J Bacteriol, February 1998, p. 605-613, Vol. 180, No. 3
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Mikrobiologie II,
Received 22 September 1997/Accepted 4 December 1997
The FhuA protein of Escherichia coli K-12 transports
ferrichrome, the antibiotic albomycin, colicin M, and microcin 25 across the outer membrane and serves as a receptor for the phages T1, T5,
80, and UC-1. FhuA is activated by the electrochemical potential of the cytoplasmic membrane, which probably opens a channel in FhuA. It
is thought that the proteins TonB, ExbB, and ExbD function as a
coupling device between the cytoplasmic membrane and the outer
membrane. Excision of 34 residues from FhuA, tentatively designated the
gating loop, converts FhuA into a permanently open channel. FhuA
contains two disulfide bridges, one in the gating loop and one
close to the C-terminal end. Reduction of the disulfide bridges results
in a low in vivo reaction of the cysteines in the gating
loop and no reaction of the C-terminal cysteines with biotin-maleimide,
as determined by streptavidin-
-galactosidase bound to
biotin. In this study we show that a cysteine residue introduced
into the gating loop by replacement of Asp-336 displayed a rather high
reactivity and was used to monitor structural changes in FhuA
upon binding of ferrichrome. Flow cytometric analysis revealed
fluorescence quenching by ferrichrome and albomycin of fluorescein-maleimide bound to FhuA. Ferrichrome did not inhibit Cys-336 labeling. In contrast, labeling of Cys-347, obtained by replacing Val-347 in the gating loop, was inhibited by
ferrichrome, but ferrichrome quenching was negligible. It
is concluded that binding of ferrichrome causes a
conformational change of the gating loop and that Cys-347 is part
of or close to the ferrichrome binding site. Fluorescence
quenching was independent of the TonB activity. The newly introduced
cysteines and the replacement of the existing cysteines by
serine did not alter sensitivity of cells to the FhuA ligands tested
(T5,
80, T1, colicin M, and albomycin) and fully supported growth on
ferrichrome as the sole iron source. Since cells of E. coli
K-12 display no reactivity to thiol reagents, newly introduced
cysteines can be used to determine surface-exposed regions of outer membrane proteins and to monitor conformational changes during their function.
*
Corresponding author. Mailing address: Mikrobiologie
II, Universität Tübingen, Auf der Morgenstelle 28, D-72076
Tübingen, Germany. Phone: (49) 7071 297096. Fax: (49) 7071 294634. E-mail: v.braun{at}uni-tuebingen.de.
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