FIG. 1.

Enrichment protocol used to identify genes important for B. anthracis germination within cultured macrophages. (A) Flowchart outlining the critical steps in the enrichment protocol. The specific details are described in Materials and Methods. Briefly, spores were prepared from a B. anthracis Sterne 7702 Himar1 library, and these were used to infect cultured macrophages (Mφ) for 90 min. After infection, the macrophages were lysed and exposed to heat in order to kill vegetative organisms and preserve spores. Surviving spores were germinated and grown in vitro using rich medium, and then new spores were prepared. These spores were used to infect cultured macrophages, which were again used to enrich for germination-defective mutants. After five rounds of enrichment, individual clones were examined by RATE reaction to determine where the transposon was inserted into the genome of the germination defective mutant. (B) Bar graph representing the percentage of heat-resistant spores represented in the population following the steps of enrichment. Aliquots were collected at each of the first three rounds of enrichment, and the total CFU and heat resistant CFU were determined and used to calculate the percentage of spores in the population.