Oxyrase

Single U2OS cells stably expressing GFP-tagged G3BP1 (Pelkmans Lab^{64 (link)}) were plated on patterned coverslips following the protocol mentioned above. After spreading for 3–4 h, the cells were switched into media containing 100 nM SiR-tubulin (Spirochrome) and 10 μM verapamil (Spirochrome) and incubated in the dye for at least 4 h. The cells were imaged at 37 °C in imaging media containing Leibovitz’s L-15 medium (ThermoFisher Scientific) with 10% fetal bovine serum, 30 μl ml^–1 Oxyrase (Sigma-Aldrich) and 10 mM lactic acid, with 0.5 mM sodium arsenite (Sigma-Aldrich). Imaging was done on a Nikon Ti2 Eclipse epifluorescent microscope with a cage incubator (Okolab), using ×100 oil objective with numerical aperture (NA) of 1.49 and a Nikon DS-Qi2 camera.

The preparation of the faecal inoculum and the batch fermentation assay were carried out as described previously [37 (link)]. Briefly, faeces from 29 healthy newly weaned crossbred pigs (Large White × Landrace) fed a cereal- and milk-based diet were pooled, aliquoted and stored at −20 °C. One day prior to the batch fermentation assay, the pooled faeces were diluted (1:5 w/v) in phosphate-buffered saline (Sigma-Aldrich, St. Louis, MO, USA) after oxygen removal using oxyrase (Sigma-Aldrich, St. Louis, MO, USA) to prepare the faecal inoculum (FI) that was stored at 4 °C anaerobically. The FI was added to the fermentation medium at a 1:10 v/v ratio (21 mL final volume). The inclusion levels of LDWB-F, LHWB-F, LDWB-N and LHWB-N in the FI/fermentation medium were 0 (control tubes), 1, 2.5 and 5 mg/mL. The batch fermentation was carried out under anaerobic conditions using oxyrase and CO₂ flushing at 39 °C for 24 h with gentle stirring (100 rpm). Sampling (5 mL fermentation broth) was performed at 0 and 24 h in duplicate. After centrifuging at 12,000× g for 5 min, the resultant pellets were stored in −20 °C until further analysis. All experiments were repeated on three independent occasions (biological replicates n = 3).

Bifidobacterium longum subsp. longum Reuter (B. longum, ATCC #15707) was cultured under anaerobic conditions in reinforced clostridial media containing 9% Oxyrase (Sigma‐Aldrich #SAE0013) at 37°C for approximately 24 h. Each day prior to gavage, the bacteria were centrifuged at 3000 × g, media aspirated, and washed in sterile PBS for a total of two times. The pellet was resuspended in sterile PBS and used for gavage.

Cells were plated onto two-well Labtek chambers the day before imaging. Live-cell imaging was performed at 37°C in a CO₂-independent medium (Life Technologies) supplemented with 20% vol/vol fetal bovine serum, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2 mM glutamine. To minimize photodamage, maximum light deposition of sample was kept <5 J/cm², and O₂ concentration in the imaging medium was lowered to ∼5% by using the Oxy Fluor enzymatic system (Oxyrase Inc). To this end, imaging medium was supplemented with Oxyrase (c_final = 0.3 U/ml) and sodium lactate (c_final = 10 mM, Sigma-Aldrich). O₂ concentration was monitored with a FireStingO₂ fiber-optic oxygen meter with a retractable needle tip (PyroScience GmbH). Cells were incubated for at least 30 min before imaging to allow O₂ to reach ∼5% and imaged up to 6 h.
Imaging conditions for the different fluorescent probes were as follows: LAP excitation at 488 nm, GaAsP emission collected between 490 and 550 nm; ATTO 633 excitation at 633 nm, emission long-pass filter 655 nm, and APD detection; ATTO 565 excitation at 561 nm, emission long-pass filter 545 nm, and APD detection; image voxel size x, y, z 90 × 90 × 400 nm, pixel dwell 8.2 µs, no averaging.

For transient reporter assays, constructs with the nopaline synthase (NOS) promoter driving the expression of the 5′UTR of ADH1 fused with the firefly luciferase (Luc) coding region were made using the pGreenll-0800 Luc plasmid (47 (link)), which also contains the 35S promoter driving Renilla luciferase (Ren) expression. The luciferase activity assays were performed following the instructions of the Dual Luciferase Reporter Assay System (Promega). The Luc/Ren ratio of each sample was calculated. For low oxygen treatment, 1:10 (v/v) Oxyrase (Sigma-Aldrich) was added after protoplast transformation and protoplasts were incubated overnight. For the quantification of reporter expression, reverse transcription and quantitative reverse transcription PCR were performed as previously described (44 (link)). The primers for the genes analyzed are listed in table S2.

Mutant or wild-type strains, with the pGLOW reporter, were grown in EVCC + Aviguard. Oxyrase (Sigma-Aldrich; St. Louis, MO) was added to the medium (1:20 final dilution) and overlaid with mineral oil to create low oxygen conditions. Salmonella starting cell density was 10⁵ CFU/mL. The lyophilized Aviguard was reconstituted as recommended by the manufacturer (Lallemand Animal Nutrition) in sterile saline with Oxyrase and used to inoculate EVCC cecal medium (1:20 dilution) (7.99 Log₁₀ viable cells/mL). The viability of the rehydrated Aviguard was determined microscopically using LIVE/DEAD BacLight stain (Molecular Probes). Sterile 96-well, black-walled polystyrene microtiter plates with a clear bottom (Fisher Scientific) were used to monitor Salmonella fluorescence with a BioTek Synergy HT 96-well fluorescence microtiter plate reader at 37°C. A filter was added to the BioTek Synergy HT fluorescent plate reader (BioTek; Winooski, VT) to record pGLOW fluorescence based on its excitation frequency (Drepper et al., 2007 (link)). Fluorescence was recorded every 30 min. Strains were run in triplicate.