The paradigm that bacterial genomes consist of a single circular chromosome is no longer valid. Linear chromosomes have been identified in Borrellia burgdorferi [1], various Streptomyces species [2, 3], Agrobacterium tumefaciens [4] and various other species. In addition, it is now appreciated that genomes of several bacterial taxa consist of multiple replicons. Most organisms with a multi- or bipartite genome structure belong to the α-Proteobacteria (including Rhodobacter sphaeroides [5, 6] and various Rhizobium [7, 8], Agrobacterium [4, 8], Brucella [9, 10] and Azospirillum [11] species) or the β-Proteobacteria. Most isolates from species belonging to the β-proteobacterial genera Burkholderia and Ralstonia harbour multiple replicons, including members of the Burkholderia cepacia complex [12–16], Burkholderia gladioli [15], Burkholderia pseudomallei [17], Burkholderia glumae [13], Burkholderia glathei [13], Burkholderia sp. LB400 [18], Ralstonia pickettii [13], Ralstonia eutropha [13] and Ralstonia metallidurans [18]. Multiple replicons may have arisen from the need to achieve higher overall replication rates [19]. The origin of these multiple replicons is at present unclear but it has been suggested that they could have their origin in gene duplication followed by divergence; in this case intrachromosomal recombinational events within a duplicated region could give rise to the formation of two stable replicons [8]. In the genus Brucella these rearrangements have occurred in the region containing the ribosomal RNA genes [10] but in theory the rearrangements can occur at any repeated sequence [20]. An additional explanation is that the presence of multiple replicons within an organism involved horizontal DNA transfer [19, 21, 22]. This hypothesis was used to explain the presence of two chromosomes in Vibrio cholerae: the small chromosome was suggested to be derived from a megaplasmid captured by an ancestral Vibrio [23, 24]. This megaplasmid probably acquired genes from diverse bacterial species before its capture by the ancestral Vibrio; subsequent relocation of essential genes from the chromosome to the megaplasmid completed its stable structure.

Ralstonia solanacearum is a soil-borne phytopathogen with an unusually broad host-range, causing bacterial wilt on a wide range of crops, including economically important crops like potato, tomato, ginger and banana [25]. Recently the genome sequence of R. solanacearum strain GMI1000 was determined [26]. It was shown that the 5.8-Mb genome is organised into two replicons, a 3.7-Mb chromosome and a 2.1-Mb megaplasmid. This bipartite genome structure is characteristic for most R. solanacearum strains [27] and derivatives of strain GMI1000 without the megaplasmid have not been obtained [26]. The larger replicon contains all the basic genes required for survival of the bacterium; the smaller replicon carries several metabollically essential genes also present on the chromosome (including a rDNA locus, a gene coding for the α-subunit of DNA polymerase III and the gene for protein elongation factor G) but also contains several genes coding for enzymes involved in primary metabolism (including amino acid and cofactor biosynthesis) not present on the chromosome. The smaller replicon also contains all the hrp genes (required to cause disease in plants) and it has been suggested that it has a significant function in overall fitness and adaptation of the organism to various environmental conditions [26]. The origin of the bipartite genome structure of R. solanacearum is not clear. To determine whether the megaplasmid was formed through intrachromosomal recombinational events within a duplicated region or was recently acquired from other organisms we compared the abundance, distribution and composition of simple sequence repeats between the chromosome and the megaplasmid of R. solanacearum GMI1000. We also compared the compositional bias of di- and tetranucleotides between both replicons.

Repeated DNA consists of homopolymeric tracts of a single nucleotide or of small or large numbers of multimeric classes of repeats. These multimeric repeats can be homogenous (i.e. built from identical units), heterogeneous (i.e. built from mixed units) or are built from degenerate repeat sequence motifs [28]. A special category of repeats are tandem repeats which are made up of periodically repeated monomeric sequences of varying length, arranged in a 'head-to-tail' configuration [29]. Several mechanisms have been proposed for the creation of tandem repeats, including 'slipped strand mispairing' in which illegitimate base-pairing during replication gives rise to addition of repeat units [30, 31]. There is growing evidence that small tandem repeats (also called simple sequence repeats or SSRs) affect gene expression. A first effect of SSRs is the mediation of phase variation through the loss or gain of one or more repeats [29]. Phase variation is the process by which many bacterial species undergo reversible phenotypic changes resulting from genetic alterations in certain loci [32, 33]. SSRs can also be involved in gene regulation by affecting spacing between flanking regions [34] or spacing between the -35 and -10 promotor regions [35]. Variation in abundance, distribution and composition of SSRs has been described [28] and it has been proposed that variation in SSR results in variation in gene expression and key phenotypes and hence provides an important target for natural selection and evolution [28, 36].

The comparison of genome-wide compositional biases as a tool to study bacterial evolution has been introduced by Karlin and co-workers [37–39]. It is thought that dinucleotide relative abundance values are constant within a genome because the factors that work on them are constant throughout the genome; and it has been postulated that the set of dinucleotide relative abundance values constitute a genomic signature that reflects the pressures of these factors [38]. Differences in genome signature between different organisms can be attributed to differences in context-dependent mutation rates generated by the replication-repair system and differences in efficiency of the replication machinery on different sequences. In addition, many DNA structural properties (including curvature, flexibility and helix stability), which may play an important role in biological processes like replication, are determined by dinucleotide arrangements [38, 40]. Tetranucleotide relative abundances are also characteristic for a given genome [39]. It has been postulated that frequent tetranucleotides may include parts of repetitive structural, regulatory and transposable elements, while low values for some palindromic tetranucleotides have been attributed to restriction avoidance [39].

To study the origin of the bipartite genome structure of R. solanacearum GMI1000 we compared the abundance, distribution and composition of simple sequence repeats and differences in compositional biases between the chromosome and the megaplasmid of R. solanacearum GMI1000.

It can be concluded that both replicons that constitute the R. solanacearum genome are very similar in respect to distribution and composition of SSRs and presence of compositional biases, although minor differences between both replicons are present. The megaplasmid carries the hrp genes required to cause disease in plants, genes coding for constituents of the flagellum and genes involved in exopolysaccharide production; it also contains 315 genes of unknown function [26]. The minor variations in SSR and compositional biases observed between both replicons may therefore be attributable to minor differences in gene expression and regulation of gene expression between both replicons. Alternatively, it is not unlikely that some of the observed differences are the result of the small sample numbers observed (for example the minor differences in tetranucleotide SSR distribution over coding and non-coding regions in both replicons [Table 3]). At present no completely sequenced and fully annotated genomes of other β-Proteobacteria with multiple replicons are available for comparison and therefore it is difficult to place the observed differences in a broader perspective. Nevertheless, the observed similarities in SSRs and compositional biases indicate that both replicons have shared a similar evolutionary history and suggest that the megaplasmid was not recently acquired from other organisms by lateral gene transfer but is a part of an ancestral R. solanacearum chromosome. Alernatively, the hypothesis of an ancient acquisition by lateral gene transfer followed by a long co-evolution with the chromosome cannot be completely ruled out.