Bosi et al. BMC Genomics 2011, 12:403 http://www.biomedcentral.com/1471-2164/12/403 cromosome(s) residing the same cytoplasm and/or with phages infecting the same cell. As a consequence, newly acquired genes can be integrated on plasmids and (even- tually) be maintained. In general, three major processes can mediate HGT among bacteria: transformation (the uptake of free DNA), transduction (DNA transfer mediated by bacter- iophages) and conjugation (DNA transfer by means of plasmids or integrative conjugative elements) [4]. How- ever, regardless of the transfer mechanism, once that DNA has entered the recipient cell it can undergo homologous recombination or homology-facilitated ille- gitimate recombination and can be successfully inte- grated into the genome of the new host. Lastly, if the newly acquired DNA confers a selective advantage to the host, it can be maintained and, possibly, spread again through the bacterial population. Importantly, it can be surmised that, at least in the first stages follow- ing the integration event (before the amelioration pro- cess can start), exogenous sequences maintain their own peculiar compositional features [e.g. GC% and dinucleo- tide relative abundance difference (δ*)] that usually differ from the rest of the “new” hosting molecule; for this reason these sequences are often defined “atypical”. Atypical (and, possibly, horizontally transferred) genes detection can be pursued by composition-based meth- ods [5,6] that involve alignment-free features, such as GC% content and/or δ* [6]. Compositionally oriented methods rely on the observation that some genome fea- tures (including GC% content and δ*) are typical for a given bacterial genome and similar between closely related genomes. Accordingly, recently acquired genes are likely to display anomalous composition, especially when they originated in distantly related species; more- over a different composition will also be observed in those cases in which amelioration process has been retarded. Interestingly, it has been proposed that the genome signature (a compositional parameter reflecting the dinucleotide relative abundance values between two different DNA strands) of plasmids does not resemble that of their host genome, probably indicating either absence of amelioration or a less stable relationship between plasmids and their host [7]. Based on composition-oriented strategies, recent ana- lyses on large sets of bacterial and archaeal chromo- somes have revealed their mosaic structure, since considerable proportions of most of them consist of horizontally acquired genes [8-10]. For example, apply- ing a Bayesian method on 116 prokaryotic complete genomes, Nakamura et al (2004) found that the average proportion of horizontally transferred genes per genome was about 12% of all ORFs, ranging from 0.5% to 25%. Similarly Cortez et al. (2009), analysing a set of 119 bac- terial and archaeal chromosomes (351111 ORFs), found Page 2 of 12 that a large fraction of them was populated by atypical genes (defined as clusters of atypical genes, CAGs) (58487, 16% of all genes). Hence, this strongly indicates that archaeal and bacterial chromosomes contain an impressive proportion of recently acquired foreign genes (including ORFans, that is open reading frames without matches in current sequence databases) coming from a still largely unexplored reservoirs [10]. Finally, the same authors found that among the identified CAGs, a large number were likely of plasmid origin [10]. These lines of evidence suggest that genetic mobility should not be merely interpreted in terms of transportation of genes bypassing the cell barriers of prokaryotes, but rather as a perpetual flow between discrete reproductive units [11], i.e chromosomes and/or MGE, including plasmids. In fact, an emerging view suggests that plasmids (and MGEs in general) should be considered as mosaics of functional blocks (modules) of genes [12,13]. Remark- ably, in a few cases, the mosaic structure of plasmids (according to compositional criteria) has been demon- strated [9,14,15] revealing interesting insights on the dis- semination of key biological traits such as antibiotic resistance, virulence and heavy metal detoxification. Accordingly, it is reasonable that the identification and the analysis of plasmid atypical genes (hereinafter PAGs) might reveal interesting insights in the (probably com- plex) network of intra- and inter-cellular gene transfer (s) that plasmids can face during their evolution. Indeed, PAGs might be either the outcome of (one or more) HGT(s) or of internal recombination events with chro- mosome(s) residing the same cytoplasm but possessing different compositional signatures. However, to the best of our knowledge, a massive ana- lysis of alien modules that may reside on plasmid mole- cules has not been undertaken up to now. Therefore, the aim of this work was to develop a statistically vali- dated computational strategy that integrates two distinct compositional measures [GC% content and δ*] to scan nearly 2000 archaeal and bacterial plasmids for the pre- sence of PAGs. Finally retrieved PAGs datasets have been analyzed, revealing interesting trends in the overall plasmid gene exchange network. Methods Analyzed genomes All the available complete plasmids, phages and chro- mosome sequences were downloaded from NCBI ftp site at (http://www.ncbi.nlm.nih.gov/Ftp/, as on February the 1st 2010). Concerning plasmids, we focused our attention only on those longer than 3 kb and harboring at least 2 ORFs, in order to be able to detect δ* and dif- ferences in GC% content among all the genes. This allowed to assemble a dataset of 1853 plasmids for a total of 128.569 ORFs. The complete list of plasmids