Figure 5.

The CXXC motifs are essential for BosR to activate rpoS expression. To mutate the CXXC motifs, specific point mutation was introduced into bosR in pOY152. The resultant shuttle vectors were introduced into the bosR mutant OY08. Spirochetes grown in BSK-II medium containing varying concentrations of IPTG were harvested at late log-phase (~10⁸ cells ml⁻¹). B. burgdorferi whole cell lysates were analyzed by immunoblot. Approximately 4 × 10⁷ spirochetes were loaded onto each lane. Concentrations of IPTG and BosR mutant names (e.g., C114S, etc.) are indicated above the image. Specific antibodies, denoted as α-, are indicated on the left. FlaB was used as a normalization control for equivalent loading. For detection of FlaB, RpoS, and OspC, cell lysate from WT stain 297 was included as a control. Immunoblots for BosR, RpoS, and FlaB detection were developed by chemiluminescence, whereas immunoblots for OspC detection were developed colorimetrically. Shown are representative images from 2 to 4 independent experiments with at least two biological replicates.