Similar proteolytic protection has been observed for plug domains of the P pili assembly usher PapC (39,40) and the OM iron-chelate transporter FhuA (41), although, in these cases, the respective plug domain is part of the same polypeptide as the β-barrel.…”

Reconstitution of bacterial outer membrane TonB-dependent transporters in planar lipid bilayer membranes

“…However, the stability and independence of the plug domain when apart from the barrel has been demonstrated, allowing us to suggest that the unfolding happens only to the plug domain. Thus, Braun et al (26) showed in vivo that separately expressed FhuA plugs and barrels can complement each other to form functional units, and Oke et al (27) showed the stability of the plug domain of TbpA from N. meningitidis when expressed and purified alone in E. coli, independent of the barrel. Most compelling, Bonhivers et al (28) demonstrated, using differential scanning calorimetry, that the plug and barrel domains of FhuA behave as autonomous domains and unfold at different temperatures, with the plug domain unfolding before the barrel domain.…”