One Enzyme, Three Metabolites: Shewanella algae Controls Siderophore Production via the Cellular Substrate Pool  Sina Rütschlin, Sandra Gunesch, Thomas Böttcher  Cell Chemical Biology  Volume 24, Issue 5, Pages 598-604.e10 (May 2017) DOI: 10.1016/j.chembiol.2017.03.017 Copyright © 2017 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2017 24, 598-604. e10DOI: (10. 1016/j. chembiol Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 1 Putative Avaroferrin Gene Cluster of S. algae and the Proposed Biosynthetic Pathway of the Three Siderophores The biosynthesis genes are highlighted in black. The origin of the diamine components is indicated by color (cadaverine, blue; putrescine, red). Cell Chemical Biology 2017 24, 598-604.e10DOI: (10.1016/j.chembiol.2017.03.017) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 2 Extracted Ion Chromatograms of LC-MS Analysis of Siderophore Production by AvbD (A) From bottom: heat-inactivated enzyme control extracted ion chromatogram (EIC) trace for linear avaroferrin precursor and avaroferrin followed by EIC at m/z 405.2344 [M + H]+ corresponding to linear avaroferrin precursor (Pre-2), and EIC at m/z 387.2238 [M + H]+ for avaroferrin (2). MS/MS fragment ions confirming the two types of avaroferrin precursor are indicated. (B) Production of all three siderophores by AvbD: traces for putrebactin (1) m/z 373.2082 [M + H]+, avaroferrin (2) m/z 387.2238 [M + H]+, and bisucaberin (3) m/z 401.2395 [M + H]+. See also Figure S2. Cell Chemical Biology 2017 24, 598-604.e10DOI: (10.1016/j.chembiol.2017.03.017) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 3 Plot of Initial Velocities Calculated for Conversion of 0.1 mM HS[5]A with Different Concentrations of HS[4]A to Bisucaberin and Avaroferrin Diamonds, bisucaberin (3); squares, avaroferrin (2). Error bars indicate SD calculated from three independent experiments. See also Figure S3. Cell Chemical Biology 2017 24, 598-604.e10DOI: (10.1016/j.chembiol.2017.03.017) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 4 Production of Siderophores by Shewanella algae Representative LC-MS traces of extracts of S. algae with the mean average relative production of all three siderophores calculated from three independent experiments under (A) no addition of cadaverine or putrescine; (B) addition of 1 mΜ cadaverine; and (C) addition of 1 mΜ putrescine. Mean values were calculated from integrated peak areas of LC-MS traces where the combined integrals of the three siderophores were set as 100%. Cell Chemical Biology 2017 24, 598-604.e10DOI: (10.1016/j.chembiol.2017.03.017) Copyright © 2017 Elsevier Ltd Terms and Conditions

Figure 5 Intracellular Substrate Pool of Putrescine and Cadaverine in S. algae (A) Amine derivatization for detection of putrescine and cadaverine by LC-MS analysis. (B) Mean ratios of putrescine and cadaverine of S. algae for an incubation time of 1 or 5 days. Values are given as percentage of the total diamine concentrations calculated from integrated peak areas of LC-MS traces, each in triplicate. Representative EICs for m/z 289.21 [M + H]+ for derivatized putrescine (4, top) and m/z 303.23 [M + H]+ for derivatized cadaverine (5, bottom) for S. algae cells grown on swarming medium supplemented with 100 μM 2,2′-bipyridine after 1 day of incubation. See also Figure S5. Cell Chemical Biology 2017 24, 598-604.e10DOI: (10.1016/j.chembiol.2017.03.017) Copyright © 2017 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2017 24, 598-604. e10DOI: (10. 1016/j. chembiol Copyright © 2017 Elsevier Ltd Terms and Conditions

Cell Chemical Biology 2017 24, 598-604. e10DOI: (10. 1016/j. chembiol Copyright © 2017 Elsevier Ltd Terms and Conditions