The incubation temperature was set to 37 °C. As positive controls for cell surface exposure, strains JG137 and JG138, producing OmcAstrep and MtrCstrep, were chosen; as a control for OM integrity under the incubation conditions, the periplasmic c-type cytochrome MtrA containing an N-terminal strep-tag (MtrAstrep) was Copanlisib mouse produced in an S. oneidensisΔmtrA background (JG50) (Schuetz et al., 2009). Cells were grown anaerobically overnight in minimal media with fumarate as an electron acceptor. At an OD578 nm of ∼0.2, 0.1 mM arabinose

was added to induce OM cytochrome and MtrA/MtrB production. After 4 h of production, cells were harvested and washed twice with mineral media without fumarate and lactate and then resuspended in HEPES buffer (100 mM, pH 7.5) containing 50 μM MgCl2 to obtain a final OD578 nm between 3 and 5. All further measurements were performed in independent duplicates in an anaerobic glove box. Specific reduction rates were obtained by normalization to the protein content of the cell suspension. Fifty microliters of the cell suspension was pipetted in a well of a microtiter plate. The assay was started through the addition of 150 μL of a solution containing 10 mM lactate and 10 mM ferric citrate. At different time points (0–30 min), the reaction was stopped by

the addition of 100 μL 3 M HCl. The Fe2+ concentration of the samples was determined using the ferrozine reagent (Viollier et al., 2000). The MFC setup used in this study features an anode I-BET-762 clinical trial and cathode chamber with a working volume of 8 mL each, separated by a Nafion-117 membrane (Quintech, Göppingen; Kloke et al., 2010). A saturated calomel reference electrode (SCE) was Thiamine-diphosphate kinase separated from the anode compartment by another Nafion membrane. Electrodes were made of graphite felt cubes (Alpha Aesar, Karlsruhe) connected to platinum wires (0.1 mm; Chempur, Karlsruhe). The anode compartment was filled with 5.5 mL mineral media containing 50 mM lactate and 0.1 mM arabinose. Five hundred microliters of a cell suspension with an OD578 nm of 4 was added to start the experiment. All MFC

experiments were performed in duplicate and conducted at a constant temperature of 30 °C. The whole setup was connected to a potentiostat (Pine Instruments, Grove City). The standard measurement protocol consisted of two phases: after a conditioning period with a constant current flux over 5 h (0.3 μA cm−3), MFC cultures were subjected to a continuous increase in current density at a rate of 1.1 μA cm−3 h−1 over 45 h (current sweep phase). The anode compartment was continuously flushed with nitrogen gas to maintain anoxic conditions. Additional terminal electron acceptors were not added. A markerless multideletion mutant in all annotated OM cytochromes of S. oneidensis was constructed to generate a strain platform that allows for analysis of OM cytochrome activity without the potential detection of redundant activities from similar proteins.