Molecular Genetics and Pathogenesis of Anthrax

National Institute Of Allergy And Infectious Diseases

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Abstract

Our lab previously demonstrated that the anthrax toxin transcriptional activator AtxA binds directly to the Sigma A-like promoter region of the pagA gene (encoding protective antigen, PA) immediately upstream of the DNA-dependent RNA polymerase (RNAP) binding site (J Bacteriol. 2019 Nov 5;201(23): e00569-19). During this reporting period, using electrophoretic mobility shift assays and in vivo analyses, we confirmed 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 of these newly studied promoters were enhanced in the presence of CO2/bicarbonate and AtxA, as previously seen for the pagA promoter, for which we identified AtxA protein as class I transcriptional activator. 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. The RNAP binding sites of the newly studied promoters show no consensus and differ from the Sigma A-like promoter region of pagA. In silico analysis of the probable AtxA binding sites in the studied promoters revealed several palindromes. Sequence alignment of these palindromes showed some similarity between the lef and cya promoters and greater similarity between the sRNA promoters, excluding XrrC. All the analyzed palindromes showed very little overlap with the palindrome of the Sigma A-like pagA promoter. It remains unclear how AtxA and RNAP identify such diverse DNA-sequences and differentially regulate promoter activation of the studied genes. During the 2023 reporting period, we also started to study the RNAP of B. anthracis. The core polymerase and its sigma A subunit were each expressed in Escherichia coli. The isolated RNAP contained all expected subunits and displayed activity as assessed by synthesis of a fluorogenic RNA aptamer. This work provides a basis for later work to obtain a cryo-EM structure of B. anthracis RNAP and for identifying of its transcriptional activation by AtxA. Also, we have purified Bacillus subtilis and E. coli RNAPs to use as controls for the newly isolated RNAP of B. anthracis.