Hemin

Hemin, δ-aminolevulinic acid (δ-ala) and 2,2’-dipyridyl (Dip) were obtained from Sigma Chemical (Sigma, China). Hemin was dissolved immediately before use in 0.1 N NaOH, Dip was dissolved in ethanol, and δ-ala was dissolved in distilled water. Hemin and δ-ala were filter-sterilized with 0.22-μm pore-size Millipore filters. E. coli strains were grown on LB medium (Sigma-Aldrich, Product Number: L3522) aerobically at 37°C. When required, δ-ala was used at a concentration of 50 μg/ml. Solid media contained 1.5% Difco agar. Iron-depleted medium for E. coli was obtained by the addition of Dip at a final concentration of 150 μM. Antibiotics for E. coli were added to the following final concentrations (μg/ml): Ampicillin (Amp), 100; Kanamycin (Km), 50; Spectinomycin (Spc), 50; Gentamicin (Gen), 20. Arabinose was added at 0.02% for induction of the pBAD promoter. IPTG was added at 0.5 mM for induction of pAM238 expression. R. anatipestifer was grown on LB plates supplemented with 5% defibrinated sheep blood [4 (link)] or TSA plates (Tryptone soy broth, TSB, containing 1.5% agar) [9 (link)] at 37°C under 5% CO₂ atmosphere.

Hemin (Sigma-Aldrich, Oslo, Norway) was dissolved in DMSO to a final concentration of 25 mM. For the crystallization of IsdG, Hemin and IsdG were mixed in a 1:1 molar ratio and incubated for 1 h at 4 °C. Excess Hemin and DMSO were removed using a Micro Bio-Spin 6 size-exclusion column (Bio-Rad, Oslo, Norway) equilibrated with 50 mM Tris-HCl, pH 7.5, and 100 mM KCl. For activity measurements, 25 mM Hemin dissolved in DMSO was diluted 4× in 50 mM Hepes, pH 7.5, and 50 mM KCl (6.25 mM), added in a 1:1 molar ratio to IsdG, and allowed to bind for 1 h at 4 °C.

For reporter studies, C. glutamicum wild type or one of the mutant strains were transformed with a reporter plasmid (Table S3). A preculture in BHI medium (25 µg/ml kanamycin) was inoculated from a fresh BHI agar plate and incubated for 8–10 h at 30°C in a rotary shaker. After that, cells were transferred into a second preculture in CGXII medium (Keilhauer et al., 1993) containing 2% (w/v) glucose and 0 µM FeSO₄ to starve the cells from iron. However, protocatechuic acid (PCA) was present in the preculture, allowing the uptake of trace amounts of iron. After growth overnight, the main culture was inoculated to an OD₆₀₀ of 1 in CGXII medium and cultivated in 48‐well Flowerplates (m2p‐labs GmbH, Aachen, Germany) at 30°C, 95% humidity, 1200 rpm. For the hemin stock solution, hemin (Sigma Aldrich, Munich, Germany) was dissolved in 20 mM NaOH to a concentration of 2.5 mM and, as an iron source, added to the medium in the desired concentrations. Growth of the cells (biomass production) was recorded as the backscattered light intensity of sent light with a wavelength of 620 nm (signal gain factor of 12). For the measurement of eYFP fluorescence, the culture was excited at 510 nm and emission was measured at 532 nm (signal gain factor of 50). Measurements were performed in 15 min intervals.

The V. cholerae strain used in this work was VC833, a multidrug-resistant V. cholerae O1 strain isolated from human feces during a cholera outbreak in Nigeria in 2010.^{21 (link)} The strain was obtained from the Bacteria Culture Collection of Environment and Health at the Fundação Oswaldo Cruz. VC833 is resistant to streptomycin, trimethoprim, trimethoprim/sulfamethoxazole, cefotin, cefuroxime, ampicillin, nalidixic acid, ciprofloxacin, sulfonamide, sulfamethoxazole, and chloramphenicol.^{21 (link)}V. cholerae was routinely grown in Brain Heart Infusion broth (BHI; Sigma-Aldrich) supplemented with hemin (5 mg/mL; Sigma-Aldrich) at 37°C with shaking (225 rpm).
Bacteroides thetaiotaomicron ATCC29741 and Bacteroides vulgatus ATCC8482 were obtained from the American Type Culture Collection. Enterocloster (previously Clostridium) citroniae FM-V5-E was isolated from a stool sample from a healthy 38-year old female.^{11 (link)}Bacteroides and E. citroniae strains were cultured in an anaerobic chamber (80% N₂, 10% H₂, 10% CO₂) in BHI (Sigma-Aldrich) supplemented with hemin (5 mg/mL; Sigma-Aldrich) at 37°C.

A total of four C. difficile strains were studied: a toxigenic pulsotype and PCR ribotype: SLO231/UK[CE]821 (ICC45) isolated in oncology hospital in Brazil; a NAP1/ST01/RT027 strain (LIBA 5756) isolated from an outbreak in a hospital in Costa Rica; a reference epidemic strain NAP1/027/ST01 (R20291) and a non-toxigenic C. difficile ATCC 700057. All C. difficile strains were grown on Brucella agar (Oxoid^® - Thermo Fisher Scientific, Basingstoke, UK), supplemented with 0.1% vitamin K, 0.1% hemin (Sigma^®, Saint Louis, USA) and 5% defibrinated sheep blood, as well as in Brain Heart infusion (BHI) broth supplemented with, 0.1% L-Cysteine, 0.1% vitamin K, 0.4% yeast extract and 0.1% hemin. The plates and tubes were incubated in anaerobic jars with an AnaeroGen™ atmosphere generation system (Oxoid^® - Thermo Fisher Scientific, Basingstoke, UK) at 37°C.