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Iron is an essential element for the growth of living cells, and although abundant in nature, under aerobic conditions and at neutral or alkaline pH it is insoluble and thus is not readily available (12, 13). Moreover, in host tissues, iron remains tightly bound to high-affinity iron-binding proteins, such as ferritin, transferrin, lactoferrin, hemoglobin, and iron-sulfur proteins. Because only 10¹⁸ M iron is in the free form (8, 12), a concentration not sufficient to sustain bacterial growth, bacteria have evolved various scavenging systems to acquire iron from the environment (6, 11). These systems utilize either cell surface molecules that make direct contact with the host iron-binding proteins (17, 19) or low-molecular-weight molecules, called siderophores, that show a higher affinity for iron than the host iron chelaters. The iron-siderophore transport mechanisms are well studied in gram-negative bacteria (6), particularly in Escherichia coli, but are understudied in gram-positive bacteria, including staphylococci. Here we report the cloning and characterization of a ferrichrome uptake operon, fhu, with three consecutive open reading frames (ORFs), designated fhuC, fhuB, and fhuD. We also show that the fhuB null mutation results in reduced ferrichrome uptake.

Staphylococcus aureus strains were grown in tryptic soy broth or defined medium (21), whereas E. coli was grown in Luria-Bertani broth with appropriate antibiotics. The iron-limited and iron-rich defined media were prepared by adding 10 µM dipyridyl (Sigma, St. Louis, Mo.) and 10 µM Fe(III), respectively (4). DNA isolation, cloning, and transformations were performed by standard methods (14, 16). DNA probes were labeled with the Prime-a-Gene labeling system (Promega Corp., Madison, Wis.). The DNA restriction and modification enzymes were obtained from Promega. DNA sequences were determined with an ABI Prism 310 automated sequencer (Perkin-Elmer, Foster City, Calif.), and the sequence data were analyzed by BLAST (1).

The S. aureus genome database at the University of Oklahoma (http://www.genome.ou.edu/staph.html) revealed the presence of a DNA fragment with strong homology to the fhuC gene of Bacillus subtilis. The partial fhuC sequence was used as a probe to identify an operon in S. aureus consisting of three consecutive ORFs (Fig. 1) with an array of GATAAT upstream sequences analogous to the so-called Fur box. The first ORF of this operon, fhuC, is homologous to the fhuA gene of B. subtilis (58% identity; 76% similarity) and the genes encoding ATP-binding proteins of other bacterial iron transport systems. Based on a hydropathy analysis that suggests a cytoplasmic location for this protein and its similarity to the ATP-binding proteins, the protein seems to belong to the family of the ATP-binding cassette transporters. The second and third genes of this operon are homologous to the fhuB (43% identity; 67% similarity) and fhuG (40% identity; 64% similarity) genes of B. subtilis, respectively. They are similar to ferrichrome permeases of other bacteria and thus belong to the FecCD family of membrane transport proteins (17).

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Organization of the S. aureus fhu operon. The directions of three ORFs, the location of the Fur box, and restriction endonuclease sites are shown. fhuB was disrupted by insertion of a kanamycin gene (Kan). The abbreviations for restriction sites are as follows: E, EcoRV; H, HindIII; K, KpnI.

To determine the role of the fhu operon, we constructed a mutation in the fhuB gene as described earlier (22). A 6.7-kb HindIII fragment containing the three reading frames was cloned in the vector pTZ18R (10). A 3.2-kb EcoRV fragment was deleted from this construct, and then a 1.5-kb kanamycin cassette was inserted at the unique KpnI site of the fhuB gene. The 4.8-kb HindIII fragment with the disrupted fhuB gene was subcloned into the HindIII site of the shuttle vector pBT2 (3), which cannot replicate in gram-positive bacteria above the nonpermissive temperature. Approximately 10 µg of this construct was electroporated into S. aureus RN4220 cells, and the transformants were selected on tryptic soy agar plates containing 100 µg of kanamycin/ml and 10 µg of chloramphenicol/ml. The transformants were then grown at 43°C, and the chloramphenicol-sensitive clones were checked by Southern blotting and PCR techniques for the replacement of the native fhuB gene with the disrupted gene. The mutation in the fhuB gene was subsequently transduced into S. aureus 8325-4 with a phage 80 lysate using the method described by Novick et al. (15). To complement mutation in trans, the 6.7-kb HindIII fragment containing the entire fhu operon was cloned into the pCU1 shuttle vector (2) and transferred into the fhuB mutant strain.

