Transcriptional regulation of the protective antigen gene of Bacillus anthracis

Bicarbonate is required for production of the major virulence factors, the toxins and capsule, of Bacillus anthracis. In this study we examined the basis for stimulation of production of protective antigen (PA), a central component of the two anthrax toxins encoded by plasmid pXO1. RNA prepared from B. anthracis grown in media with and without added bicarbonate was probed for PA mRNA. Data showed that bicarbonate was required for increased transcription of the PA gene (pag) in minimal medium. Transcription of pag was low in rich medium and could not be stimulated by the addition of bicarbonate. To characterize further the factors required for transcriptional regulation of pag, the promoter region of pag was fused to the chloramphenicol acetyltransferase gene (cat-86) of vector pPL703 and transformed by electroporation into pXO1+ (Tox+) and pXO1- (Tox-) strains of B. anthracis. Analysis of chloramphenicol acetyltransferase produced by the pag-cat-86 fusion in each of these backgrounds confirmed the results obtained by hybridization. Data obtained with this fusion also revealed that the large toxin plasmid, pXO1, found in virulent strains of B. anthracis, was required for stimulation of transcription of pag by bicarbonate. This result suggests the existence of a trans-acting factor that is involved in the activation of pag transcription.