Gemini fluorescent plate reader

Effect of DMB on the PMF was measured by an EtBr accumulation assay as outlined elsewhere [29 (link)]. Strain JE9426 (wild-type) was grown overnight (∼20 h) in LB and sub-cultured into (5% v/v) NCE minimal medium as described under culture media and growth studies section). Cultures were grown with shaking (180 rpm) at 37°C to an optical density at 600 nm of ∼0.5 and treated for 15 min with various concentrations of DMB or other bases under the same conditions. Carbonyl cyanide m-chlorophenylhydrazone (CCCP; 50 μM final) was used as positive control for the assay. Cultures (200 μL) were transferred into a black, round-bottom 96-well microtiter plate, and EtBr was added to a final concentration of 6.25 μM. Relative fluorescence was monitored immediately after addition of EtBr using a BioTek Gemini fluorescent plate reader for 4 min at 530 nm and 600 nm excitation and emission wavelengths, respectively. The rate of relative fluorescence was determined by simple linear regression and significance was determined by paired t test using Prism (GraphPad, version 9) software.

The effect of CobS overproduction and CobC and CobS coexpression on membrane permeability was examined by a modified ethidium bromide accumulation assay as outlined elsewhere (48 (link)). Briefly, starter cultures of E. coli C41(λDE3) harboring empty cloning vector or plasmids encoding CobS^WT or CobS^D82A alone or both CobC^WT and CobS^WT proteins were grown overnight in LB containing antibiotic at 37°C with shaking at 150 rpm. Starter cultures were subcultured (1% [vol/vol] inoculum) into 198 μL of LB plus antibiotic in a 96-well microtiter plate (Falcon) and incubated in a plate reader (BioTek EON) at 37°C with orbital shaking. At an OD₆₃₀ of ∼0.4, IPTG was added to a final concentration 0, 0.5, or 1 mM to induce expression of cobS⁺ alone or both cobS⁺ and cobC⁺. Cells were incubated for 30 min as described above. Cultures (150 μL) were transferred into the wells of a black, round-bottom 96-well microtiter plate, and ethidium bromide was added to a final concentration of 6.25 μM. Relative fluorescence (excitation, 530 nm; emission, 600 nm) was monitored immediately upon addition of ethidium bromide using a BioTek Gemini fluorescent plate reader for 180 s. The rate of relative fluorescence (relative fluorescence units [RFU]/s) was determined by linear regression. Significance was determined by unpaired Student's t test using Prism version 8 (GraphPad) software.