Bacillus anthracis plasmid pXO1 carries genes for three anthrax toxin proteins, pag (protective antigen), cya (edema factor), and lef (lethal factor). Expression of the toxin genes is enhanced by two signals: CO2/bicarbonate and temperature. The CO2/bicarbonate effect requires the presence of pXO1. I hypothesized that pXO1 harbors a trans-acting regulatory gene(s) required for CO2/bicarbonate-enhanced expression of the toxin genes. Characterization of such a gene(s) will lead to increased understanding of the mechanisms by which B. anthracis senses and responds to host environments. A regulatory gene (atxA) on pXO1 was identified. Transcription of all three toxin genes is decreased in an atxA-null mutant. There are two transcriptional start sites for pag. Transcription from the major site, P1, is enhanced in elevated CO2. Only P1 transcripts are significantly decreased in the atxA mutant. Deletion analysis of the pag upstream region indicates that the 111-bp region upstream of theP1 site is sufficient for atxA-mediated increase of this transcript. The cya and lef genes each have one apparent transcriptional start site. The cya and lef transcripts are significantly decreased in the atxA mutant. The atxA mutant is avirulent in mice. The antibody response to all three toxin proteins is significantly decreased in atxA mutant-infected mice. These data suggest that the atxA gene product activates expression of the toxin genes and is essential for virulence. Since expression of the toxin genes is dependent on atxA, whether increased toxin gene expression in response to CO2/bicarbonate and temperature is associated with increased atxA expression was investigated. I monitored steady state levels of atxA mRNA and AtxA protein in different growth conditions. The results indicate that expression of atxA is not influenced by CO2/bicarbonates. However, the levels of atxA mRNA and AtxA protein are higher at 37¡C than 28¡C.But, increased atxA expression does not directly attribute to increased pag expression at high temperature. Expression of pag in strains overproducing AtxA is significantly decreased compared to a wildtype strain. Moreover, recombinant AtxA does not bind to the pag upstream DNA. These data suggest that an additional factor(s) is involved in regulation of pag, and that the relative amounts of such a factor(s) and AtxA is important for optimal pag expression. Four proteins in B. anthracis extract were co-immunoprecipitated with tagged AtxA. Amino-terminal sequence of one protein has been determined and found highly homologous to chaperonins of GroEL family. Studies are under way to examine the role of this GroEL-like protein in regulation of toxin genes.

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Regulatory Factors and Control of Anthrax Toxin Gene Expression