Bosi et al. BMC Genomics 2011, 12:403 http://www.biomedcentral.com/1471-2164/12/403 ORFs for each plasmid. Data obtained are summarized in Figure 9 which shows the different trends for CMPs and NCMPs. This holds true also when PAGs dataset retrieved at lower thresholds (i.e. 70%, 80% and 90%) were analyzed (Additional File 11) The high number of PAGs retrieved in CMPs provide strong support to the idea that plasmids have played (and are still playing) a central role in microbial evolu- tion [11]. In fact, our data suggest that, by visiting dif- ferent cells, CMPs can undergo recombination event(s) with the host’s DNA molecules more frequently than NCMPs; consequently, they can probably acquire pieces of exogeneuos DNA that, in turn, can be further spread within the whole microbial communities. This idea is also partially supported by the finding that CMPs, on average, possess more and longer PAGs clusters in respect to NCMPs as reported in Additional file 12. Conclusions Within the complex evolutionary network of plasmids, new functional blocks are added and exchanged. How- ever, to date, information on plasmids atypical regions is available only for a very limited number of plasmids and/or microorganisms. In this work we have developed a computational pipe- line to detect compositionally atypical ORFs that reside on plasmids and performed a large scale analysis of them. Implementing our strategy on a dataset of nearly 2000 plasmids we have identified 8065 PAGs, almost 6% of all the analyzed ORFs. Accordingly, these PAGS are likely the outcome of (one or more) HGT event(s), although it must be mentioned the hypothesis that, at least part of them, may derive from events of internal Figure 9 PAGs in conjugative plasmids. Abundance of PAGs in conjugative/mobilizable plasmids (CMPs) and not conjugative/ mobilizable plasmids (NCMPs). Page 10 of 12 recombination with chromosome(s) inhabiting the same cytoplasms but that, in some cases, may possess differ- ent compositional features in respect to the correspond- ing plasmids (as suggested in [7]). It is worth of noticing that the total amount of retrieved PAGs is, on average, lower than that estimated for chromosomes (10-15%) [10,20]. This might be due to the high confidence interval (C.I. 95%) applied during our PAGs retrieval pipeline (Figure 1), that might have led to a partial understimatation of the actual amount of atypical ORFs that are integrated on plasmids. Indeed, applying the same pipeline for PAGs retrieval with lower CI thresholds allowed to assemble larger PAGS datasets (embedding 14731, 27201 and 38950 sequences at 70%, 80% and 90% CIs, respectively). Nevertheless analyses on these (lower confidence) assembled datasets revealed overlapping trends, suggesting that the possible exclusion of some false negatives did not influence the general conclusions that can be drawn on PAGs and, more in general, on plasmid-mediated HGT. Overall, we found that PAGs are not uniformly distributed among the sampled plasmids dataset. Indeed most of the plas- mids harbor a few atypical genes or do not possess any atypical gene at all, wehreas PAGs enriched plasmids are progressively more rare. This finding is in partial agreement with previous findings on horizontal flow of plasmid genes [34] and suggests that all plasmids may not contribute equally to the overall horizontal flow of genes but, instead, some of them may occupy more cen- tral positions in the overall network of HGT events. In particular, this role might be covered by conjugative plasmids that have been shown to possess, on average, a higher amount of atypical regions in respect to not mobilizable ones. Interestingly, plasmids size does not sensibly correlate with PAGs content. This result may provide important evolutionary insights, suggesting that the acquisition of exogeneous DNA (i.e. HGT) might not be the only force driving the plasmids assembly and the expansion of their coding capabilities. Indeed, it might be possible that other molecular mechanisms play a role in this process, such as gene duplication (possibly followed by evolutionary divergence) (Maida et al. unpublished data). Moreover, the fact that for a fraction of the iden- tified PAGs it was not possible to retrieve an associated function, together with the observation that PAGs encoded proteins are, on average, shorter than not-aty- pical ones, points towards the presence of a fraction of pseudogenes within PAGs. Accordingly, these might have originated from unsuccessful HGT events, one of the most likely source of pseudogenes within prokaryo- tic genomes [39]. The automated functional annotation we have per- formed has revealed that, among the annotated PAGs,