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 transactivator of 
anthrax toxin synthesis. Here we show that transcription of atxA 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 
atxA-null mutant in which the atxA-coding sequence on pXO1 is replaced with an 
omega km-2 cassette. Transcription of all three toxin genes is decreased in the 
absence of atxA. The pag gene possesses two apparent transcription start sites, 
P1 and P2; only transcripts with 5' ends mapping to P1 are decreased in the 
atxA-null mutant. Deletion analysis of the pag promoter region indicates that 
the 111 bp region upstream of the P1 site is sufficient for atxA-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 atxA-null mutant. The atxA-null mutant is avirulent in 
mice. Moreover, the antibody response to all three toxin proteins is decreased 
significantly in atxA-null mutant-infected mice. These data suggest that the 
atxA gene product also regulates toxin gene expression during infection.