Supernatant siderophore

units were normalized to culture optical density. CH5424802 nmr Siderophore preparations Siderophore concentrates were prepared by growing S. aureus strains with aeration in TMS with 0.1 μM EDDHA. Culture supernatants were harvested at 15 and 40 hours after initial culturing. Cells were pelleted by centrifugation and supernatants were lyophilized. The freeze-dried supernatant was extracted with methanol (one-fifth the original supernatant volume), and then passed through a Whatman No. 1 filter paper to remove insoluble material followed by rotary evaporation. The methanol-extracted material was solubilized in water to 5% of the original supernatant volume. The resulting preparations were stored at -20°C. Siderophore plate-disk diffusion assays Siderophore growth promotion assays were performed essentially as described [9]. Briefly, S. aureus strains were seeded into TMS agar (1 × 104 cells ml-1) containing 10 μM EDDHA. Ten-μL aliquots of culture supernatant concentrates (as prepared above) were added to sterile paper disks which were then placed onto the TMS agar plates. Growth promotion was quantified by measuring the diameter of growth around the disc after 36 h at 37°C. Computer analyses DNA sequence analysis, oligonucleotide primer design BIRB 796 order and sequence alignments were performed either using programs available through NCBI or using Vector NTI Suite software package (Informax, Bethesda,

MD). Graphs were generated using GraphPad Prism 4.0. Results The S. aureus sbn operon contains genes predicted to encode L-Dap biosynthesis enzymes Original studies on the structural elucidation of staphyloferrin B revealed that it contained citric acid, α-ketoglutaric acid (α-KG), 1,2-diaminoethane (Dae), and L-2,3-diaminopropionic acid (L-Dap) [15] (Figure 1A). The unusual nonproteinogenic amino acid L-Dap serves a critical role for the siderophore in terms of iron-coordination, since a carboxyl group oxygen and the nitrogen atom on the primary amine of L-Dap contribute two of the six iron-ligands used to obtain the distorted octahedral geometry in the ferric-staphyloferrin B complex [28] (Figure

1A). In the proposed biosynthetic pathway, L-Dap Ureohydrolase is twice incorporated into the staphyloferrin B molecule, as the amine nucleophilic substrate for the type A and type C NIS synthetases SbnE and SbnF, respectively [17]. While SbnE condenses the first molecule of L-Dap to citrate, the action of the decarboxylase SbnH removes the carboxyl group from the L-Dap residue to give rise to the Dae portion of staphyloferrin B [17]. SbnF then condenses a terminal L-Dap onto a citryl-Dae intermediate within the staphyloferrin B structure [17]. Since L-Dap plays such a pivotal role in iron-coordination for staphyloferrin B, and since the biosynthesis of this siderophore requires two units of L-Dap per unit of staphyloferrin B, we were interested in elucidating the genetic requirement for L-Dap biosynthesis in S. aureus.