Bosi et al. BMC Genomics 2011, 12:403 http://www.biomedcentral.com/1471-2164/12/403 Page 9 of 12 Figure 8 PAGs clusters. PAGs clusters size distribution. Clusters longer than 8 genes are not shown here. (503) are bi-cistronic clusters, while another important fraction (119) is represented by arrays of three genes. Longer clusters were quite rare and we found only 26, 21 and 5 clusters embedding 4, 5 and 6 genes, respec- tively. This trend is in partial agreement with previous findings on cromosomal clusters of atypical genes [10] and suggests that PAGs clusters are quickly fragmented and/or eroded following integration. Alternatively, this could suggest that the transfer and integration of shorter clusters is somehow favoured in respect to longer ones. Finally, as shown in Additional file 10, short gene arrays seem to be more frequent also when PAGs were retrieved al lower CIs. Among identified PAGs clusters, some possess a par- tially documented evolutionary history, mainly driven by HGT/recombination events as, for example, mercury resistance gene cluster(s). In fact, mercury resistance genes (mer) have been usually found embedded in a sin- gle compact operon [42] that, in turn, has been sug- gested to represent an aberrant mercury resistance transposon (namely TndPKHLK2) that, in some cases, has lost those genes responsible for its transposition [43]. The analysis of PAGs clusters allowed the identifi- cation of (at least) 10 different mer clusters (see, Addi- tional file 10) that showed a different composition in respect to the source molecule, thus revealing the pivo- tal role of HGT in the spreading of this metabolic ability across bacteria belonging to (sometimes) very different taxonomical units and inhabiting separate ecological niches. Moreover, other gene clusters (or part thereof) coding for important biological traits (i.e. antibiotic resistance, host invasion and cobalamin biosynthesis) were retrieved. For example invasion-associated genes encoding proteins involved in invasion of mammalian cells were found in atypical clusters on plasmids retrieved from different specie of Shigella genus (see Additional file 10) providing further support to the idea that one (or more) HGT envent(s) played a role in spreading this feature within representatives of this genus [32,44]. Similarly, plasmid mediated HGT seems to have contributed to the spreading of other key meta- bolic traits in microbial representatives, as, for example, cobalamin biosynthesis [45] and tetracycline resistance for which very similar (atypical) gene clusters were retrieved from very distantly related microorganisms, including Geobacter, Halorubrum, Methylibium, Methy- lobacterium and Deinococcus representatives in the case of cobalamin biosynthesis and Escherichia, Klebsiella, Aeromonas, Serratia, Yersinia and Enterobacter in the case of tet genes (see Additional file 10). PAGs and conjugative plasmids Among plasmids, conjugative ones have been defined “vessels” of the communal gene pool [11]. Indeed, this class of plasmids possesses the ability to “visit” different cells and, in principle, undergo genetic rearrangements (such as homologous recombination) with other plas- mids and/or other informative molecules (phages and chromosomes). For this reason, conjugative plasmids might be expected to possess a higher amount of atypi- cal genes in respect to plasmids that are not (or are less) mobilizable. To test this hypothesis, we performed a comparative analysis of the amount of PAGs belonging to conjugative and non-conjugative plasmids. The conju- gative/mobilizable plasmids (hereinafter referred to as CMPs) embedded in our dataset were identified by searching for their propagation-related genes adopting the following strategy: 1) a dataset of genes involved in the propagation of conjugative plasmids (tra and mob like genes) was assembled probing a search on the ACLAME database http://aclame.ulb.ac.be/; 2) a BLAST search with each of the sequence of our dataset against the tra/mob-like database previously assembled (setting a minimum e-value of 1e-20) was carried out and then 3) the number of tra/mob-like genes was determined for each plasmid. In this way we were able to divide our plasmid dataset into (693) conjugative/mobilizable plas- mids (CMPs, possessing at least one gene involved in conjugation and/or mobilization) and (1160) not conju- gative/mobilizable plasmids (NCMPs, i.e. plasmids that do not possess any tra or mob related genes). Data obtained revealed that, on average, CMPs harbor a higher number of PAGs than that exhibited by NCMPs and that the distribution of PAGs in these two classes of plasmids were significantly different (Mann-Whitney test, p-value < 0.001). Indeed, CMPs possess, on average, 5.7 PAGs, whereas NCMPs posssess only 2.9 atypical