Chelex 100

S. pneumoniae D39 strain was routinely cultured in Todd-Hewitt broth (Oxiod, UK) containing 0.5% yeast extract (THY) or grew on Columbia agar (Difco, USA) containing 5% sheep blood (Ruite, China) at 37°C with 5% CO₂. When necessary, appropriate antibiotics were added to media: erythromycin (Erm) at 0.2 μg/mL, chloramphenicol (Cm) at 4 μg/mL, spectinomycin (Spec) at 100 μg/mL, tetracycline (Tet) at 3.5 μg/mL. The iron-restricted medium was produced by adding 5% Chelex-100 (Bio-Rad) to THY for 8 h with continuous agitation, followed by filter sterilization to remove the Chelex-100 and supplementation with 100 μM CaCl₂ and 2 mM MgCl₂. The iron content in the medium after Chelex-100 treatment was determined by inductively coupled plasma mass spectrometry (ICP-MS, Thermo Scientific, USA). When required, 20 μM FeCl₃, hemin, or Fch was added to the iron-restricted medium.

Apo-BcII and apo-VIM-2 were generated using an adaptation of a previous method [17] (link). Thus, di-Zn(II) binding enzyme solutions were dialysed against three changes of > 100 volumes of an EDTA-containing solution (50 mM HEPES pH 7.5, 200 mM NaCl, 20 mM EDTA, 2 mM TCEP·HCl) over 24 h. EDTA was removed by a second dialysis of three changes of > 100 volumes of a metal-free solution (50 mM HEPES pH 7.5, 100 mM NaCl, 2 mM TCEP·HCl, Chelex 100) over 24 h. All dialyses were carried out at 4 °C using Slide-A-Lyzer® Dialysis Cassettes (Thermo Scientific). The concentrations of the apo-enzymes were determined using a NanoDrop spectrophotometer measuring absorption at 280 nm (ε = 29,450 M^− 1 cm^− 1 and ε = 31,400 M^− 1 cm^− 1 for BcII and VIM-2, respectively). All subsequent concentrations used in assays were based on this measurement.