CLONING AND CHARACTERIZATION OF A GENE WHOSE PRODUCT IS A TRANSACTIVATOR OF ANTHRAX TOXIN SYNTHESIS

The 184-kb Bacillus anthracis plasmid pXO1, which is required for viru lence, contains three genes encoding the protein components of anthrax toxin, cya (edema factor gene), lef (lethal factor gene), and pag (pr otective antigen gene). Expression of the three proteins is induced by bicarbonate or serum. Using a pag-lacZ transcriptional construct to m easure pag promoter activity, we cloned in Bacillus subtilis a gene (a txA) whose product acts in trans to stimulate anthrax toxin expression . Deletion analysis located atxA on a 2.0-kb fragment between cya and pag. DNA sequencing identified one open reading frame encoding 476 ami no acids with a predicted M(r) of 55,673, in good agreement with the v alue of 53 kDa obtained by in vitro transcription-translation analysis . The cloned atxA gene complemented previously characterized Tn917 ins ertion 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 transcri ption of pag but also that of cya and lef. Beta-Galactosidase synthesi s from the pag-lacZ transcriptional fusion construct introduced into a n 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.