Presence of the siderophores pyoverdine and pyochelin in the extracellular medium reduces toxic metal accumulation in Pseudomonas aeruginosa and increases bacterial metal tolerance

Fig. S1. Effect of metals on the growth of P. aeruginosa strains able or not able to produce PVD and/or PCH. A. Pseudomonas aeruginosa PAO1 and PAD07 (a strain unable to produce PVD and PCH) were grown overnight with or without various metals at a concentration of 10 μM (grey and sky blues bars for PAO1 and PAD07 respectively) and a concentration of 100 μM (black and navy blue bars for PAO1 and PAD07 respectively) in succinate medium (6 g l⁻¹ K₂HPO₄, 3 g l⁻¹ KH₂PO₄, 1 g l⁻¹ (NH₄)₂SO₄, 0.2 g l⁻¹ MgSO₄, 4 g l⁻¹ succinic acid, 3.1 g l⁻¹ NaOH, pH 7.0). B. PAD07 (navy blue bars, a strain unable to produce PVD and PCH) and PAO6297 (green bars, a strain able to produce only PVD and not PCH) and CDC5(pPVR2) (yellow bars, a strain able to produce only PCH and not PVD) cells were grown overnight in the presence or absence of various metals at a concentration of 100 μM. A and B. The cultures were incubated for 20 h and the OD at a wavelength of 600 nm was measured. A culture in the absence of metal was used as reference (100%) for each strain. Data are the average of triplicates. The black line indicates the level of 0% growth inhibition. AlCl₃ (Sigma), CdCl₂.2.5H₂O (Probalo), CoCl₂.6H₂0 (Strem), CrCl₂ (Strem), CuCl₂.2H₂O (Strem), EuCl₃ (Aldrich), FeCl₃ (Prolabo), Ga(NO₃)₃.24H₂O (Strem), MnSO₄ (Prolabo), NiCl₂.4H₂O (Strem), Pb(NO₃)₂ (Aldrich), SnCl₂ (Prolabo), TbCl₃.6H₂O (Strem), ZnCl₂ (Alfa Aesar) and Tl₂SO₄ (Fluka) were sources of the metals ions used in this study (referred to as metals). Stock solutions of metals were prepared at concentrations of 50 mM in 0.5 N HCl or 0.5 N HNO₃.

Fig. S2. Metals incorporated into P. aeruginosa PAD07 cells in the presence or absence of siderophores. Pyochelin and PVD were harvested from, respectively, CDC5(pPVR2) and PAO6297 cultures filtered through a 0.22 μm pore size nitrocellulose membrane (PCH has an absorbance at a wavelength of 310 nm (ξ = 4200 l mol⁻¹ cm⁻¹) and PVD at 400 nm (ξ = 19000 l mol⁻¹ cm⁻¹). Pyoverdine- and PCH-deficient PAD07 cultures with an OD₆₀₀ of 1 were incubated at 29°C for 45 min, in the presence of 5 μM metals (navy blue bars) and the experiment was repeated with either 100 μM PCH (yellow bars) or 50 μM PVD (green bars). The cells were harvested, washed, and the metal content determined by ICP-AES as described previously (Braud, 2009 #831). The data are the average of three experiments.

Fig. S3. PCH production by CDC5(pPVR2) cells in the presence of 100 μM of metals. CDC5(pPVR2) (cells unable to produce PVD) were grown with 100 μM of metals for 14 h at 29°C. The extracellular medium was separated from the cells by filtration through cellulose acetate membranes (pore size: 0.45 μm). The pH of this solution was adjusted to 2–3 by progressive addition of citric acid and PCH was extracted with dichloromethane. Pyocheline concentration was determined by measuring the absorbance at 310 nm (ξ = 4200 l mol⁻¹ cm⁻¹). The black line indicates the level corresponding to 0% inhibition in PCH production with and without iron respectively. The data are the average of three experiments. The results are expressed as a function of PCH produced (μg ml⁻¹) to bacterial growth (OD₆₀₀ nm).

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Filename	Format	Size	Description
EMI4_126_sm_Fig_S1a.tif		2969K	Supporting info item
EMI4_126_sm_Fig_S1b.tif		3003K	Supporting info item
EMI4_126_sm_Fig_S2.tif		2415K	Supporting info item
EMI4_126_sm_Fig_S3.tif		2091K	Supporting info item
EMI4_126_sm_legend.doc		23K	Supporting info item

Please note: Wiley-Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.