The Bacillus anthracis toxin genes, cya, lef, and pag, can be viewed as a 
regulon, in which transcription of all three genes is activated in trans by the 
same regulatory gene, atxA, in response to the same signal, CO2. In atxA+ 
strains, toxin gene expression is increased 5- to 20-fold in cells grown in 5% 
CO2 relative to cells grown in air. CO2-enhanced toxin gene transcription is not 
observed in atx4-null mutants. Here, we used two independent techniques to 
obtain evidence for additional CO2-induced atxA-regulated genes. First, total 
protein preparations from atxA4+ and atxA isolates grown in 5% CO2 and in air 
were examined by two-dimensional electrophoresis. Comparison of the resulting 
protein patterns indicated that synthesis of non-toxin proteins is influenced by 
growth in elevated CO2 and the toxin gene regulator, atxA. Second, we generated 
random transcriptional lacZ fusions in B. anthracis with transposon Tn917-LTV3. 
Transposon-insertion libraries were screened for mutants expressing CO2-enhanced 
atxA-dependent beta-galactosidase activity. DNA sequence analysis of transposon 
insertion sites in 17 mutants carrying CO2- and atxA-regulated fusions revealed 
10 mutants carrying independent insertions on the 185-kb toxin plasmid pXO1 
which did not map to the toxin genes. The tcr-lacZ fusion mutants (tcr for toxin 
coregulated) were Tox+, indicating that these genes may not be involved in 
anthrax toxin gene activation. Our data indicate a clear association of atxA 
with CO2-enhanced gene expression in B. anthracis and provide evidence that atxA 
regulates genes other than the structural genes for the anthrax toxin proteins.