Dl lactate

EC‐Oxyrase stock solution (Sigma #SAE0010, 30 units/ml) was diluted (0–3 μl oxyrase solution in 200 μl final volume, i.e., 0:200, 1:200, 2:200, or 3:200) in culture media supplemented with 10 mM DL‐lactate (Sigma), which serves as the substrate for the oxyrase enzyme. The media were mixed and kept at 37°C for 2 h to ensure sufficient removal of oxygen. The resulting media were added into cells, and the expression of inducible FP was induced at the same time. The same experimental and data analysis procedures as above were then applied for these cells, except that a frame rate of 1 per 10 min was used instead of 1 per 6 min.

Stool samples were suspended 1:1 (w/v) in EBB/T and incubated anaerobically (10% H₂, 10% CO₂, 80% N₂) in a Don Whitley Scientific DG250 Workstation (LA Biosystems, Waalwijk, The Netherlands) at 37 °C with 100 μM sodium 3-(3,4-dihydroxyphenyl)-DL-lactate (39363, Sigma). Samples were taken at 0, 20, and 45 h and analyzed on HPLC-ED as described below.

Strains used in this study are listed in Table S1 in the supplemental material. Strains were cultured routinely on YPD agar (1% yeast extract, 2% Bacto peptone, 2% glucose, and 2% Bacto agar) at 30°C. For β-glucan masking experiments, strains were grown at 30°C, 200 rpm, overnight in yeast nitrogen base without amino acids (BD Difco; 6.7 g/liter) containing the appropriate supplements, plus 2% glucose (YNB-Glu) and then diluted to an OD₆₀₀ of ∼0.1 in fresh YNB-Glu with and without 2% d/l-lactate (Sigma) and grown 5 h at 30°C, 200 rpm. To prepare hypoxic cultures, cells were inoculated into YNB-Glu in a screw-cap conical flask under nitrogen (16 (link)). Cells treated with castanospermine (Cayman Chemical) were grown as described above for β-glucan masking experiments with the addition of castanospermine (in dimethyl sulfoxide [DMSO]) to a final concentration of 1 μM.

For all live-cell fluorescence-imaging experiments, FluoroBrite DMEM (Gibco) with 10% FBS was used and supplemented with a 1:100 ratio of Oxyfluor (Oxyrase, Mansfield, OH, USA) with 10 mM DL-lactate (Sigma-Aldrich, St. Louis, MO, USA) as a substrate to reduce photobleaching and phototoxicity. Cells were imaged with a spinning-disk confocal scan head (Yokogawa CSU-X1, Sugar Land, TX, USA) automated Nikon Ti2 (Melville, NY, USA) microscope frame using a 40X APO-Chromat silicone oil objective (N.A. 1.15) to reduce spherical aberration in 3D. A Lun laser launch provided 405, 442, 488, 514, 561, and 647 laser lines. The primary dichroics (442/514/647and 405/488/568/647) were from Semrock (Rochester, NY, USA). Images were captured using a Prime 95B back-thinned CMOS camera (Photometrics, Tucson, AZ, USA) in 16-bit mode. A motorized Z-piezo stage (PI [Physik Instrumente], Auburn, MA, USA) was used to rapidly capture Z-stacks every 2 μm over a Z-distance of 60 μm. An environmental chamber surrounding the microscope-maintained cells at a constant 37 °C, with 10% CO2 and approximately 50% humidity (Okolab, Sewickley, PA, USA). We used NIS elements to control all hardware.