BMC Genomics 2005, 6:103 http://www.biomedcentral.com/1471-2164/6/103 observation that group II introns target specific genes or gene domains [34] despite the lack of experimental data available concerning the insertion of this particular element. Similarly, the three plasmids possessed separate versions of the VirD4 homolog. The CDS encoding this gene was found to be interrupted in a distinct manner on both pXO2 and pAW63, while it appeared to be intact on pBT9727. As indicated previously, the pAW63-borne homolog (CDS 14 and 15) was interrupted by the puta- tive ORF-less group II intron B.th.I2, while the cause of the disruption of the pXO2-borne homolog was a frameshift due to the deletion of two nucleotides in a C-rich stretch located at two-thirds of the original protein. Likewise, homologs for the putative cell-surface protein encoded by CDS 26 of pAW63 were found on both pXO2 and pBT972, albeit in two different forms. While pBT9727 was found to possess a presumably intact version of the gene, the pXO2-borne version appeared to be disrupted by a single nucleotide deletion causing a frameshift two- thirds into the original protein. This was confirmed to be a genuine feature and not a sequencing artifact by com- paring the three available sequences of pXO2 [Gen- Bank:NC_002146, GenBank:NC_007323, GenBank:NC_003981] which had identical frameshifts in the corresponding locus. Finally, the three homologs of the putative adhesin (CDS 13 on pAW63) were found to differ in a very interesting way. While the first half of the protein was well conserved in all three versions, the second half containing the repeated motifs was almost identical between pXO2 and pAW63, although pXO2 lacked several repetitions of the basic units, and pBT9727 possessed completely different basic units which nevertheless gave rise to highly similar secondary structure predictions. Sequence variability is highest in the replication control center area and is associated with the presence of multiple mobile genetic elements The mobile DNA-based borders of the replication control center of each plasmid were found to be comparable as follows. The putative prophage structure bordering the replicon (CDS 27 to 45 on pAW63) was found on all three plasmids, although the second part of this structure was poorly conserved, to the point of being absent from pXO2. The other side of this region was delineated in all three cases by variants of the site-specific recombinase found on pAW63 (CDS 61). The area between the repli- con and this recombinase was highly variable between the three plasmids, with pAW63 featuring the most CDSs therein (CDS 50 to 60). Regarding the regulatory elements present in this region, it was observed that while both the putative pheromone receptor (CDS 51) and the DNA-binding protein (CDS 52) identified on pAW63 were also present as close homologs on the two other plasmids, the putative tran- scriptional activator RapD (CDS 57) was not found in any form on either of them. On the other hand, both pBT9727 and pXO2 were found to possess almost identical copies of two other genes that were not present on pAW63: a homolog of the DNA-damage repair gene uvr from B. sub- tilis, and the gene encoding the transcriptional activator TetR. A recent recombination event caused pAW63 to exchange part of its replicon It is interesting to note that while the origin of replication and main replication protein Rep63A (CDS 47) were found to be highly conserved across all three plasmids, no homologs were found for the two other replication-asso- ciated CDSs of pAW63 (CDS 48 and 49), but two CDSs of similar size and orientation were found in equivalent locations relative to the common backbone on each of the other two plasmids, suggesting they share a functional homology with the two pAW63 CDSs, despite their lack of sequence similarity. Furthermore, these pBT9727- and pXO2-borne pairs of CDSs were found to share a high level of sequence identity with each other, suggesting that the original corresponding part of the pAW63 replicon had been exchanged for its present form at some point fol- lowing the divergence of pXO2 and pAW63. In support of this idea, a similar pattern of resemblance/difference was observed for a series of partially palindromic iterons present in the vicinity of the replication genes which were previously believed to be implicated in the replication process. While the iteron sequences found on pBT9727 and pXO2 were by no means identical, they did share sim- ilar repeated motifs that were completely different from those identified on pAW63. The discovery of further repe- titions of these various motifs as well as several additional palindromes in key locations of the sequence have led to the elucidation of a complex structure of palindromic and/or iterative elements serving as node points for recombination events that may have been responsible for major divergences in synteny between the three plasmids in the replicon area, as illustrated in Figure 3. Comparative evidence supports the existence of a 37 kb pathogenicity island (PAI) on pXO2 The 37 kb region of DNA located after the replicon on pXO2 was found to exhibit most of the characteristics of a bacterial pathogenicity island (PAI) as defined by Hacker and Kaper [35]. All of the genes carried by pXO2 that are known to play a capital role in the course of anthrax infec- tion, such as the capsule genes, were clustered in this region, along with the regulatory genes acpA and acpB, Page 9 of 14 (page number not for citation purposes)