TRANSPORT ACTIVITY OF FHUA, FHUC, FHUD, AND FHUB DERIVATIVES IN A SYSTEM FREE OF POLAR EFFECTS, AND STOICHIOMETRY OF COMPONENTS INVOLVED INFERRICHROME UPTAKE

The Escherichia coli fhu operon, composed of the fhuA, C, D, and B gen es, is essential for the utilization of ferric siderophores of the hyd roxamate type and for the uptake of the antibiotic albomycin. We have had difficulty studying the effects of missense mutations in individua l plasmid-encoded transport genes because appropriate test strains wer e not found: all isolated chromosomal mutations in either one of the f hu genes (with a complete loss of function) negatively influenced the expression of other fhu genes in the operon. In order to analyze Fhu m utant proteins in a system free of polar effects, we constructed a pla smid-encoded gene cassette system by introducing unique restriction si tes that allowed precise cloning of individual fhu genes. The fhu cass ette 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 me mbrane (via FhuA, TonB, ExbB, D) rather than transport across the cyto plasmic membrane (via FhuC, D, B) was rate limiting. The stoichiometry of the components involved in the uptake of iron(III) hydroxamates se ems to be important for proper functioning.