B. anthracis IVIAT Figure 2. B. anthracis species-specific autolysis effects following addition of exogenous putative autolysins N-acetylmuramoyl-L- alanine amidases BA0485 and BA2446: (A) reduction in A595 optical density, (B) reduction in colony forming units, and (C, D) morphological changes of B. cereus (C) or B. anthracis (D). B. anthracis (Sterne strain), B. cereus (ATCC 14579), B. subtilis (168), and E. coli (DH5a) vegetative cells were resuspended in 20 mM sodium phosphate buffer containing BA0485 and BA2446 (final concentration 2 uM) or buffer alone (BA0485, pH 7.0; BA2446, pH 8.7). (A) A595 readings were recorded every 2 min. and reported as a percentage of starting A595. Buffer-only controls were not different for cultures containing B. cereus, B. subtilis, or E. coli. (B) Dilutions of cells were plated prior to addition of BA0485 and BA2446 proteins and then again following 20 min of incubation with buffer alone or buffer containing exogenous BA0485 or BA2446, and CFU are reported as percentage of the starting CFU. (C, D) BA0485, BA2446, or buffer alone were added to cell suspensions of B. anthracis, B. cereus, B. subtilis, and E. coli, and incubated for 20 min. Bacterial morphological changes were only evident following protein addition to B. anthracis (D); representative B. cereus samples are shown (C). Gram stains were performed and all images were captured at 10006. doi:10.1371/journal.pone.0001824.g002 Plus-RIL strains (Stratagene, La Jolla, CA) were grown in Luria Bertani (LB) media supplemented with appropriate antibiotics. in pET30abc using DNA from B. anthracis RP42, 9131, and 9131(pXO2) and previously described techniques [3,6,41,42]. Construction of an inducible B. anthracis genomic DNA expression library We constructed a BL21(DE3)-based inducible expression library including Sau3A derived 0.5–1.5 kb B. anthracis genomic fragments Adsorbing convalescent macaque serum to B. anthracis grown in vitro Healthy rhesus macaques (Macaca mulatta) in the weight range of 2.6 kg to 4.5 kg were quarantined for a minimum of 6 weeks prior PLoS ONE | www.plosone.org 6 March 2008 | Volume 3 | Issue 3 | e1824