Trace vitamin supplement

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.

WT and ΔSPT cultures of B. thetaiotaomicron and B. ovatus were grown in 5 mL of minimal media [M9 salts (Teknova), 1% vitamin K1-hemin solution (Becton Dickinson), 1% trace mineral supplement (ATCC), 1% trace vitamin supplement (ATCC), 2% lactose)] supplemented with 10μM of deuterium (D4)-labelled alanine (Sigma) or 10μM of unlabeled alanine (Sigma). After 48 hrs, resulting cultures were pelleted by centrifugation at 8,000 rpm for 10 min and cell density was normalized. Lipids were extracted using isopropanol and sent for lipidomic analysis.