BMC Genomics 2005, 6:103 http://www.biomedcentral.com/1471-2164/6/103 an opportunistic pathogen which has been implicated in food poisoning [2] and endophtalmitis [3], B. anthracis, the etiological agent of anthrax [4], and B. thuringiensis which produces δ-endotoxin crystals that are toxic to insect larvae [5]. These subspecies are thought to have emerged from a common ancestor following a series of genetic rearrangements mediated inter alia by mobile DNA elements (transposons, insertion sequences and phages), in synergy with various mechanisms of horizon- tal gene transfer (conjugation, transduction or transfor- mation), leading to the acquisition of virulence genes. This is exemplified by the presence of large virulence plas- mids in B. anthracis (pXO1 and pXO2) [6], emetic strains of B. cereus [7] and B. thuringiensis that carry the genes responsible for the main phenotypic properties by which these bacteria can be distinguished. While the inter- and intra-molecular movements of mobile elements can obviously have major consequences for the organization and composition of the host genome, it is probably the mechanism of conjugation that best ena- bles the dispersion of these elements throughout the gene pool. Several conjugation systems have been described in Gram-negative bacteria, all of them involving the forma- tion of a sex pilus to bring the participants in close con- tact, followed by the actual transfer of genetic material via a type IV secretion system (T4SS) [8]. There is much less data available concerning conjugation among Gram-pos- itive bacteria, but the present consensus distinguishes four main transfer strategies, the most common of which seems to be that of the so-called broad host range plas- mids. At present, the best characterized of these are the multiresistance plasmids pSK41 [9] and pGO1 [10] from Staphylococcus, pRE25 [11] from Enterococcus, pMRC01 [12] from Lactococcus and pIP501 [13] from Streptococcus as recently reviewed by Grohmann and coworkers [14]. The broad-host-range conjugative plasmid pAW63 was identified in B. thuringiensis serovar kurstaki, where it dis- plays an efficient ability to conjugate in liquid medium, both for its own transmission (around 10-3 transconju- gants per donor in broth mating, up to a frequency of 1:1 between kurstaki strains) as well as that of small mobiliz- able plasmids [15]. Moreover, heterologous conjugation experiments have shown that it is also capable of transfer to its cousins B. thuringiensis serovar israelensis and B. cereus, as well as to Bacillus sphaericus, Bacillus licheniformis [15] and less closely related species such as Listeria innocua and Enterococcus faecalis (A. Wilcks, pers. comm.; G. Van der Auwera and J. Mahillon, unpublished). In the present study, the complete sequence determina- tion of pAW63 has led to a functional map of the plasmid yielding insights into its conjugative apparatus, which includes T4SS-like components, as well as its resemblance to other large plasmids of Gram-positive bacteria. Of par- ticular interest is the extensive homology shared between pAW63 and pXO2, the second virulence plasmid of B. anthracis [16], as well as pBT9727 from the pathogenic strain B. thuringiensis serovar konkukian strain 97-27 [17]. Results pAW63 is a 71,777 bp circular molecule The complete nucleotide sequence of pAW63 was deter- mined to be 71,777 bp long with a G+C content of 33.8%. Analysis of the coding content and organization revealed few intergenic regions, and a total of 76 coding sequences (CDSs) were identified (Fig. 1), of which all but 8 were found to be in the same orientation (defined as counter- clockwise). Interestingly, these 8 CDSs were all located in close proximity to each other, and one of these (CDS 47) corresponded to a component of the pAW63 replicon, which was isolated and sequenced in a previous study [18] while several others were later assigned putative func- tions as mobile genetic elements as detailed further in the text. The sequence was found to have an 81% coding ratio with an average CDS length of 768 bp. The largest CDS identified was CDS 26, weighing in at 4,248 bp. Several CDSs were associated to distinct spikes in G+C composi- tion and will be discussed individually. The BLAST simi- larity searches showed that most of the 76 predicted CDSs encoded proteins with similarity to proteins from other organisms, although 36 of these were hypothetical pro- teins and only 26 could be attributed biological functions, while 14 of the predicted genes did not have any known homologs at all. The 26 genes encoding proteins with dis- cernible functions were assigned to functional categories according to a classification scheme adapted from Riley [19]. The physical details and relevant BLAST hit results (scored by percentage of amino acid identity) of all 76 CDSs are summarized in Table 1. pAW63 generic relationships Most of the significant similarity results originated from Gram-positive bacteria species such as Enterococcus spp., Streptococcus spp., Staphylococcus spp., Lactococcus spp. and Listeria spp. in addition to Bacillus halodurans, Bacillus sub- tilis, Bacillus licheniformis and the members of the B. cereus sensu lato group. It is of particular interest to note that out of the 76 CDSs predicted on pAW63, 50 CDSs showed strong similarity (between 48.8 and 98.7% amino acid identity, with an average of 81.1%) to CDSs found on pXO2 from B. anthracis [GenBank:NC_007323] and 49 CDSs showed strong similarity (between 43.8 and 97.4% amino acid identity, with an average of 70.5%) to pBT9727 from B. thuringiensis serovar konkukian [Gen- Bank: CP_000047], with 42/76 (55.3%) of these CDSs being shared by all three plasmids. This was consistent with previous observations of sequence similarities between these plasmids [18,20,21]. Page 2 of 14 (page number not for citation purposes)