Given the lack of original records, it is unclear whether the NRS-356 variant in this study might have passed through Camp Detrick and been returned to the University of Wisconsin. However, given the date on the label and the general secrecy of operations at Camp Detrick during the Second World War we consider this possibility Orbifloxacin unlikely. During development of BGas a simulant for B. anthracis, strains were selected that exhibited the most desirable characteristics, those being rapid growth, high spore yield, and experimental reproducibility. Without being aware of the nature of the genetic alterations in their ”optimized” strains, BW Benzethonium Chloride workers at Camp Detrick selected a mutant that provided dramatically higher total and relative spore yields, and generated consistent experimental results. These strains were adopted into the inventories of numerous biodefense laboratories and have been used for many decades in simulations of decontamination and dispersal. By applying a combination of genomic and biochemical profiling techniques, our data demonstrate that the BG isolates were ”enhanced” by researchers at Camp Detrick during the development of the organism as a simulant. The selection of a strain with the desired properties appears to have occurred in at least two discrete steps, as shown by the genome sequences and metabolic profiles. The initial step appears to have been the adaptation of a strain to growth in corn steep liquor, an acidic medium rich in protein and lactate. The robust growth of the Detrick strains relative to 1942 in low-pH medium containing high lactate levels is likely due to mutations in mmgD, or a short-chain 3-oxoacyl- reductase, or both. The most likely candidate for a mutation in the Detrick isolates that increases growth is the frameshift in mmgDthat occurred following the divergence from the 49822 lineage and results in an altered C-terminus. The mmgD geneencodes a 2-methylcitrate synthase that is expressed in the mother cell at the intermediate stages of sporulation. A null mutation in mmgD had no perceptible effect on sporulation, although other TCA-cycle enzymes when mutated led to a loss of sporulation. The effects of the frameshift mutation on sporulation and cellular physiology on the function of the enzyme are not clear at this time. We speculate that the frameshift mutation alters the substrate specificity of MmgD in favor of citrate, thus increasing the flux of lactate-derived intermediates through the tricarboxylic acid cycle. Evidence for this possibility includes the observations that 2-methylcitrate synthases can have partial citrate synthase activity and that the B. subtilis mmgD gene can complement a gltA mutant of E. coli. Alternatively, alteration of 
function of mmgD may have predisposed the lactate-adapted strain to acquisition of a hypersporulating phenotype, which is not readily isolated or stable in B. subtilis; however the presence of a hypersporulating phenotype in an independently evolved lineage of BG indicates that the species may have an intrinsic predisposition to evolving such a phenotype in vitro. The ”military” strains also grow more readily on media containing D,L-diaminopimelic acid, a major component of bacterial peptidoglycan. Corn steep liquor is derived from the incubation of corn in water at 42�C55uC, during which a lactic fermentation by a community of wild organisms including numerous uncharacterized Bacillus spp. occurs. Total bacterial counts at the conclusion of CSL production can be quite high, thus the availability of such compounds for growth is not surprising. Another potential source of meso-DAP could be bacterial autolysis during sporulation. The relative roles of each of the alleles in growth on lactate and/or meso-DAP is the subject of current investigation in our laboratory. The second step in the development of BG as a simulant appears to have been the deliberate selection of a hypersporulating variant. Importantly, the selection of a strain optimized for spore yield resulted in the fixation of a new spo0F allele that has no counterpart among the available.