Identification and characterization of a germination operon on the virulence plasmid pXOI of Bacillus anthracis

The spores of Bacillus anthracis, the agent of anthrax disease, germinate w ithin professional phagocytes, such as murine macrophage-like RAW264.7 cell s and alveolar macrophages. We identified a cluster of germination genes ex tending for 3608 nucleotides between the pag and atxA genes on the B. anthr acis virulence plasmid pXOI. The three predicted proteins (40, 55 and 37 kD a in size) have significant sequence similarities to B. subtilis, B. cereus and B. megaterium germination proteins. Northern blot analysis of total RN A from sporulating cells indicated that the gerX locus was organized as a t ricistronic operon (gerXB, gerXA and gerXC). Primer extension analysis iden tified a major potential transcriptional start site 31 bp upstream from the translation initiation codon of gerXB. Expression of the gerX operon was s tudied using a gerXB-lacZ transcriptional fusion. Expression began 2.5-3 h after the initiation of sporulation and was detected exclusively in the for espore compartment. A gerX null mutant was constructed. It was less virulen t than the parental strain and did not germinate efficiently in vivo or in vitro within phagocytic cells. These data strongly suggest that gerX-encode d proteins are involved in the virulence of B. anthracis.