BACKGROUND: Upon infection of a mammalian host, Bacillus anthracis responds to 
host cues, and particularly to elevated temperature (37°C) and bicarbonate/CO2 
concentrations, with increased expression of virulence factors that include the 
anthrax toxins and extracellular capsular layer. This response requires the 
presence of the pXO1 virulence plasmid-encoded pleiotropic regulator AtxA. To 
better understand the genetic basis of this response, we utilized a controlled 
in vitro system and Next Generation sequencing to determine and compare RNA 
expression profiles of the parental strain and an isogenic AtxA-deficient strain 
in a 2 × 2 factorial design with growth environments containing or lacking 
carbon dioxide.
RESULTS: We found 15 pXO1-encoded genes and 3 chromosomal genes that were 
strongly regulated by the separate or synergistic actions of AtxA and carbon 
dioxide. The majority of the regulated genes responded to both AtxA and carbon 
dioxide rather than to just one of these factors. Interestingly, we identified 
two previously unrecognized small RNAs that are highly expressed under 
physiological carbon dioxide concentrations in an AtxA-dependent manner. 
Expression levels of the two small RNAs were found to be higher than that of any 
other gene differentially expressed in response to these conditions. Secondary 
structure and small RNA-mRNA binding predictions for the two small RNAs suggest 
that they may perform important functions in regulating B. anthracis virulence.
CONCLUSIONS: A majority of genes on the virulence plasmid pXO1 that are 
regulated by the presence of either CO2 or AtxA separately are also regulated 
synergistically in the presence of both. These results also elucidate novel 
pXO1-encoded small RNAs that are associated with virulence conditions.