ABSTRACTCholesterol-dependent cytolysins (CDCs) are secreted, pore-forming toxins that are associated with pathogenesis in a variety of gram-positive bacteria.Bacillus anthracisproduces anthrolysin O (ALO), a CDC that is largely responsible for the hemolytic activity of culture supernates when the bacterium is cultured in appropriate conditions.B. cereusandB. thuringiensis, species closely related toB. anthracis, produce CDCs with significant amino acid sequence homology to ALO. Transcription of theB. cereusandB. thuringiensisCDC genes is controlled by PlcR, a transcription regulator that requires a pentapeptide derived from thepapRgene product for binding to a consensus sequence (PlcR box) and transcriptional activation of downstream genes. A PlcR box precedes theB. anthracis alogene, and theB. anthracisgenome contains threeplcR-like genes, one of which harbors a nonsense mutation that is predicted to result in a truncated, nonfunctional protein. We detected mRNA ofalo,papR, and the threeplcR-like genes in spleens ofB. anthracis-infected mice, indicating gene expression in vivo. Analysis ofalotranscription in batch culture revealed a potential transcription start located between the PlcR box and the translational start. Nevertheless, steady-state levels ofalotranscripts and ALO protein were unaffected by deletion ofpapRor disruption of the PlcR box. Our data indicate that despite the presence of the transcriptionally activeplcRandpapRgenes inB. anthracisand a PlcR box in the promoter region of thealogene,aloexpression is independent of this control system.