The anthrax toxin activator gene atxA is associated with CO2-enhanced non-toxin gene expression in Bacillus anthracis

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 tl ans 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 atxA-null mutants. Here, we used two independent techniques to obtain evidence for additional CO2-induced atxA-regulated genes, First, total protein preparations from atxA(+) 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 (fcr 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 atx4 regulates genes other than the structural genes for the anthrax toxin proteins.