Cultivation and Manipulation of E. coli and R. anatipestifer

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.