Identification and characterization of RgtA sRNA in B. pertussis. (A) Visualization of the dRNA-seq normalized data set (±treatment with terminal exonuclease) showing the region between the BP2735 and BP2736 genes. Graphs display sequencing depth of the positive (dark red) and negative (light red) strands. (The different color intensities depict the different library replicates.) Gene annotations are depicted as green arrows. Red bar above the picture denotes the genomic position of RgtA RNA. (B) Detection of RgtA by northern blot analysis. Total RNA isolated from B. pertussis Tohama I (lane 2) and ΔrgtA (lane 3) strains was separated on 8% PAA-8M urea gel, transferred to membrane, and probed with biotinylated oligo specific to RgtA. Biotinylated Century-Plus RNA ladder was loaded as a molecular size marker (lane 1). (C) Determination of the transcriptional start site of RgtA by primer extension analysis. The product of the primer extension reaction (PE, lane 5) was separated together with sequencing reactions performed with the primer used for primer extension (lanes 1–4) on a 6% polyacrylamide–8 M urea gel. Only the relevant part of the autoradiograph is presented. The sequence of the coding strand is shown on the left with the transcriptional start site (asterisk) and the plausible −10 sequence (vertical line). (D) Nucleotide sequence map of the rgtA locus. The transcriptional start site identified by primer extension and dRNA-seq analysis (rectangular arrow, +1), plausible −35 and −10 promoter sequences (underlined), putative RisA binding sites (gray boxes), and two possible terminators (convergent arrows) are depicted.
