FIGURE 2.

Membrane-permeable acids activate transcription of RpoS-dependent virulence factors and the oxidative stress regulon. Gene expression and immunoblot analysis of B. burgdorferi strain B31-A3 in response to membrane-permeable acids. (A) qRT-PCR analysis of key regulators of the Rrp2–RpoN–RpoS/BosR signal transduction cascade, rrp2, rpoN, rpoS, and bosR. (B) B. burgdorferi strain B31-A3/pBSV2G-rpoSp-lacZ_Bb containing a shuttle vector with the rpoS promoter fused to lacZ_Bb was grown in the (–) absence or (+) presence of acetate, benzoate, lactate, or propionate and β-galactosidase activity in cell lysates was analyzed. Brackets with asterisks indicate a statistically significant difference between the means with p-values <0.05 determined using a two-way ANOVA. (C) Immunoblot analysis of RpoS, BosR, and the BosR-regulated factor NapA/BB690 in B. burgdorferi in response to benzoate. Flagellin is included as a loading control and the position of molecular weight standards is indicated on the right in kDa. (D) qRT-PCR analysis of RpoS-dependent genes ospC, bba66, bba34, bbj24 and BosR-regulated genes napA/bb690 and cdr/bb728. Expression levels were normalized to the eno gene encoding enolase and are represented as transcript copies per 100 eno copies. Asterisks denote a statistically significant difference of the mean determined using a two-way ANOVA with p < 0.05. All experiments were performed using at least three biological replicates with the mean and standard deviation displayed in (A,B,D) and a representative immunoblot presented in (C).