Survival of <i>B</i>. <i>burgdorferi</i> during starvation conditions <i>in vitro</i> depends on <i>rel</i>_Bbu.

<p>Strains were grown to late log phase in BSK + RS (0 h) before shifting to RPMI for 24 h or 72 h. Wild-type (WT), <i>rel</i>_Bbu mutant (<i>rel</i>_Bbu^<i>-</i>), and <i>rel</i>_Bbu^<i>-</i> pBS-<i>flacp</i>-<i>rel</i>_Bbu cells were stained with PI and visualized by DIC and fluorescence microscopy. Images are overlays of DIC and fluorescence images with PI shown in blue. Arrowheads are RBs that did not stain with PI and arrows point to PI-stained RBs. Scale bar = 10 μm. (B) The percentage of live spirochetes as quantified by PI staining following incubation in RPMI for various times (24 h to 96 h). Black squares represent WT in BSK + RS, black circles represent WT in RPMI, white squares represent <i>rel</i>_Bbu^- in BSK + RS, white circles represent <i>rel</i>_Bbu^- in RPMI, gray squares represent <i>rel</i>_Bbu^<i>-</i> pBS-<i>flacp</i>-<i>rel</i>_Bbu in BSK + RS, and gray circles represent <i>rel</i>_Bbu^<i>-</i> pBS-<i>flacp</i>-<i>rel</i>_Bbu in RPMI. Values are the means of three independent experiments with at least 100 spirochetes counted for each time point and error bars represent SEM. (C) Wild-type (black bars), <i>rel</i>_Bbu^- (hatched bars) and <i>rel</i>_Bbu^- pBS-<i>rel</i>_Bbu (gray bars) strains were grown in BSK + RS to late log phase before shifting to RPMI. To quantify the number of live <i>B</i>. <i>burgdorferi</i> at the time points indicated, samples of each culture were plated in semi-solid BSK, allowed to grow for two weeks and colonies enumerated to yield colony forming units (CFUs). Each value represents the mean of three independent experiments normalized to the initial number of colonies before shifting to RPMI (0 h). Error bars represent SEM.