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

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 Tn917-LTV1 transposon insertion library after selection on iron-limited media containing aerobactin and streptonigrin. Chromosomal DNA flanking the Tn917-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.

The Role of FhuD2 in Iron(III)-Hydroxamate Transport in Staphylococcus aureus

“…Solid media were obtained by the addition of 1.5% (w/v) Bacto-agar (Difco). Tris-minimal succinate was the iron-deficient medium used throughout (9). FeCl 3 (50 M) was added to Tris-minimal succinate media as required.…”

“…Siderophore Plate Bioassays-Siderophore plate bioassays were performed essentially as previously described (9). Siderophores were used at concentrations of 50 M except for aerobactin (100 M) and rhodotorulic acid (14 mM).…”

“…1 by an asterisk), and the functional consequences on iron(III)-hydroxamate transport were assessed using a siderophore plate bioassay (9). Table III summarizes the mutations and the resultant growth phenotypes, which can be divided into three general groups: (i) mutations that drastically affect transport of all iron(III)-hydroxamates, (ii) mutations that have a differential effect on the transport of the various iron(III)-hydroxamates, and (iii) mutations that do not affect iron(III)-hydroxamate transport.…”

“…Staphylococcus aureus is a Gram-positive human pathogen that possesses an ABC transporter (FhuCBG,FhuD1,FhuD2) for the import of iron(III)-hydroxamates (8,9). FhuB and FhuG constitute the membrane-spanning components, whereas FhuC is the ATP-binding protein and two acylated proteins, FhuD1 and FhuD2, are thought to serve as the binding proteins for the system.…”

IruO Is a Reductase for Heme Degradation by IsdI and IsdG Proteins in Staphylococcus aureus

“…Consequently, S. aureus has evolved multiple strategies for iron acquisition (7). S. aureus produces two siderophores, staphyloferrin A (8,9) and staphyloferrin B (10), and has a transport system that can co-opt hydroxamate-type siderophores produced by other bacteria (11). S. aureus can also obtain heme from host heme-containing proteins hemoglobin and haptoglobin, transport it across the bacterial cell envelope, cleave the porphyrin ring, and release iron for use by the cell with the well characterized ironregulated surface determinant (Isd) 3 system (12).…”