BMC Genomics 2005, 6:103 http://www.biomedcentral.com/1471-2164/6/103 [8,26]. Briefly, the T4SS can be summarized as a two-step mechanism [27] which involves the replication of the plasmid DNA by effector proteins followed by the translo- cation of a single-stranded molecule across the cell enve- lope into the recipient cell. The transfer is powered by the action of a molecular pump and the DNA is thought to travel through a channel formed by a core protein com- plex, while the necessary close cell-to-cell contact is medi- ated by surface structures. Analysis of the pAW63 sequence (Fig. 1) showed that CDSs 14 and 15 were in fact the two halves of an inter- rupted CDS encoding a homolog of the VirD4 compo- nent, which has been shown to function as a coupling protein between the DNA strand replication machinery and the transmembrane transfer complex and power- house. Interestingly, the 935 bp space between them was found to be completely devoid of coding sequences and showed a distinctly higher G+C content than was consid- ered average for the rest of the plasmid. CDS 9 displayed 37% identity with VirB4, a membrane associated N-tri- phosphatase which is thought to play a role in providing the energy needed to power the actual DNA transfer. CDS 22 displayed 35% identity to VirB11, a subunit implicated in both Type II and Type IV secretion systems that was shown to be necessary for conjugation, although its exact role remains unclear. Structural analyses have shown it to possess a transmembrane domain as well as an ATPase domain, and recent research suggests that the subunits are arranged as a dynamic hexameric assembly associated with the inner membrane and functioning as a gating component [28]. pAW63 encodes homologs to components of Gram-positive conjugation systems from diverse species Another 10 CDSs were classified as conjugative on the basis of their homologies, this time to genes from Gram- positive conjugation systems. Most notably, CDS 63 showed a distinctly high G+C content and was found to encode a topoisomerase displaying 45% identity to the TrsI/TraI protein from the Lactococcus lactis conjugative plasmid pMRC01 [12]. This component of the lactococcal conjugative system is thought to function as a relaxase, a role equivalent to that of VirD2 in the T4SS machinery. CDSs 67 and 70 both displayed significant homology to LtrC-like (from the Lactococcal transfer ORF C) putative conjugative elements from Streptococcus, Staphylococcus, Lactococcus and Listeria species (with an average 30% iden- tity), although they had different 'most relevant BLAST hits', as shown in Table 1. The function of the LtrC ele- ment itself, which was identified in the tra region of the lactococcal conjugative plasmid pRS01 [29], has not yet been determined. CDSs 6 and 8 were predicted to be the two halves of an interrupted protein with significant resemblance to ORF 14 from the enterococcal conjugative transposon Tn916, a self-transmissible molecule encoding tetracycline resist- ance [30]. The function of Tn916 CDS 14 has not yet been elucidated, but further results of the similarity search dis- playing comparable identity levels included a soluble lytic murein transglycosylase from E. faecium as well as the invasion-associated extracellular protein Iap from L. monocytogenes. Furthermore, three CDSs (CDS 13, 16 and 26) were pre- dicted to encode proteins that may be involved in the establishment of intimate cell-to-cell contact, either as aggregation substances secreted into the medium, or as cell surface determinants. CDS 13 showed a distinctly high G+C content in its second half, which contained two tracts of distinct repeated units that were predicted to cor- respond to a series of helix-turn-helix motifs followed by a series of pleated sheets in the protein product. It also dis- played 25% identity with the adhesin AidA from the Type V secretory pathway of E. faecium, which relies on an autotransporter system for cytolysin secretion. The adhesin itself is primarily implicated in the pheromone- mediated enterococcal mating process as an aggregation substance, but it has also been shown to act as an adhe- sion factor in the course of infection of eukaryotic cells by pathogenic E. faecalis strains and as such it may be consid- ered a virulence determinant [31]. CDS 26 was the largest CDS found on the plasmid (4.2 kb) and showed little sig- nificant resemblance to any known sequences except for a positive match (20% identity) with a 3.3 kb long cell-sur- face protein from the B. cereus reference strain ATCC14579 and another (23% identity) with a 2.37 kb long Rhs family protein (for Recombination hot spot) from Bifidobacterium longum. Rhs products are thought to possess properties typical of cell surface proteins and often present internal rearrangements that lead to anti- genic variation [32]. CDS 16 was unlike any known bacte- rial sequences (apart from those of plasmids pBT9727 and pXO2) but the putative 643 residue protein was predicted to have a secondary structure consisting essentially of hydrophilic helices, suggesting it may function as a sur- face-associated or free-acting aggregation substance. Finally, the remaining CDSs correspond to ATPases pre- dicted to be involved in segregation and DNA repair (CDSs 3, 10, 62 and 76), as well as a predicted membrane- bound hydrolase (CDS 68). pAW63 and mobile genetic elements Five CDSs were readily identified as mobile genetic ele- ments on the basis of their homologies. Standing apart from the other four by both character and location, CDS 7 was found positioned right between the two fragments Page 6 of 14 (page number not for citation purposes)