BMC Genomics 2005, 6:103 http://www.biomedcentral.com/1471-2164/6/103 The comparison of the tra regions of pAW63, pBT9727 and pXO2 has provided several valuable indications on the relative importance of specific genes in the conjugative process. While pAW63 is known to be fully functional and quite efficient as a conjugative plasmid, pXO2 is believed to be incapable of autonomous transfer although it has been shown to be mobilizable by the conjugative plasmid pXO14 from B. thuringiensis toumanoffi [46], suggesting that pXO2 carries a set of conjugative genes that is incom- plete or disrupted in one or several key components. Both pXO2 and the newly sequenced plasmid pBT9727 were now shown to possess a set of potential conjugation genes closely related to those of pAW63. The transfer capabili- ties of pBT9727 have not yet been assayed, but its sequence features point towards partial conjugative func- tion at the very least, and possibly full self-transmissibil- ity. For these reasons, further studies will focus on loci exhibiting discrete variations in order to expose their spe- cific contribution to the transfer process. For instance, the unique interruptions found in the pXO2 homolog of the VirD4 element and in that of the putative cell surface pro- tein encoded by CDS 26 on pAW63 may prove to be par- ticularly significant, especially if pBT9727 is shown to be fully conjugative. From a broader point of view, the combination of the phylogenetic analysis of the three proteomes with the elu- cidation of the structural features delineating the major sequence differences in the replication control center has made it possible to roughly reconstruct the molecular evo- lution of the plasmid trio. The revelation that pBT9727 was the first to diverge from the common stem, leaving pXO2 and pAW63 to branch off from an intermediate form some time later, carries several important implica- tions for the ecology of the B. cereus s.l. family. At a very basic level, it highlights the mixed lineage of these plas- mids, as attested by their distinct genetic properties (con- jugation versus virulence) contrasting with the nearly perfect conservation on all three of the minireplicon of pXO2 [47]; plasmids which were isolated from strains that are treated quite differently, namely as a biopesticide versus a dangerous pathogen. This is particularly relevant in the light of recent incidents linking B. cereus sensu stricto strains to cases of anthrax-like pathologies [21,48,49], as well as the discovery that the cereulide genetic determi- nants of emetic B. cereus are located on a plasmid [7,50]. Most significantly, it makes a strong case for the pXO2 capsule genes region to be officially designated a patho- genicity island, a hypothesis further validated by the struc- tural features typical of mature PAIs that were identified in this study. The recognition of pXO2 as a PAI-equipped vir- ulence plasmid would also be consistent with the observa- tion that most of the major known pathogens possess such PAIs, many of these carried by virulence plasmids, and it would put pXO2 on par with pXO1, which is known to carry its own anthrax toxin-bearing PAI. In addi- tion, it should be noted that the configuration of IS231- related IRs observed on pXO2 (Fig. 2), which is quite likely due to the insertion of the IS231L element into the PAI after its acquisition by pXO2, entails that a region rep- resenting almost the entire PAI, containing all the capsule genes and their associated regulatory elements, can tech- nically be considered an IS231-derived Mobile Insertion Cassette (MIC). As such, this cassette could undergo a transposition event provided a functional IS231 trans- posase is supplied in trans. Incidentally, this opportunity for virulence genes to be directly mobilized by a mobile genetic element from within a larger PAI is paralleled in pXO1 by the presence in its PAI of a Class II transposon, TnXO1, which carries the anthrax germination operon gerX as its passenger genes [51]. Methods Bacterial isolates and PCR amplification B. thuringiensis strain HD73 containing pAW63 was grown in liquid LB medium at 30°C. 1 ml cultures grown over- night were centrifuged into a pellet, washed twice in ddH20 and resuspended in 250 µ l ddH20. Freshly pre- pared aliquots of 5 µ l of resuspended cell solution were used as template in each subsequent 50 µ l PCR reaction. PCR amplification was performed using 'Hi-Fi polymer- ase mix' from Fermentas according to the manufacturer's specifications. For target fragments of over 3 kb it was nec- essary to adjust the MgCl2 concentration of the reaction mix as well as adding dimethylsulfoxide (DMSO) to opti- mize yield and specificity. Further details can be obtained upon request. Cloning procedures made use of One Shot® TOP10 Electrocomp™ E. coli from Invitrogen, and trans- formation was performed according to the manufacturer's specifications. Sequencing strategy The sequencing strategy took advantage of the previously observed similarity between pAW63 and the virulence plasmid pXO2 from B. anthracis [18,20]. Further investi- gation (C. Kuske, pers. comm.) yielded thirty short sequences (400 bp long on average) corresponding to regions of pAW63 which had been shown to hybridize to pXO2. This data was used to construct a backbone sequence on the basis of which primer pairs could be designed to amplify the intervening regions. The resulting amplicons were purified on gel using Qiagen QiaQuick Gel Purification Kit according to the manufacturer's spec- ifications. Amplicons smaller than 5 kb were directly sequenced from the purified PCR sample, those between 5 and 10 kb were cloned into sequencing vectors, and those larger than 10 kb were each subcloned into separate libraries. All cloning procedures were performed using the Topo XL PCR cloning kit of Invitrogen according to the manufacturer's specifications. Amplicons were individu- Page 12 of 14 (page number not for citation purposes)