Identification of a Positively Charged Platform in <i>Staphylococcus aureus</i> HtsA That Is Essential for Ferric Staphyloferrin A Transport

In response to iron starvation, <i>Staphylococcus aureus</i> secretes both staphyloferrin A and staphyloferrin B, which are high-affinity iron-chelating molecules. The structures of both HtsA and SirA, the ferric-staphyloferrin A [Fe­(III)-SA] and ferric-staphyloferrin B [Fe­(III)-SB] receptors, respectively, have recently been determined. The structure of HtsA identifies a novel form of ligand entrapment composed of many positively charged residues. Through ionic interactions, the binding pocket appears highly adapted for the binding of the highly anionic siderophore SA. However, biological validation of the importance of the nine SA-interacting residues (six arginines, one tyrosine, one histidine, and one lysine) has not been previously performed. Here, we mutated each of the Fe­(III)-SA-interacting residues in HtsA and found that substitutions R104A, R126A, H209A, R306A, and R306K resulted in a reduction of binding affinity of HtsA for Fe­(III)-SA. While mutation of almost all proposed ligand-interacting residues decreased the ability of <i>S. aureus</i> cells to transport ⁵⁵Fe­(III)-SA, <i>S. aureus</i> expressing HtsA R104A, R126A, R306A, and R306K showed the greatest transport defects and were incapable of growth in iron-restricted growth media in a SA-dependent manner. These three residues cluster together and, relative to other residues in the binding pocket, move very little between the apo and closed holo structures. Their essentiality for receptor function, together with structural information, suggests that they form a positively charged platform that is required for initial contact with the terminal carboxyl groups of the two citrates in the Fe­(III)-SA complex. This is a likely mechanism by which HtsA discerns iron-bound SA from iron-free SA

An ABC Transporter with Two Periplasmic Binding Proteins Involved in Iron Acquisition in <i>Pseudomonas aeruginosa</i>

Pyoverdine I is the main siderophore secreted by <i>Pseudomonas aeruginosa</i> PAO1 to obtain access to iron. After extracellular iron chelation, pyoverdine-Fe uptake into the bacteria involves a specific outer-membrane transporter, FpvA. Iron is then released in the periplasm by a mechanism involving no siderophore modification but probably iron reduction. The proteins involved in this dissociation step are currently unknown. The pyoverdine locus contains the <i>fpvCDEF</i> operon, which contains four genes. These genes encode an ABC transporter of unknown function with the distinguishing characteristic of encompassing two periplasmic binding proteins, FpvC and FpvF, associated with the ATPase, FpvE, and the permease, FpvD. Deletion of these four genes partially inhibited cytoplasmic uptake of ⁵⁵Fe in the presence of pyoverdine and markedly slowed down the <i>in vivo</i> kinetics of iron release from the siderophore. This transporter is therefore involved in iron acquisition by pyoverdine in <i>P. aeruginosa</i>. Sequence alignments clearly showed that FpvC and FpvF belong to two different subgroups of periplasmic binding proteins. FpvC appears to be a metal-binding protein, whereas FpvF has homology with ferrisiderophore binding proteins. <i>In vivo</i> cross-linking assays and incubation of purified FpvC and FpvF proteins showed formation of complexes between both proteins. These complexes were able to bind <i>in vitro</i> PVDI-Fe, PVDI-Ga, or apo PVDI. This is the first example of an ABC transporter involved in iron acquisition <i>via</i> siderophores, with two periplasmic binding proteins interacting with the ferrisiderophore. The possible roles of FpvCDEF in iron uptake by the PVDI pathway are discussed