BMC Genomics 2005, 6:103 http://www.biomedcentral.com/1471-2164/6/103 Table 1: CDSs of pAW63: homologies and comparison with pXO2 and pBT9727 (Continued) #059 #060 #061 #062 #063 #064 #065 #066 #067 #068 #069 #070 #071 #072 #073 #074 #075 #076 + 54988 + 56252 + 57303 - 58650 - 60821 - 61323 - 62731 - 63383 - 64659 - 65290 - 66240 - 67475 - 67681 - 68132 - 68628 - 69124 - 70679 - 71773 55699 56870 57834 58203 58679 60942 61429 62798 63633 64738 65313 66302 67471 67703 68151 68695 69197 70744 237 206 177 149 714 127 434 195 342 184 309 391 70 143 159 143 494 343 - site-specific recombinase, resolvase Clostridium thermocellum (54%) phage site-specific recombinase B. cereus ATCC 14579 (64%) CAAX N-terminal protease B. cereus ATCC 14579 (35%) DNA topoisomerase, TrsI/TraI Lactococcus lactis (45%) - conserved hypothetical protein B. cereus G9241 (35%) AbiQ abortive infection mechanism L. lactis (34%) signal transduction histidine kinase E. faecium (38%) putative membrane-bound hydrolase Bacillus licheniformis (32%) - ATPase involved in DNA repair E. faecium (42%) - - - - - ATPase involved in DNA repair E. faecium (36%) - - - - #101 - #103 - #104 - - - - - - - #107 90% #109 - #110 83% #111 77% #112 62% #113 80% - - - - - 49% - 96% - - #065 69% #066 73% #071 68% #072 74% - - - - #073 28% #074 83% #075 92% #076 60% #077 94% #078 75% #079 69% - - - - #079 50% #080 83% * CDS numbers are abbreviated forms originating from their formal designations: pAW63-###, GBAA_pXO2_0###, pBT9727_0### Replication and regulatory functions are grouped in an 8 kb 'control center' All the elements that were putatively identified as being directly involved in plasmid replication, copy control or other regulatory processes were found to reside within an 8 kb region of the plasmid containing 12 CDSs (CDS 46 to 57), of which 6 could not be assigned to a functional category. This region was delineated by mobile genetic elements or remnants thereof, and contained five of the eight CDSs that were found in clockwise orientation, the other three being located in its gene mobility-associated flanking sequences. The 4.1 kb replicon of pAW63 was characterized in a pre- vious study [18] and classified as belonging to the pAMβ1 family of theta-replicating conjugative plasmids, with which it shares a similar cis-functioning origin of replica- tion (ori). The largest of the four CDSs contained in this region, Rep63A (CDS 47), displayed strong similarity to the replication proteins of several plasmids in this family, as well as 96% amino acid identity with the RepS protein of the pXO2 replicon [18]. Rep63B, the second largest CDS of the replicon (CDS 48), displayed strong similarity with copy control proteins RepB and PrgP from the Ente- rococcus faecalis conjugative plasmids pAD1 [22] and pCF10 [23], respectively [18]. Although the two smaller CDSs (49 and 50) did not display any significant homol- ogies, the authors noted that one of these, CDS 49, showed properties (location, size, orientation and hydrophilicity) that likened it to repC and prgO, which are putative genes encoding stability functions on pAD1 and pCF10, respectively. Upon further sequencing of the plasmid, three additional putative regulatory elements were identified in close prox- imity to the replicon. These elements (CDS 51, 52 and 57) were homologs of genes that are highly conserved throughout the B. cereus group as well as in some less closely related species. CDS 51 and 52 displayed signifi- cant homologies to an oligopeptide ABC transporter func- tioning as a pheromone binding protein, and to a DNA- binding protein, respectively. The clockwise-oriented CDS 57 showed a distinctly low overall G+C content and was found to encode a protein with 94% identity to RapD, a response regulator aspartate phosphatase acting as a tran- scriptional activator involved in the regulation of sporula- tion [24]. The conjugative functions of pAW63 are grouped in a 42 kb tra region All 15 CDSs assigned putative conjugative functions were found to be located within a 42 kb region of the plasmid (CDS 1 to 26 and 62 to 76; hereafter referred to as the tra region). Every one of the 41 CDSs predicted within this region was in the same counter-clockwise orientation, suggesting an operon-style transcription scheme may be in place, which would be consistent with the genetic organization of most known conjugative systems [25]. pAW63 encodes three homologs of Gram-negative T4SS components Three CDSs were found to encode proteins with signifi- cant similarity to components of the Vir secretion system, originally characterized in Agrobacterium tumefaciens, which is the archetypal model for the Type IV Secretion System (T4SS) that is believed to carry out the conjugative process in Gram-negative bacteria, as recently reviewed Page 5 of 14 (page number not for citation purposes)