BMC Genomics 2005, 6:103 http://www.biomedcentral.com/1471-2164/6/103 RFeiglautrioen3al diagram of the replicon region of the three plasmids Relational diagram of the replicon region of the three plasmids. Comparison of the replicon region of each plasmid reveals a complex structure of palindromic and/or iterative elements serving as node points for recombination events. Sequence segments are represented by thick horizontal lines joined by solid diagonal lines. The background striping highlights 'shared' versus 'unique' sequence segments as indicated by the text legend on the left hand side. Horizontal dashes are spacers to indicate a shorter length or lack of corresponding segment. Node points putatively involved in recombination events are represented by green circles; iterative and/or palindromic sequence units by half arrows; CDSs by rectangles, above the sequence line to indicate clockwise orientation, or below to indicate counter- clockwise orientation. which are directly associated with their expression during the development of the pathology [36]. Furthermore, this region had distinctive genetic properties from the rest of the plasmid, such as a lower G+C content (30.9% against 34.3%), and more variation in the orientation and phase of coding sequences. It was also found to be rich in mobile genetic elements (some cryptic or defective), such as multiple inactivated copies of the IS231 transposase [16,37] and a vestigial class II transposon. The comparison of the sequences flanking the proposed PAI on pXO2 and their equivalents on pAW63 and pBT9727, as described above (Fig. 3), revealed the exist- ence of specific nodes consisting of iterative and/or palin- dromic sequences that could be held responsible for the acquisition of the PAI region through a past recombina- tion event, though not excluding the possibility of later rearrangements having taken place within this region. pXO2 and pBT9727 possess copies of Insertion Sequence IS231L in different loci, causing a visible interruption in synteny The results of the three-way BLAST Score Ratio Analysis of this complex plasmid trio highlighted a group of five CDSs that were not present on the pAW63 sequence itself but were shared with a high degree of similarity between pBT9727 and pXO2. Their location was different on either plasmid with respect to the common backbone, thus rep- resenting a significant interruption in the synteny of the plasmids. It appeared that this small group had been pre- viously described on pXO2 as an IS231-derived [38] Insertion Sequence (IS) and designated IS231L [37], although it was assumed to be incapable of autonomous transposition due to a frameshift in the CDS encoding the transposase. However, the copy of the IS231L element harboured by pBT9727 was found to have an uninter- rupted and therefore supposedly functional transposase, as well as nearly perfect Inverted Repeats (IR), the canon- ical 20 bp boundaries of this type of mobile element matching those of the pXO2-borne IS231L element. Interestingly, the copy of IS231L found on pBT9727 was found to be nested within the Tn1546-related [39] left IR of a novel Class II transposon belonging to the Tn3 fam- ily, encoding a single cryptic CDS and tentatively named TnBt9727. Likewise, the pXO2-borne copy of IS231L was nested within the TnARS1-related [GenBank AY780525] right IR of a vestigial class II transposon. No correspond- ing left IR was found for this element, but an IS3 family transposase was found in close proximity to the putative right IR, suggesting a possible structural association. Furthermore, the presence of two other CDSs with homol- ogy to the IS231 transposase in the PAI of pXO2 prompted a search for other instances of IRs matching those of IS231L in the vicinity of these CDSs. A well-con- served copy of the right IR was indeed discovered near the Page 10 of 14 (page number not for citation purposes)