BMC Genomics 2005, 6:103 http://www.biomedcentral.com/1471-2164/6/103 end of the PAI beside a second CDS encoding an IS231 transposase-like gene (Fig. 2). Discussion A large proportion of the pAW63 sequence was observed to possess significant similarity to plasmids originating from several different bacterial species harbouring funda- mentally different conjugative and replication systems. The resulting hybrid combination of genes probably opens up the mating scope of the plasmid. The physical arrangement of the plasmid was identified as a composite structure consisting of a 42 kb tra region encoding conju- gation functions, organised in typical operon fashion, and an 8 kb control center encoding the replication and regu- lation functions, joined by mobile genetic elements or remains thereof (Fig. 1). Analysis of the replication control center of pAW63 revealed that it possessed copy control proteins similar to those found on the enterococcal conjugative pheromone plasmids pAD1 and pCF10. However, pAW63 conjugative transfer among B. thuringiensis strains has been shown to be independent from recipient-encoded pheromones [15]. It has been suggested that in addition to their role in the transfer process, host-produced pheromones may play a role in the replication of the pAD1 and pCF10 plasmids [23]. Given a similar copy control mechanism, the appar- ent independence of pAW63 from pheromone signalling may account for the greater host range of pAW63 com- pared to pAD1 and pCF10, by removing the requirement for a compatible pheromone system in the recipient mat- ing partner. It was therefore very interesting to find near the pAW63 replicon two CDSs respectively encoding an ABC transporter-type pheromone receptor, homologous to those found in Enterococcus and Listeria species, and a close homolog of RapD, a transcriptional activator from B. thuringiensis serovar morrisoni. At this point it is unclear whether the function of these elements is restricted to either the plasmid copy control or the regulation of con- jugation, or whether these processes are directly linked by common regulatory pathway components. Nonetheless, the presence of a putative pheromone-sensing mechanism on pAW63 suggests that the conjugative transfer process of the plasmid may follow alternate or optionally additive regulatory pathways depending on the species of the mat- ing cells. Regarding basic conjugative function, the presence on the plasmid of genes related to the T4SS system corroborates similar findings [40] concerning the transfer system of the streptococcal plasmid pIP501, which bears a tra region containing VirD4, VirB4 and Vir B11 homologs. Interest- ingly, all three of the pAW63 and pIP501 T4SS-like pro- teins correspond to components of the system that operate within the cell or in association with the inner membrane to prepare the plasmid DNA for transfer and provide motor function, and no homologs have been found on the plasmid to the 'outer' components. This could be explained by the idea that the inner part of the Gram-positive cell wall structure is sufficiently similar to that of the Gram-negative hosts for some of the 'inner' components, designated as effector and transporter mole- cules, of both transfer systems to share their main charac- teristics and possibly a common evolutionary history derived from replication mechanisms. Conversely, it seems reasonable to assume that major differences in the cell wall structure and composition, such as the presence of a thick peptidoglycan layer and the absence of an outer membrane, would be mirrored by the presence of specific protein components responsible for the 'external' steps of the conjugative process. This includes sensing of recipi- ents, cell to cell attachment and the bridging of cell walls to effect DNA transfer, and requires the assembly of a core complex as well as cell surface structures. With respect to the latter, several putative cell surface- or membrane-asso- ciated proteins, such as the adhesin encoded by CDS 13 and the very large protein encoded by CDS 26, have already been identified in the tra region of pAW63, although further studies will be necessary to work out their specific roles in the conjugative process. The presence of introns B.th.I1 and B.th.I2 within two genes of which at least one (the VirD4 homolog) may be considered essential to the conjugative process, poses the question of the functionality of the interrupted genes, as well as that of possible collateral effects caused by these elements. Regarding maintenance of functionality of the host genes, current models of group II intron splicing as well as experimental research show that intron excision during transcription leads to the production of a complete and functional protein [41]. In addition, this type of mobile element is thought to insert preferentially into cer- tain conjugative genes such as relaxases, and it has been suggested that such an insertion actually enhances the effi- ciency of the conjugative system [34,42]. Furthermore, the relationship between B.th.I1, which encodes its own reverse transcriptase, and B.th.I2, which does not, deserves further investigation. While such a configuration has rarely been observed among eubacteria, ORF-less introns that were closely related to an intron encoding a reverse transcriptase located in the same genome have been identified in cyanobacteria and archeae [43]. It has been proposed that these CDS-less elements were deriva- tives of their 'complete' RT-encoding relatives [44]. Signif- icantly, several of these 'degenerate' elements were shown to be mobile, raising the possibility that mobility may be supplied in trans [41] as is the case for the Mobile Inser- tion Cassette MIC231 trans-activated by IS231 trans- posase [37,45]. Page 11 of 14 (page number not for citation purposes)