0.2 m pore size membrane

Plasma heme was measured using 1-Step™ Turbo 3,3',5,5'-Tetramethylbenzidine (TMB) enzyme-linked immunosorbent assay (ELISA) Substrate (Thermofisher scientific). This method is based on the oxidation of TMB by the pseudoperoxidase activity of heme. For this assay, hemin chloride porcine (designated as heme, Sigma-Aldrich) was diluted in 0.1 M NaOH (Merck) and the pH was adjusted to 7.8–8.2 (29 (link)). Subsequently, this solution was filtered through 0.2 µm pore size membrane (Whatman). Hereafter, serial dilutions (4.0–2.0–1.5–1–0.5–0.25–0 µM) of a 2 mM heme stock were made in 20 mM HEPES (Sigma-Aldrich) + 1% bovine serum albumin (BSA, Millipore), pH 7.4. Next, plasma samples (patients 10×, controls 2.5×) were diluted in 20 mM HEPES + 1% BSA, pH 7.4, where after 20 µl of every sample and 80 µl of the turbo TMB substrate was added to a microtiter plate (Greiner Bio-One™) and incubated for 10 min in the dark. The reaction was stopped with 100 µl 2 M sulfuric acid (Sigma-Aldrich) and absorbance was read at 450 nanometer (nm) with Victor 3 V multilabel plate reader (PerkinElmer).

Strains carrying plasmids were grown overnight in LB with ampicillin. They were then subcultured into fresh media with antibiotics and grown to mid-log phase. When cultures reached an OD₆₀₀ of ~0.3, 0.5 mM IPTG was added, and samples were harvested at different time points. RNA was extracted by the hot phenol method as described in reference 72.
Northern blot analysis was carried out as described in reference 73. Briefly, 7 µg total RNA (for DicF) or 10 µg total RNA (for xylR and pykA mRNAs) was run on acrylamide gels and 1% agarose gels using 1× Tris-acetate-EDTA (TAE) or 1× MOPS (morpholinepropanesulfonic acid) buffer, respectively. RNA in acrylamide gels was transferred to a 0.2-µm-pore-size membrane (Whatman) in 0.5× TAE buffer by electrophoresis. RNA in agarose gels was transferred by capillary transfer using 20× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Following transfer, the membranes were probed overnight with biotinylated DNA oligonucleotides (IDT) complementary to the respective RNAs. Detection was carried out according to the instructions for the Brightstar Biodetect kit (Ambion).