Anthrax toxin gene expression in Bacillus anthracis is dependent on the presence 
of atxA, a trans-acting regulatory gene located on the resident 185-kb plasmid 
pXO1. In atxA+ strains, expression of the toxin genes (pag, lef, and cya) is 
enhanced by two physiologically significant signals: elevated CO2/bicarbonate 
and temperature. To determine whether increased toxin gene expression in 
response to these signals is associated with increased atxA expression, we 
monitored steady-state levels of atxA mRNA and AtxA protein in cells cultured in 
different conditions. We purified histidine-tagged AtxA [AtxA(His)] from 
Escherichia coli and used anti-AtxA(His) serum to detect AtxA in protein 
preparations from B. anthracis cells. AtxA was identified as a protein with an 
apparent size of 56 kDa in cytoplasmic fractions of B. anthracis cells. Our data 
indicate that atxA expression is not influenced by CO2/bicarbonate levels. 
However, the steady-state level of atxA mRNA in cells grown in elevated 
CO2/bicarbonate at 37 degrees C is five- to sixfold higher than that observed in 
cells grown in the same conditions at 28 degrees C. A corresponding difference 
in AtxA protein was also seen at the different growth temperatures. When atxA 
was cloned on a multicopy plasmid in B. anthracis, AtxA levels corresponding to 
the atxA gene copy number were observed. However, this strain produced 
significantly less pag mRNA and protective antigen protein than the parental 
strain harboring atxA in single copy on pXO1. These results indicate that 
increased AtxA expression does not lead to a corresponding increase in pag 
expression. Our data strongly suggest that an additional factor(s) is involved 
in regulation of pag and that the relative amounts of such a factor(s) and AtxA 
are important for optimal toxin gene expression.