BMC Genomics 2005, 6:103 http://www.biomedcentral.com/1471-2164/6/103 of the interrupted Tn916 ORF 14-like element and dis- playing 44% identity to IepA, a group II intron-encoded protein from Bacillus megaterium which further BLAST results revealed to be a reverse transcriptase and maturase. This immediately led to the conclusion that CDS 7 was part of a group II intron that had inserted itself in the uninterrupted ancestor of CDSs 6 and 8, a finding sup- ported by the skewed G+C content profile corresponding to the site of the proposed insertion. The element was ten- tatively named B.th.I1 (for B. thuringiensis Intron #1). Fur- thermore, a closer examination of the 935 bp fragment of seemingly non-coding DNA with high G+C content found interrupting the VirD4 homolog (CDSs 14 and 15) revealed that the extremities of this insert were nearly identical to those of the B.th.I1 intron mentioned above, indicating the presence in this locus of a second retroele- ment presumably derived from the first. This second ele- ment was tentatively named B.th.I2 (for B. thuringiensis Intron #2). Three other of these CDSs (CDS 58, 60 and 61) were found in close proximity to each other by the regulation side of the proposed 'control center' region, as mentioned previously, and were predicted to encode various types of DNA recombinases. Finally, CDS 45 was found flanking the replication side of the replication control center and showing similarity to a prophage helix-turn-helix protein from the B. cereus strains G9241 and ATCC 14579. The 10 kb region down- stream of CDS 45 was found to encode 18 rather short CDSs (≈300 bp on average) with no significant homolo- gies in a configuration reminiscent of typical bacteri- ophage gene structure, which may correspond to a prophage integrated within the plasmid sequence. Intriguingly, while the first 4 kb of this region showed a G+C content in accordance with the rest of the plasmid, the following 6 kb segment showed a distinctly low G+C content along most of its length. As a counterpoint to the considerable amount of phage- derived genetic material tentatively identified on the plasmid, CDS 66 displayed significant similarity to AbiQ, a single-protein abortive infection mechanism from L. lac- tis which limits phage dissemination by shutting down the lytic cycle and leading the infected host cells to their death [33]. pAW63 shares a common backbone with pXO2 from B. anthracis and pBT9727 from B. thuringiensis konkukian Similarities between pAW63 and the virulence plasmid pXO2 from B. anthracis had been observed previously [18,20] and further investigation (C. Kuske, pers. comm.) had yielded thirty short sequences (400 bp long on aver- age) corresponding to regions of pAW63 which had been shown to hybridize to pXO2. This data was used in this study as the basis for the sequencing of pAW63, in a strat- egy that involved the outlining of a backbone sequence from which primers pairs were designed to amplify and individually sequence the intervening regions. These premises had guaranteed at least several hits to the virulence plasmid from B. anthracis, pXO2, and the final sequence of pAW63 certainly did not disappoint. Indeed, a preliminary alignment of their sequences showed that aside from a 37-kb region found only on pXO2 corre- sponding to the region containing the anthrax capsule genes and associated regulatory elements, they possessed a highly similar genetic makeup. Furthermore, the BLAST results had also revealed the existence of a third closely related plasmid, pBT9727, which was recently published along with the genome sequence of its host strain B. thur- ingiensis serovar konkukian strain 97-27 [Gen- Bank:NC_005957] and shows a comparable overall gene organization and coding content to that of the other two. These findings were consolidated by performing a BLAST Score Ratio Analysis of the predicted proteomes of the three plasmids, the results of which indicated a high level of synteny, with the pAW63 and pBT9727 pair showing the highest ratio of shared CDSs although the associated identity percentages were the poorest. Regarding the CDSs shared by each of these plasmids with pXO2, pBT9727 possessed the highest number of these but for the most part pAW63 showed higher identity percentages (Tables 1 and 2). A global phylogenetic analysis was performed on the three proteomes by systematically aligning the 42 homologous predicted proteins shared by all three plas- mids together along with their most relevant common BLAST hit result and building a tree from the four sequences (data not shown). Results were consistent for all quartets examined and suggested that pBT9727 had been the first to branch off from the common ancestor, while pAW63 and pXO2 diverged later. The detailed alignment of the three sequences shown in Figure 2 clearly illustrates the extent of the synteny observed between these plasmids. The tra region of pAW63 finds its equivalent on both pXO2 and pBT9727, with high levels of identity and contrasting discrete variations Both pXO2 and pBT9727 were found to possess an approximately 42 kb long region that was almost identical to the tra region of pAW63 with respect to gene structure and organization, as illustrated in Fig. 2. All of the CDSs identified on pAW63 as possible conjugative genes were found to have highly similar homologs on both of these plasmids (see percentages in Tables 1 and 2), although several displayed key differences as detailed below. Page 7 of 14 (page number not for citation purposes)