Cross-talk to the genes for Bacillus anthracis capsule synthesis by atxA, the gene encoding the trans-activator of anthrax toxin synthesis

The two major virulence factors of Bacillus anthracis are the tripartite toxin and the polyglutamate capsule, which are encoded by genes on the large plasmids, pXO1 and pXO2, respectively. The genes atxA, located on pXO1 and acpA, located on pXO2, encode positive trans-acting proteins that are involved in bicarbonate-mediated regulation of toxin and capsule production, respectively. A derivative strain cured of pXO1 produced less capsular substance than the parent strain harbouring both pXO1 and pXO2, and electroporation of the strain cured of pXO1 with a plasmid containing the cloned atxA gene resulted in an increased level of capsule production. An acpA-null mutant was complemented by not only acpA but also the atxA gene. The cap region, which is essential for encapsulation, contains three genes capB, capC, and capA, arranged in that order. The atxA gene stimulated capsule synthesis from the cloned cap region. Transcriptional analysis of cap by RNA slot-blot hybridization and primer-extension analysis revealed that atxA activated expression of cap in trans at the transcriptional level. These results indicate that cross-talk occurs, in which the pXO1-located gene, atxA, activates transcription of the cap region genes located on pXO2. We identified two major apparent transcriptional start sites, designated P1 and P2, located at positions 731 bp and 625 bp, respectively, upstream of the translation-initiation codon of caps. Transcription initiated from p1 and P2 was activated by both atxA and acpA, and activation appeared to be stimulated by bicarbonate. Deletion analysis of the upstream region of the cap promoter revealed that activation by both atxA and acpA required a DNA segment of 70 bp extending upstream of the P1 site. These results suggest that cross-talk by atxA to the genes encoding capsule synthesis is caused by the interaction of the atxA gene product with a regulatory sequence upstream of cap.