Anaerobic chamber

The assay followed a procedure described earlier11 (link). In brief, recombinant T. hominis proteins were expressed in E. coli with a His-tag and purified by Ni-NTA affinity chromatography followed by gel filtration (Äkta Purifier System 10, Column 16/60 Superdex 200 pg, GE Healthcare). Samples for the in vitro Fe/S-cluster synthesis assay were prepared in an anaerobic chamber (Coy Laboratory Products, Ann Arbor, MI, USA). Protein solutions and reagents were incubated under anaerobic conditions over night at 10 °C before the experiments. The 300 μl standard reaction contained 2.5 μM ThNfs1–ThIsd11, 3 μM ThYfh1, 3 μM ThYah1, 0.3 μM human FdxR and 100 μM of either ThIsu1 or CtIsu1 in buffer R (35 mM Tris–HCl, pH 8, 150 mM NaCl, 0.2 mM MgCl₂, 20 μM PLP, 0.5 mM NADPH, 0.5 mM sodium ascorbate, 0.3 mM FeCl₂). The reaction was transferred to a CD cuvette, sealed tightly and placed at 30 °C in a CD spectrophotometer equipped with an automatic stirring device (J-815, Jasco). After 2 min of temperature equilibration the Fe/S-cluster synthesis reaction was initiated by anaerobic addition of 0.5 mM cysteine. The CD signal change at 431 nm was recorded. Subsequently, full spectra were recorded from 300 to 650 nm. Initial rates were estimated by a linear fit to the initial 4.5 min of the reaction. Evaluation of the data were carried out using Origin 8 G software.

Naïve (previously not exposed to antibiotics) or antibiotic-treated (vancomycin and metronidazole in drinking water for a week) mice were euthanized and the cecal contents were collected in water, PBS or solutions of interest at 100 mg/ml. The suspension was first centrifuged at 3,500 g for 10 min and serially filtered through 0.45-μm and 0.22-μm filters. For the study in S1B Fig, the cecal suspension was incubated for 24 h in an anaerobic chamber (Coy Laboratory Products) with 2.8–4.0% hydrogen before filtration. In some experiments, the filtrates were autoclaved through a liquid cycle for 15 min. The inoculum was prepared by diluting a fresh culture of bacteria at late exponential phase (OD₆₀₀ = 0.8–1.0) with PBS and 20 μl of the inoculum (~ 10⁴ CFU) was added to 180 μl of the cecal filtrates on a 96-well plate. For the competitive study, wild type and each mutant strains were mixed at 1:1 (~5 x 10³ CFU of each strain) for inoculation. The plate was incubated at 37°C and the bacterial growth was monitored by track-plating serial dilutions of the cultures on LB agar plates without or with rifampicin (in addition to carbenicillin and neomycin).

The inoculum for batch fermentation was prepared by growing the mutants in MTC medium overnight at 55 °C in an anaerobic chamber (COY Laboratory Products, Grass Lake, MI). The fermentation was carbon limited and carried out in 27 mL Balch tubes with 10 mL of MTC medium containing 5 g L⁻¹ of cellobiose as the carbon source, supplemented with 5 mM sodium acetate where noted, under a N₂ headspace sealed with butyl rubber stoppers. The tubes were inoculated with 0.5% v/v culture and incubated at 55 °C. The fermentation products were determined after 53 h of growth. Final cellobiose concentration was usually <0.5 mM, suggesting that fermentation activity was complete. Fermentations were performed at least two times with three independent biological replicates each. The “No Acetate” data were previously reported [11 (link)], which were generated simultaneously with the “Added Acetate” data reported here.

Clostridium autoethanogenum WT and ΔagrD1D2 cells were grown in duplicate in 750 mL serum flasks containing 400 mL PETC with 50 mM D-fructose. Cells were harvested at early to mid-exponential phase at OD₆₆₀ 0.4–0.45 in an anaerobic chamber (COY laboratory products, USA). Cultures were centrifuged at 3,000 × g for 5 min and following removal of the pellets of each strain were flash frozen in liquid N₂ and stored at -80 °C. Cell pellets were resuspended thoroughly in a combination of 750 μL of BugBuster protein extraction reagent (Merck Millipore, United States) with 750 μL 50 mM TRIS–HCl and 0.5 mM MgCl₂, pH 7.0, this mixture was prepared anaerobically. 10 mg/mL of lysozyme, and 1 mg/mL of DNase (Sigma Aldrich) was added to the mixtures and gently aspirated. The lysis mixture was placed into a pre-evacuated 15 mL Hungate tube and sealed. The tube was placed onto a rocker at room temperature at 50 RPM for one hour. Tubes were placed in the anaerobic chamber and each tube’s mixture was pipetted into a 2 mL cryovial (Sarstedt, Germany) and spun at 20,000×g at 4 °C for 20 min. The clear supernatant was injected into a 5 mL serum flask vacuum filled with pure N₂ and stored on ice ready for analysis. For full specific activity measurement methods followed published procedures^{35 (link),81 (link)}, for full methodology see Supplementary information, Sect. 2.