Iron uptake by S. aureus strain 8325-4, its fhuB mutant, and the fhuB mutant complemented with the native gene, were performed as described earlier with slight modifications (9, 17). Cells were grown overnight at 37°C in iron-free defined medium (iron in the medium was chelated by adding 100 µM dipyridyl), diluted in 50 ml of fresh iron-free defined medium, and grown to an optical density at 580 nm of 0.6 to 0.7. The cells were subsequently harvested and washed twice with transport buffer (1 g of KH₂PO₄, 2 g (NH₄)₂SO₄, 4 g of NaCl, 17.9 g of tricine, 5 mg of MgSO₄ · 7H₂O, 3 mg of CaCl₂, 5.7 mg of nitrilotriacetate, and 10 g of glucose in a total volume of 1 liter, pH 8.0). The cells were resuspended in transport buffer to an optical density of 1.0 at 580 nm. Radiolabeled iron purchased as ⁵⁹FeCl₃ in 0.1 N HCl from Amersham Pharmacia Biotech, Inc. (Piscataway, N.J.) [specific activity, 697 MBq/mg of Fe(III)] was used to prepare a 500-fold stock of siderophore (0.2 mM) to give a metal/ligand ratio of 1:10. A 5.0-ml cell suspension was placed in a 50-ml disposable tube to which labeled siderophore was added to a final concentration of 0.4 µM. Samples (0.5 ml) were taken out at specific time points (0, 5, 10, and 15 min) and filtered through presoaked 0.45-µm-pore-size filters. The filters were washed with 20 ml of ice-cold transport buffer containing 20 nM FeCl₃ and air dried, and the radioactivity was counted with a Beckman scintillation counter. These experiments were performed in triplicate. The iron uptake studies were also carried out by adding ⁵⁹FeCl₃ to a final concentration of 0.04 µM to the same density of cells in the transport buffer as described above.

In these assays, the fhuB mutant strain showed an impaired ferrichrome-iron uptake compared to the wild-type bacterium (Fig. 2A). However, the fhuB mutant complemented with the wild-type gene was able to import iron-ferrichrome to the normal levels (Fig. 2A). In the studies involving radiolabeled FeCl₃ directly, the wild type, the fhuB mutant, and the complemented fhuB mutant strains acquired iron with roughly similar efficiencies (Fig. 2B), suggesting that the fhu operon is specific for ferrichrome-utilizing iron uptake. However, the overall growth kinetics of the mutant did not show any appreciable change compared to those of the wild-type bacterium whether grown in defined medium, defined medium lacking iron, or complex medium (data not shown). This might be supportive of the multiple reports of a very low requirement for iron in S. aureus growth (5, 7, 20).

View larger version (16K): [in this window] [in a new window]   FIG. 2.   Ferric ion uptake by S. aureus 8325-4 (), S. aureus fhuB mutant (), and S. aureus fhuB mutant complemented () strains. The uptake studies were carried out as described in the text. The values are averages of three independent experiments. (A) Iron (59Fe)-ferrichrome complex was used as a substrate in the transport study. (B) Ferric chloride (59Fe) was used as a substrate in the uptake study.

Recently, Sebulsky et al. (18) reported a transposon Tn917 insertional mutant in S. aureus with impaired uptake of the ferric hydroxamate complexes. Further characterization of this mutant suggested that the transposon had inserted into the same chromosomal locus reported earlier (23) and characterized in this manuscript. Their transposon insertion mutant is in the fhuG gene (18), which is fhuD (the third ORF of the fhu operon) in our nomenclature. It is therefore important to note that mutations in both the permease homologues fhuB (present study) and fhuD (18) result in similar phenotypes of impaired iron hydroxamate uptake.

In conclusion, we have identified an S. aureus operon with three genes that constitutes a transport system which specifically utilizes siderophores to trap iron from the environment. Further characterization of the genes of this locus might help devise strategies to control iron metabolic pathways and in turn to design effective control mechanisms against this important human pathogen.

Nucleotide sequence accession number. The nucleotide sequence reported here has been submitted to GenBank under accession number AF132117.