Oleic acid

The WT and ΔfakA mutant strains were grown to mid-exponential and stationary phase in 5 mL of TSB at 37 °C with shaking in Falcon tubes. Each strain was grown in the presence and absence of 20% human serum (v/v) or the fatty acid mix: oleic acid (18:1), linoleic acid (18:2), and arachidonic acid (20:4) (Nu-Chek Prep, Inc., Elysian, MN), each at a final concentration of 100 μM. Cultures were pelleted by centrifugation, washed, and resuspended in normal saline at a McFarland reading of 0.9. Cell membrane fluidity was measured by polarizing spectrofluorometry using a BioTek Synergy H1 plate reader (BioTek Instruments, Winooski, VT) with the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH).

The WT and ΔfakA mutant strain were grown to stationary phase in 7 mL of MHB50 at 37 °C with shaking for 24 hours in Falcon tubes. Both strains were grown in the presence and absence of the fatty acid mix: oleic acid (18:1), linoleic acid (18:2), and arachidonic acid (20:4) (Nu-Chek Prep, Inc., Elysian, MN), each at a final concentration of 100 μM. Cultures were pelleted by centrifugation, resuspended in 7 mL MHB50 containing the fluorogenic dye, 2’,7’-dichlorodihydrofluorescein diacetate (H₂DCFDA) at a concentration of 10 μM, and incubated for 45 minutes at 37°C protected from light. Cultures were pelleted by centrifugation, washed with saline, and resuspended in 7 mL of MHB50. Cells were added in triplicate to a black Nunc 96-well flat-bottom microplate in the presence or absence of the fatty acid mix with a final volume of 200 μL. Reactive oxygen species were measured by fluorescence readings (λ excitation=485 nm, λ emission=535 nm) using a BioTek Synergy H1 plate reader set at 37 °C for 8 hours.