Cloning and characterization of a gene whose product is a trans-activator of anthracis toxin synthesis

The 184-kb Bacilus anthracis plasmid pXO0, which is required for virulence, contains three genes encoding the protein components of anthrax toxin, cya (edema factor gene), kef (lethal factor gene), and pag (protective antigen gene). Expression of the three proteins is induced by bicarbonate or serum. Using a pagklacZ transcriptional construct to measure pag promoter activity, we cloned in BaciUus subtilis a gene (alxA) whose product acts in trans to stimulate anthrax toxin expression. Deletion analysis located aIxA on a 2.0-kb fragment between cya and pag. DNA sequencing identified one open reading frame encoding 476 amino acids with a predicted Mr of 55,673, in good agreement with the value of 53 kDa obtained by in vitro transcription-translation analysis. The cloned airA gene complemented previously characterized Tn917 insertion mutants UM23 tp29 and UM23 tp32 (J. M. Hornung and C. B. Thorne, Abstr. 91st Gen. Meet. Am. Soc. Microbiol. 1991, abstr. D-121, p. 98), which are deficient in synthesis of all three toxin proteins. These results demonstrate that the atxA product activates not only transcription of pag but also that of cya and lef. 3-Galactosidase synthesis from the pag-lacZ transcriptional fusion construct introduced into an insertion mutant (UM23 tp62) which does not require bicarbonate for toxin synthesis indicated that additional regulatory genes other than atxA play a role in the induction of anthrax toxin gene expression by bicarbonate. Bacillus anthracis causes the highly infectious diseas