Structural and Functional Analysis of Toxin and Small RNA Gene Promoter Regions in Bacillus anthracis

It was previously demonstrated that anthrax toxin activator (AtxA) binds directly to the σ^A-like promoter region of pagA (encoding protective antigen, PA) immediately upstream of the RNA polymerase binding site. In this study, using electrophoretic mobility shift assays and in vivo analyses, we identified AtxA-binding sites in the promoter regions of the lef and cya genes (encoding lethal and edema factors, respectively) and of two Bacillus anthracis small RNAs (XrrA and XrrB). Activities of all four newly studied promoters were enhanced in the presence of CO₂/bicarbonate and AtxA, as previously seen for the pagA promoter. Notably, the cya promoter was less activated by AtxA and CO₂/bicarbonate conditions. The putative promoter of a recently described third small RNA, XrrC, showed a negligible response to AtxA and CO₂/bicarbonate. RNA polymerase binding sites of the newly studied promoters show no consensus and differ from the σ^A-like promoter region of pagA. In silico analysis of the probable AtxA binding sites in the studied promoters revealed several palindromes. All the analyzed palindromes showed very little overlap with the σ^A-like pagA promoter. It remains unclear as to how AtxA and DNA-dependent RNA-polymerase identify such diverse DNA-sequences and differentially regulate promoter activation of the studied genes. IMPORTANCE Anthrax toxin activator (AtxA) is the major virulence regulator of Bacillus anthracis, the causative agent of anthrax. Understanding AtxA's mechanism of regulation could facilitate the development of therapeutics for B. anthracis infection. We provide evidence that AtxA binds to the promoters of the cya, lef, xrrA, and xrrB genes. In vivo assays confirmed the activities of all four promoters were enhanced in the presence of AtxA and CO₂/bicarbonate, as previously seen for the pagA promoter. The cya and lef genes encode important toxin components. The xrrA and xrrB genes encode sRNAs with a suggested function as cell physiology regulators. Our data provides further evidence for the direct regulatory role of AtxA that was previously shown with the pagA promoter.