Identification and characterization of a membrane permease involved in iron-hydroxamate transport in Staphylococcus aureus

Staphylococcus aureus was shown to transport iron complexed to a variety of hydroxamate type siderophores, including ferrichrome, aerobactin, and desf errioxamine. An S. aureus mutant defective in the ability to transport ferr ic hydroxamate complexes was isolated from a Tn917-LTV1 transposon insertio n library after selection on iron-limited media containing aerobactin and s treptonigrin. Chromosomal DNA banking the Tn917-LTV1 insertion was identifi ed by sequencing of chromosomal DNA isolated from the mutant. This informat ion localized the transposon insertion to a gene whose predicted product sh ares significant similarity with FhuG of Bacillus subtilis. DNA sequence in formation was then used to clone a larger fragment of DNA surrounding the f huG gene, and this resulted in the identification of an operon of three gen es, fhuCBG, all of which show significant similarities to ferric hydroxamat e uptake (fhu) genes in B. subtilis. FhuB and FhuG are highly hydrophobic, suggesting that they are embedded within the cytoplasmic membrane, while Fh uC shares significant homology with ATP-binding proteins. Given this, the S . aureus FhuCBG proteins were predicted to be part of a binding protein-dep endent 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 insertio nally inactivated fur gene showed maximal levels of ferric hydroxamate upta ke even when the cells were grown under iron-replete conditions. By using t he Fur titration assay, it was shown that the Fur box sequences upstream of fhuCBG are recognized by the Escherichia coli Fur protein.