Gram-positive siderophore-shuttle with iron-exchange from Fe-siderophore to apo-siderophore by Bacillus cereus YxeB

Small molecule iron-chelators, siderophores, are very important in facilitating the acquisition of Fe(III), an essential element for pathogenic bacteria. Many Gram-negative outer-membrane transporters and Gram-positive lipoprotein siderophore-binding proteins have been characterized, and the binding ability of outer-membrane transporters and siderophore-binding proteins for Fe-siderophores has been determined. However, there is little information regarding the binding ability of these proteins for apo-siderophores, the iron-free chelators. Here we report that Bacillus cereus YxeB facilitates iron-exchange from Fe-siderophore to apo-siderophore bound to the protein, the first Gram-positive siderophore-shuttle system. YxeB binds ferrioxamine B (FO, Fe-siderophore)/desferrioxamine B (DFO, apo-siderophore) in vitro. Disc-diffusion assays and growth assays using the yxeB mutant reveal that YxeB is responsible for importing the FO. Cr-DFO (a FO analog) is bound by YxeB in vitro and B. cereus imports or binds Cr-DFO in vivo. In vivo uptake assays using Cr-DFO and FO and growth assays using DFO and Cr-DFO show that B. cereus selectively imports and uses FO when DFO is present. Moreover, in vitro competition assays using Cr-DFO and FO clearly demonstrate that YxeB binds only FO, not Cr-DFO, when DFO is bound to the protein. Iron-exchange from FO to DFO bound to YxeB must occur when DFO is initially bound by YxeB. Because the metal exchange rate is generally first order in replacement ligand concentration, protein binding of the apo-siderophore acts to dramatically enhance the iron exchange rate, a key component of the Gram-positive siderophore-shuttle mechanism