Trace mineral supplement

Minimal mucin media was made with 0.5% porcine mucin (M2378, Sigma), 100 mM KH₂PO₄ (pH 7.2), 15 mM NaCl, 8.5 mM (NH₄)₂SO₄, 4 mM L-cysteine, 1% vitamin K1-hemin solution (Becton Dickinson), 100 μM MgCl₂, 1.4 μM FeSO₄·7H₂O, 50 μM CaCl₂, 1% trace mineral supplement (ATCC), and 1% vitamin supplement (ATCC). Minimal M9 media was purchased (Teknova) and supplemented with 0.5% tryptone, plus or minus 0.25% porcine mucin (M2378, Sigma). Brain heart infusion medium (BHI) was purchased (Becton Dickinson) as agar or broth powder and made according to manufacturer’s instructions and supplemented with 1% vitamin K1-hemin solution (Becton Dickinson). BHI plus medium was supplemented with 5% heat inactivated fetal bovine serum, 1% vitamin K1-hemin solution (Becton Dickinson), 1% trace mineral supplement (ATCC), 1% vitamin supplement (ATCC), 3 mM D-(+)-cellubiose, 3 mM D-(+)-maltose, 6 mM D-(+)-fructose) and 4 mM L-cysteine. Media were filter sterilized using a Millipore Express filter unit (0.22-μm pore diameter) with the exception of porcine mucin, which was autoclaved in dH₂0 and added after sterile filtration. A flexible anaerobic chamber (Coy Laboratory Products) containing 20% CO₂, 5% H₂, and 75% N₂ and maintained at 37°C was used for all anaerobic microbiology steps.

Cultures of B. thetaiotaomicron VPI-5482 Δtdk and Δtdk Δspt were diluted 1:100 from overnight cultures into 5 mL of BHI medium, supplemented with 1% Vitamin K1-hemin (Becton Dickinson) or minimal medium [1× M9 salts (Teknova), 1% sterile-filtered fetal bovine serum (Sigma-Aldrich), 1% vitamin K1-hemin solution (Becton Dickinson), 1% trace mineral supplement (ATCC), 1% trace vitamin supplement (ATCC), 1 g/L D-(+)-cellobiose (Sigma-Aldrich), 1 g/L D-(+)-maltose (Sigma-Aldrich), 1 g/L D-(+)-fructose (Sigma-Aldrich) and 0.5 g/L L-cysteine (Sigma-Aldrich)] and grown to exponential phase (OD₆₀₀ ~0.4) in anaerobic conditions. Each culture was pelleted in the anaerobic chamber, supernatants were decanted, and pellets were resuspended in 500 μL Trizol. Trizol suspensions underwent a step of bead-beating using ~500 μL of 0.1 mm silica beads (BioSpec). RNA was extracted with the Direct-Zol RNA MiniPrep Plus (Zymo Research) according to manufacturer’s instructions.