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P. gingivalis [WT (ATCCBAA-308) and KgPΔIg-B] was inoculated from stocks into 20 ml of prereduced modified TSB medium [TSB + yeast extract (5 mg/ml), l-cysteine (0.5 mg/ml), hemin (5 μg/ml), and vitamin K1 (1 μg/ml)] and incubated at 37°C for 48 hours anaerobically in a Coy type C vinyl chamber. Prereduction of all solutions used was done by transferring the liquids to an anaerobic chamber for >16 hours immediately after autoclaving. On the day of the experiment, the primary culture was diluted to obtain OD of 0.2 to 0.25 using a Siemens MicroScan turbidity meter in the prereduced modified TSB medium and incubated for 6 hours to reach log phase (OD of approximately 0.5 to 0.6) at 37°C. Then, the bacteria were collected by centrifuging at 4000 rpm for 10 min and washed. Pellets were diluted to 3 × 108 to 5 × 108 CFU/ml, and 10 ml of these diluted cultures was transferred into conical tubes and centrifuged. The resultant pellet was resuspended using 10 ml of defined medium to assess growth. The defined medium consists of the following: salt base supplement [10.0 mM NaH2PO4, 10.0 mM KCI, 2 mM citric acid, 1.25 mM MgCl2, 20.0 μM CaCl2, 0.1 μM Na2MoO4, 25.0 μM ZnCl2, 50.0 μM MnCl2, 5.0 μM CuCl2, 10.0 μM CoCl2, and 5.0 μM H3BO3 (pH 7.0)] with 20 mM α-ketoglutarate, 3% BSA, hemin (5 μg/ml), and vitamin K (1 μg/ml). Fifty microliters of 100× COR388 stock prepared in DMSO was added to the bacterial suspension (3 × 108 to 5 × 108 CFU/ml) for each strain with a final concentration of 500 nM. Vehicle cultures were treated with 0.1% DMSO. The bacteria were incubated at 37°C in the anaerobic chamber, and OD was measured at 0, 21, and 43 hours to generate a time course of culture growth.

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