Transport activity of FhuA, FhuC, FhuD, and FhuB derivatives in a system free of polar effects, and stoichiometry of components involved in ferrichrome uptake |
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Authors: | A Mademidis and W K?ster |
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Institution: | (1) Mikrobiologie/Membranphysiologie, Universit?t Tübingen, Auf der Morgenstelle 28, 72076 Tübingen, Germany, DE |
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Abstract: | The Escherichia colifhu operon, composed of the fhuA, C, D, and B genes, is essential for the utilization of ferric siderophores of the hydroxamate type and for the uptake of the antibiotic
albomycin. We have had difficulty studying the effects of missense mutations in individual plasmid-encoded transport genes
because appropriate test strains were not found: all isolated chromosomal mutations in either one of the fhu genes (with a complete loss of function) negatively influenced the expression of other fhu genes in the operon. In order to analyze Fhu mutant proteins in a system free of polar effects, we constructed a plasmid-encoded
gene cassette system by introducing unique restriction sites that allowed precise cloning of individual fhu genes. The fhu cassette operon expressed in a chromosomal fhu deletion mutant enabled us to evaluate the transport activity of mutated FhuA, FhuC, FhuD or FhuB derivatives. In addition,
we found that transport across the outer membrane (via FhuA, TonB, ExbB, D) rather than transport across the cytoplasmic membrane
(via FhuC, D, B) was rate limiting. The stoichiometry of the components involved in the uptake of iron(III) hydroxamates seems
to be important for proper functioning.
Received: 20 October 1997 / Accepted: 22 December 1997 |
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Keywords: | E coli fhu operon Polar effect ABC transporter Iron TonB |
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