FIG 3.
The C. burnetii dot/icm locus is regulated by PmrA. (A) Linkage and predicted operon structures of C. burnetii dot/icm genes. Operons with upstream PmrA regulatory elements are colored. Operon-specific promoters are indicated with an arrow below the first gene of each predicted operon. icmF is truncated due to a frameshift (icmFFS). Antibodies specific for proteins encoded by boldface genes were used in immunoblotting, described below. (B) Luciferase activities of the lux operon transcriptionally fused to the promoter regions of control genes (left) and dot/icm genes (right). Assays were conducted after 4 days of growth in axenic medium of wild-type C. burnetii and the ΔpmrA mutant expressing lux fusions. Bioluminescent readings are expressed as relative light units (RLU). Results are expressed as the means of results from two biological replicates from three independent experiments. Error bars indicate the standard deviations from the means, and asterisks indicate a statistically significant difference (P < 0.0001) between wild-type C. burnetii and the ΔpmrA mutant. (C) Immunoblots of lysates of wild-type C. burnetii (lane 1), the ΔpmrA mutant (lane 2), and the complemented mutant (lane 3) probed with anti-IcmD (αIcmD), -IcmK, and -DotA antibodies. Probing for EF-Ts was conducted as a loading control.