By phylogenetic, molecular and clinical criteria, CDC 684 is a B. anthracis and its attenuated phenotype must be due to differences within its genome relative to those of other closely related B. anthracis strains. The marked degree of attenuation of CDC 684, (with an LD50 of >1 × 108 spores by the i.m. route in the guinea pig) compares with LD50 values of 175 and 306 spores reported for the virulent Ames and Vollum-1B strains [16, 17]. It therefore renders a comparative genomics approach highly informative and suggests that either subtle SNP differences and/or a dramatic and massive inversion within this chromosome are responsible for the attenuation.
Whole genome sequencing and comparative analysis indicates that there are 51 chromosomal and < 6 plasmid SNP that are unique to CDC 684 in a comparison to Vollum. The possibility that one or more of these rare SNPs may have an important role in the attenuation of CDC 684 remains a viable option. These data have defined a new CDC 684 lineage emanating from the original Vollum branch, Figure 1. Twenty-seven of these SNPs would be translated into non-synonymous mutations in putative gene functions. None of these SNPs, however, are in genes considered to be virulence factors found in opportunistic B. cereus pathogens that include a variety of hemolysins, non-hemolytic enterotoxins, monomeric entertoxins and phospholipases . The remaining 23 SNPs include 11 synonymous SNPs, 7 SNPs in pseudogenes, and 5 intra-genic SNPs. Only one of these intra-genic SNPs is located in a region within a promoter region (-7 bp) in a L-serine dehydratase gene (GBAA_4361).
What has not been excluded from this new lineage are 15 B. anthracis isolates that currently share the nodal position between the CDC 684 and Vollum lineages (see Figure 1). A sequencing effort to identify CDC 684 specific SNP that are either shared or still unique among the presumably virulent 15 isolates would point to phenotype altering SNP. Any chromosomal and plasmid SNP that are still unique to CDC 684 would be candidates for having positions in genes or regulatory regions with roles that govern known or unknown functions that are necessary in a virulent organism. There is, as yet, no clear notion whether or how any of these SNPs could cause the dramatic change in the virulence or growth properties of CDC 684.
The role of the chromosome of B. anthracis in the overall etiology of the disease anthrax is still poorly understood. It is becoming evident that the regulatory functions of the virulent plasmids (pXO1 and pXO2) work in concert with certain chromosomal regulatory functions in a virulent organism, e.g. the regulation of the pXO1 atxA gene by chromosomal sigma factors or plasmid genes involved in a signal-transduction pathway that inhibits sporulation . These and other recent studies  make it difficult to dismiss any of the CDC 684 non-synonymous mutations as candidates for a role in the attenuated phenotype without further analysis.
An alternative hypothesis to explain the attenuation of CDC 684 is a role for the large 3.3 Mbp inversion within its chromosome. While this inversion does not appear to have altered the fine-scale order of the individual genes, it has changed the orientation of the genes within the inversion with respect to the genes outside of the inversion. This change in the orientation has been illustrated by whole genome alignments  and by an analysis of the GC skewing and the location of dif sites of the CDC 684 genome and that of several B. anthracis and B. cereus sub-group isolates (Figure 4, Table 4). These analyses indicate that the spatial relationship between the origin of replication and the termination of replication in CDC 684 has been perturbed by the massive inversion. The comparative growth data (Figure 5) clearly supports the idea that chromosomal replication may be altered in CDC 684 by exhibiting an extended lag phase and a longer growth rate.
The longer DNA synthesis time needed to complete chromosomal replication may be sufficient, alone, to explain the slower cellular growth rate of CDC 684. In the asymmetrical CDC 684 chromosome, the longer leading strand distance is 3.783 Mbp vs. 2.615 Mbp for Vollum and all characterized wild type B. anthracis strains. This is a ~38% larger chromosomal distance to replicate and, assuming everything else remains constant, this will take that much longer to complete the entire chromosome. The mid log doubling time difference between the wild type strains (~80 min) and CDC 684 (~80 min) is ~45%. The similarity between the 38% long replication distance and 45% longer growth rate is striking. This observation suggests that the displaced ter region remains the site for replication termination and that the asymetrical longer leading strand replication distance in CDC 684 becomes limiting for growth in vitro.
Historical accounts suggest that there are strong tendencies to conserve the basic relationship between the position of the ori and ter sites in enteric bacteria . Following the discovery of the dif sites and related specific recombinases, it was proposed that the topological relationship between the ori and the ter/dif site must be maintained at 0° and 180°, respectively, for normal chromosomal segregation to occur . This was suggested because mutations in the Xer recombinase genes or the dif site or the displacement of the dif site to other regions of the chromosome had adverse effects on cell division.
More recently whole genome sequence comparisons between several distinct species also suggest that there is conservation in the spatial orientation between the ori and ter sites over broad groups of bacteria [39–41]. Dot plots of conserved DNA and protein sequences between pairs of species produce characteristic X-shaped patterns suggesting that large chromosomal rearrangements often revolve around and maintain the distances between the origin and the terminus.
This study illustrates a case where the naturally conserved 180° orientation of the ori and ter sites has been modified by a large chromosomal inversion in a strain of B. anthracis, CDC 684. We suggest that the consequence of the altered spatial relationship between the ori and ter sites from 180° to 120° has caused the change in growth kinetics of this isolate (Figure 5). We also suggest that this change appears to alter the length of time that CDC 684 takes to replicates its chromosome. Whether this change has also altered the virulent phenotype of this isolate is yet to be determined.