FIG 3.

(A) Growth curves of the resT knockout mutant GCB2127. B. burgdorferi cultures were grown in BSK II medium with and without IPTG at 35°C, and spirochetes were counted every 24 h. After 48 h, the cultures were diluted with an equal volume of medium to avoid entry into stationary phase and were counted before and after dilution at the 48-h time point. Growth of the wild type, B31-A (GCB908), was compared to growth of the resT-inducible strain (GCB2127). (B) Spirochetes from cultures at the indicated times following IPTG washout were stained with the LIVE/DEAD BacLight staining kit (Invitrogen) to determine if cells were alive or dead. Spirochetes were analyzed by fluorescence microscopy for staining with Syto-9 (live) or propidium iodide (dead). Ten fields of view were analyzed for each time point. Living spirochetes were also enumerated by dilution in duplicate of the cultures and plating in BSK II medium plus IPTG in 96-well microtiter plates. The percent living cells for counting was based upon the total number of spirochetes counted by dark-field microscopy. At each time point, the mean percentage of living cells ± the standard error is plotted for both the LIVE/DEAD staining and plating methods.