Identification and Characterization of a Membrane Permease Involved in Iron-Hydroxamate Transport in Staphylococcus aureus

Abstract

ABSTRACT Staphylococcus aureus was shown to transport iron complexed to a variety of hydroxamate type siderophores, including ferrichrome, aerobactin, and desferrioxamine. An S. aureus mutant defective in the ability to transport ferric hydroxamate complexes was isolated from a Tn 917 -LTV1 transposon insertion library after selection on iron-limited media containing aerobactin and streptonigrin. Chromosomal DNA flanking the Tn 917 -LTV1 insertion was identified by sequencing of chromosomal DNA isolated from the mutant. This information localized the transposon insertion to a gene whose predicted product shares significant similarity with FhuG of Bacillus subtilis . DNA sequence information was then used to clone a larger fragment of DNA surrounding the fhuG gene, and this resulted in the identification of an operon of three genes, fhuCBG , all of which show significant similarities to ferric hydroxamate uptake ( fhu ) genes in B. subtilis . FhuB and FhuG are highly hydrophobic, suggesting that they are embedded within the cytoplasmic membrane, while FhuC shares significant homology with ATP-binding proteins. Given this, the S. aureus FhuCBG proteins were predicted to be part of a binding protein-dependent transport system for ferric hydroxamates. Exogenous iron levels were shown to regulate ferric hydroxamate uptake in S. aureus . This regulation is attributable to Fur in S. aureus because a strain containing an insertionally inactivated fur gene showed maximal levels of ferric hydroxamate uptake even when the cells were grown under iron-replete conditions. By using the Fur titration assay, it was shown that the Fur box sequences upstream of fhuCBG are recognized by the Escherichia coli Fur protein.