Discovery of an Iron-Regulated Citrate Synthase in Staphylococcus aureus  Johnson Cheung, Michael E.P. Murphy, David E. Heinrichs  Chemistry & Biology  Volume 19, Issue 12, Pages 1568-1578 (December 2012) DOI: 10.1016/j.chembiol.2012.10.003 Copyright © 2012 Elsevier Ltd Terms and Conditions

Chemistry & Biology 2012 19, 1568-1578DOI: (10. 1016/j. chembiol. 2012 Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 1 Chemical Structures and Biosynthetic Gene Clusters for Citric Acid-Containing SB, Achromobactin, and Vibrioferrin Siderophores (A) Chemical structures. Fundamental components of each siderophore are labeled. (B) Biosynthetic gene clusters. The name of the siderophore whose production is directed by each cluster is listed on the left along with the bacterial genome from which the information is taken. Where present, transporter-encoding loci are also indicated. The sbnG homologs in each cluster (absent in the V. parahaemolyticus cluster) are highlighted with an asterisk. See also Table S1. Chemistry & Biology 2012 19, 1568-1578DOI: (10.1016/j.chembiol.2012.10.003) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 2 Citrate Synthase Activity of SbnG (A) SbnG hydrolyzes acetyl-CoA in the presence of oxaloacetate, but not glyoxylate. Top panel, SbnG was incubated with glyoxylate and acetyl-CoA and the amount of HS-CoA generated was determined using the DTNB assay (see Experimental Procedures). Bottom panel, the reactions were the same as those in the top panel, except that oxaloacetate was substituted for glyoxylate. Error bars indicate SD from the mean of three replicates. (B) Liquid chromatography-electrospray ionization-mass spectrometry analysis of reactions for formation of mass ion species 191.1, corresponding to that of citric acid. Full reaction contained SbnG, oxaloacetate, and acetyl-CoA and other reactions shown are those where one component, as indicated, was omitted. (C) Tandem mass spectrometry of 191.1 ion from either a citric acid standard or the full SbnG reaction. See also Figures S1–S5. Chemistry & Biology 2012 19, 1568-1578DOI: (10.1016/j.chembiol.2012.10.003) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 3 SbnG-Generated Citrate Can Be Used by Siderophore Synthetases to Make SB In Vitro (A) Disk diffusion growth promotion assays were performed by spotting enzyme reaction mixtures (via two-pot reaction strategy, see Supplemental Experimental Procedures) onto sterile paper disks before being placed onto TMS agar plates seeded with S. aureus. The diameter of growth around the disk was measured (in mm) 36 hr after incubation. Error bars indicate standard deviation from the mean of three replicates. Dashed line represents the diameter of the disk. The experiment was repeated using S. aureus sirA mutant (SirA is the Fe-SB receptor), and no growth was observed for any of the four samples. (B) Liquid chromatography-electrospray ionization-mass spectrometry analysis of SB generated from a two-pot reaction strategy. The mass ion of [M+H]− = 447.1 Da (eluted at 6.33 min) is observed only in the top spectrum (full reaction) but is absent in the bottom two spectra, where reactions lacked either oxaloacetate or acetyl-CoA, as indicated. Samples are normalized to one another based on common ions present in the samples, such as that of HEPES. Error bars represent SD from the mean of three replicates. Chemistry & Biology 2012 19, 1568-1578DOI: (10.1016/j.chembiol.2012.10.003) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 4 Identification of SbnG Inhibitors Enzyme assays were carried out as described in Experimental Procedures. Percent activity is reported as a percentage of the activity relative to an assay lacking the indicated components. Error bars indicate standard deviation from the mean of five replicates. See also Figures S6 and S7 and Table S2. Chemistry & Biology 2012 19, 1568-1578DOI: (10.1016/j.chembiol.2012.10.003) Copyright © 2012 Elsevier Ltd Terms and Conditions

Figure 5 SbnG Behaves as a Hexamer in Solution (A) Size-exclusion chromatography of SbnG. The chromatogram was obtained from size-exclusion analysis of purified SbnG using a Superdex 200 column at a flow rate of 0.2 ml/min. The running buffer consisted of 50 mM HEPES, pH 7.4, and 150 mM NaCl. The absorbance was recorded at 280 nm. The elution volumes of molecular mass standards are shown at the top of the chromatogram. The inset is a calibration curve for the Superdex 200 column. The elution volume of the standards is represented as a ratio of the elution volume of the protein standard to the void volume of the column. The ratio of the elution volume of SbnG is shown, and corresponds to a molecular mass of 188 kDa. (B) Sedimentation equilibrium analysis of SbnG. In the bottom panel, the absorbance at 280 nm is plotted as a function of the radial position. The circles represent the sedimentation equilibrium data obtained for 10 μM SbnG at a rotor speed of 10,000 rpm. The nonlinear least squares best fit to a single species (hexamer model) according to Equation (1) is represented by the solid line. The calculated molar mass was 170 kDa and is consistent with the theoretical molar mass of hexameric SbnG that is 174 kDa. In the top panel, the residuals for the nonlinear least squares best fit are plotted as a function of radial position. Error bars represent SD from the mean of three replicates. Chemistry & Biology 2012 19, 1568-1578DOI: (10.1016/j.chembiol.2012.10.003) Copyright © 2012 Elsevier Ltd Terms and Conditions