Iron-hydroxamate transport intoEscherichia coli K12: Localization of FhuD in the periplasm and of FhuB in the cytoplasmic membrane

Abstract

The fhuB, fhuC and fhuD genes encode proteins which catalyze transport of iron(III)-hydroxamate compounds from the periplasm into the cytoplasm of Escherichia coli. The fhuB, C, D genes were cloned downstream of a strong phage T7 promoter and transcribed by T7 RNA polymerase. The overexpressed FhuD protein appeared in two forms of 31 and 28 kDa and was released upon conversion of vegetative cells into spheroplasts, suggesting synthesis of FhuD as a precursor and export into the periplasm. The very hydrophobic FhuB protein was found in the cytoplasmic membrane. These properties, together with the previously found homologies in the FhuC protein to ATP-binding proteins, display the characteristics of a periplasmic binding protein dependent transport system across the cytoplasmic membrane. The molecular weight of FhuB and the sequence of fhuC, as previously published by us, was confirmed. FhuB exhibited double the size of most hydrophobic proteins of such systems and showed homology between the amino- and carboxy-terminal halves of the protein, indicating duplication of an original gene and subsequent fusion of the two DNA fragments.