The sole Spo0Fsequence that differs at position is that of B. clausii, in which tyrosine replaces histidine. Notably, the spo0F mutation is distinct from a separate spo0F mutation present in the in vitro passaged 1013 isolates. Given that the amino acid sequence of B. atrophaeus Spo0F is identical to that of B. subtilis but for two conservative substitutions, it is likely to have very similar if not identical biochemical properties. Detrick-1 and 1942 likely represent one of the two R colony morphotypes described by Hayward et al., whereas the hypersporulating F morphotypes likely arose due to the emergence of the spo0F mutation. However, the possibility that Detrick-1 represents a reversion mutant at this locus from Detrick-2 cannot formally be excluded, but since it represented the dominant morphotype in the 1952 Detrick vial we believe this is unlikely. The presence of the spo0F allele in the ATCC 9372 strains suggests thatthesestrains wereacquired by ATCCafterthis mutationappeared within the Detrick lineage. Experiments to verify the roles of each allele in modulating sporulation are currently in progress. Preliminary results indicate that transformation of B. subtilis Dspo0F with B. atrophaeus DNA and selection of spo+ cells dramatically alters colony morphology independently of the spo0F allele introduced; additional studies to verify theeffectsofeachallelearecurrentlyinprogress. The H101R and A98P allelesare likely to alter the response to signals promoting sporulation. Aspo0F allele results in a sporulation-proficient strain that throws off sporulation-deficient papillae, and the same mutation has been shown to suppress the spo2 phenotype of a strain containing a defective kinA allele. H101 has been proposed as a potential metal-binding site with particular affinity for Cu2+. Binding of Cu2+ at this site may modulate interaction with one or more sensor kinases that promote sporulation. Substitution of positively charged arginine at this position could potentially mimic the binding of a metal cation in the loop containing H101, resulting in altered sporulation of the strains due to a change in the interaction with the kinases governing sporulation. It is unclear why, given the proposed 
role of divalent Cu2+ in suppressing sporulation, H101R would result in a hypersporulation phenotype. The mechanistic relationship between spo0F and the hypersporulation phenotype will be tested in future experiments. Both variants in the 1013 lineage possess an A98P allele in spo0F. Although the presence of several other mutations within this lineage confounds the attribution of the hypersporulating phenotype to this allele at this time, the presence of a mutation in the same gene as another hypersporulating mutant is Diperodon highly suggestive. The effect of proline substitution at position 98 on Spo0F functionis not immediately obvious, but the relatively inflexible proline residue can disrupt alpha-helices in protein structures. The 1013-1 lineage exhibits a hypersporulating phenotype even more pronounced than spo0F strains in the ”military” lineage. The observation that hypersporulating phenotypes have emerged during cultivationof two independent B. atrophaeus lineages point to the possibility that certain in vitro selection Orbifloxacin pressures may actually favor hypersporulating variants. The selection pressures acting on the sporulation pathwayare highlighted by the sheer number of mutations discovered within the entire data set that occur in proteins known to play roles in sporulation. Nine of the 38 mutations found in all lineages were in genes that directly or indirectly regulate either entry into stationary phase or sporulation; this number exceeds the number that would be expected if mutations were to occur by chance, since less than 5% of B. subtilis genes are dedicated to regulatory processes of any kind. In addition to the mutations found within the ”military” lineage, the two variants of ATCC 49822 shown in Figure 2 differ by mutations in rpoB which also plays a role in entry into sporulation.