Journal: Microbiology Spectrum

Article Title: A Third Way of Energy Conservation in Acetogenic Bacteria

doi: 10.1128/spectrum.01385-22

Figure Lengend Snippet: Hydrogen-dependent methylene-THF reduction in cytoplasmic membranes of S. ovata . (A) In-gel assay of hydrogenase activity of solubilized membrane protein. Amounts of 20 μg of solubilized membrane proteins were separated by native PAGE under anoxic conditions. Proteins with hydrogenase activity were stained (red, indicated as S7 [sample 7]) by incubation of the gel with triphenyltetrazolium chloride (0.4 μM) and methyl viologen (0.2 μM) under an atmosphere of 3% hydrogen in the anaerobic chamber before the gel was stained with Coomassie brilliant blue. The protein complex with hydrogenase activity (S7) was analyzed by MALDI-TOF MS (Table S2). (B) Formation of methyl-THF from methylene-THF by the cytoplasmic membrane of S. ovata with H 2 as the electron donor. THF and formaldehyde were mixed in 1.8-mL anoxic glass cuvettes filled with reaction buffer to form methylene-THF. The gas phase was exchanged to 100% H 2 (10 5 Pa overpressure), and the reaction was started by the addition of 83 μg membrane protein. The reaction was stopped at the time points indicated, and the amount of methyl-THF formed was determined by HPLC (black squares). As controls, the assay was performed under 100% N 2 (black triangle) or membrane protein was replaced with cytoplasmic protein (open square).

Article Snippet: NAD(P) + , NAD(P)H, FAD, FMN, methyl viologen, benzyl viologen, methyl-tetrahydrofolate, tetrahydrofolate, formaldehyde, and Tris-ATP were obtained from Sigma-Aldrich Chemie GmbH (Taufkirchen, Germany).

Techniques: Activity Assay, Membrane, Clear Native PAGE, Staining, Incubation