Transcription of the major Bacillus anthracis virulence genes is triggered by CO2, a signal believed to reflect the host environment. A 180 kb plasmid, pXO1, carries the anthrax toxin genes and the genes responsible for their regulation, pagR and atxA; the latter encodes a major trans-activator. It has long been known that pXO1 genes have major effects on the physiology of B. anthracis, probably through regulatory cross-talk between plasmid and chromosomal genes. Accordingly, we found that the chromosomal S-layer genes, sap and eag, are regulated by pXO1 genes so that only eag is significantly expressed in the presence of CO2. This effect results from the product of pagR acting as the most downstream element of a signalling cascade initiated by AtxA. In vitro evidence showed that PagR is a transcription factor that controls the S-layer genes by direct binding on their promoter regions. This work provides evidence that AtxA is a master regulator that co-ordinates the response to host signals by orchestrating positive and negative controls over genes located on all genetic elements.