Chelex 100

A polyacrylamide hydrogel consisting of 15% acrylamide (Merck, Belgium) and 0.3% agarose derived cross linker (DGT Research Ltd., UK) was used as a diffusive hydrogel in a DGT probe. The recipe for making the diffusive gel can be found elsewhere^{63 (link)}. chelex-100 was used to prepare the resin gels according to the recipe obtained from DGT research Ltd: 1.6 gram chelex-100 resin was added to 4 mL of gel solution (mixed in advance and stored in a refrigerator at 4 °C), which is a mixture of acrylamide (40%, Merck, Belgium), acrylamide cross-linker (2%, DGT Research Ltd, UK) and MilliQ water. 24 µL ammonium persulfate (1%, Merck, Belgium) and 6 µL N,N,N,N-tetraethylenediamine (TEMED, Merck, Belgium) were added to the mixture and the mixed solution was casted into a glass assembly separated with a fixed thickness of Teflon spacer (0.04 cm). The assembly was placed in an oven at 45 °C for 1 h, and then the gel was peeled from the glass plates and hydrated in MilliQ water for at least one day before use.

Amicon ultra centrifugal filters were obtained from Merck-Millipore (Merck Millipore, Germany). Bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid, dipotassium Salt) and iodoacetamide-fluorescein (IAF) were from Molecular Probe (Life Technologies do Brasil Ltda). Bromophenol blue, tetramethylethylenediamine (TEMED), molecular weight marker Kaleidoscope, glycerol, glycine, acrylamide were obtained from Bio-Rad Laboratories (CA, USA). Acids and solvents (HPLC grade) were from J.T. Baker (Avantor Performance Materials, Mexico). All other reagents were from Sigma (St. Louis, MO). All aqueous solutions were prepared with ultrapure water purified by a Direct-Q3 system (Merck Millipore, Germany) and treated with Chelex 100 before use.

Mtb H37Rv (ATCC) was initially grown in Middlebrook 7H9 medium (7H9) supplemented with 10% (v/v) OADC enrichment (Becton Dickinson Microbiology Systems, Spark, MD, United States), 0.5% (v/v) glycerol and with or without Tyloxapol 0.05% (v/v; Sigma) prior to inoculation in a MM consisting of KH₂PO₄ 1 g/l, Na₂HPO₄ 2.5 g/l, asparagine 0.5 g/l, ferric ammonium citrate 50 mg/l, CaCl₂ 0.5 mg/l, ZnSO₄ 0.1 mg/l, with or without Tyloxapol 0.05% (v/v), containing 0.1% (v/v) glycerol, pH 7.0. Cultures were grown in 7H9 at 37°C until they reached an OD of 0.6 and then washed in in phosphate-buffered saline (PBS) + 0.05% Tyloxapol and transferred to MM. Low iron minimal medium (LIMM) was used for culture under iron-controlled conditions. To remove metal ions MM was treated with 5% Chelex-100 (Bio-Rad, Hercules, CA, United States) for 24 h, at 4°C with gentle agitation. Chelex-100 was removed by filtration through 0.22 μm filter (Millipore, Burlington, MA, United States) and the medium was supplemented with sterile ZnCl₂ 0.5 mg/l, of MnSO₄ 0.1 mg/l, and of MgSO₄ 400 mg/l. The amount of residual Fe in this medium, determined by atomic absorption spectroscopy is ∼1 μM. High iron MM (MM) was prepared by supplementing LIMM with 50 μM FeCl₃.

Bacterial strains and plasmids used in this study are listed in Table 1. M. tuberculosis strains were recovered from frozen stocks on Middlebrook 7H10 agar (Difco) supplemented with 0.5% (vol/vol) glycerol, 0.05% Tween 80, and 10% (vol/vol) albumin-dextrose-NaCl complex (ADN). Fe-depleted minimal medium (MM) was used for iron starvation experiments. MM contains 0.5% (wt/vol) asparagine, 0.5% (wt/vol) KH₂PO₄, 2% glycerol, 0.05% Tween 80, and 10% ADN, and its pH was adjusted to 6.8 with NaOH. To remove metal ions, MM was treated with 5% Chelex-100 (Bio-Rad) for 24 h at 4°C with gentle agitation. The Chelex-100 resin was removed by filtration through a 0.22-μm filter (Millipore), and the medium was supplemented with 0.5 mg of sterile ZnCl₂ liter⁻¹, 0.1 mg of MnSO₄ liter⁻¹, and 40 mg of MgSO₄ liter⁻¹. The amount of residual Fe in this medium, determined by atomic absorption spectroscopy, is ~1 µM. The M. tuberculosis mprA mutant used in this study was a kind gift from Issar Smith (PHRI, Newark, NJ), and it was obtained by insertion of a Kan^r cassette (aph) in the mprA gene via homologous recombination using the suicide plasmid PSM270 (72 (link)). Disruption of the gene was confirmed by PCR analysis (see Fig. S7 in the supplemental material).