The atxA gene product activates transcription of the anthrax toxin genes and is essential for virulence

Summary Bacillus anthracis plasmid pXO1 carries the structural genes for the three anthrax toxin proteins, cya (edema factor), lef (lethal factor), and pag (protective antigen). Expression of the toxin genes by B. anthracis is enhanced during growth under elevated levels of CO2. This CO2 effect is observed only in the presence of another pXO1 gene, atxA, which encodes a trans‐activator of anthrax toxin synthesis. Here we show that transcription of atx A does not appear to differ in cells grown in 5% CO2 compared with cells grown in air. Using a new efficient method for gene replacement in B. anthracis, we constructed an atx A‐null mutant in which the atx A‐coding sequence on pXO1 is replaced with an ωkm‐2 cassette. Transcription of all three toxin genes is decreased in the absence of atx A. The pag gene possesses two apparent transcription start sites, P1 and P2; only transcripts with 5′ ends mapping to P1 are decreased in the atx A‐null mutant. Deletion analysis of the pag promoter region indicates that the 111 bp region upstream of the Pi site is sufficient for atx A‐mediated activation of this transcript. The cya and lef genes each have one apparent start site for transcription. Transcripts with 5′ ends mapping to these sites are not detected in the afxA‐null mutant. The atx A‐null mutant is avirulent in mice. Moreover, the antibody response to all three toxin proteins is decreased significantly in atx A‐null mutant‐infected mice. These data suggest that the atx A gene product also regulates toxin gene expression during infection.