Liquid medium growth and membrane depolarization assays in Clavibacter michiganensis

The antimicrobial activity of SAGs was evaluated against the bacterial strain C. michiganensis subsp. sepedonicus C5 growing in liquid LB medium at 120 rpm and 26°C with an initial OD₆₀₀ adjusted to 0.1 (7 log colony forming unit per ml) and OD₆₀₀ measurements taken every half-hour. SAGs were added to growing bacteria after 1.5 hours of growth to a final concentration of 60 μg/ml. Membrane depolarization of C. michiganensis was monitored as changes in fluorescence emission intensity of the membrane-potential-sensitive dye diSC3^5,49. C. michiganensis was grown in LB liquid medium to mid log phase growth (OD₆₀₀ = 0.5) where after cells were harvested by centrifugation and washed once (5 mM glucose and 20 mM HEPES pH 7.3) and resuspended to a concentration of OD₆₀₀ of 0.05 in the same buffer. Aliquots of 100 µl were placed into quartz cuvettes containing 2.0 ml of 5 mM glucose, 100 mM KCl and 5 mM Na HEPES buffer pH 7.2. After addition of 0.4 µM diSC3⁵, cuvettes containing bacterial cells were incubated at 26°C until a stable reduction of fluorescence (around 5 min), indicating incorporation of the dye into the bacterial membrane, was achieved. SAGs (60 μg/ml), Valinomycin (1 μM plus 100 mM KCl) or water were thereafter added and the dye fluorescence increase was recorded at 622 nm (excitation wavelength) and 670 nm (emission wavelength) with a RF-5301PC spectrofluorometer (Shimadzu, Kyoto, Japan) at 0, 0.5, 1, 2 and 4 min. The antibiotic Valinomycin, a well-known K⁺-selective ionophore that changes the bacterial membrane potential, was used as positive membrane-destabilizing control. A SAGs dose-response assay on membrane permeability of C. michiganensis was performed measuring the fluorescence after 1 minute at different concentrations (0, 7, 15, 30, 60 and 90 μg/ml) of SAGs. Experiments were repeated at least three times under identical experimental conditions.