b GDC 0449 Number of pSfr64a ORFs in each category, with highest similarity to ORFs from pRet42d. c Number of pSfr64a ORFs in each category, with highest similarity to ORFs from pRet42a. d Number of pSfr64a ORFs in each category, with highest similarity VX-689 chemical structure to ORFs from the chromosome of NGR234. Among the ORFs shared between pSfr64a and pRet42a, the self-transmissible plasmid of CFN42, most are related to conjugative transfer (20 ORFs), only two were ascribed to macromolecular metabolism. Interestingly, both are related to DNA metabolism, one was classified as a putative nuclease, and the other as a probable DNA methylase. In Figure 3, it can be appreciated

that the genomic region shared between pRet42a and pSfr64a is markedly colinear. Colinearity is disrupted by the absence of an homolog to the regulatory gene cinR of pRet42a, and the presence of pSfr64a ORFs 147 and 148, which encode hypothetical proteins. The correspondence between pSfr64a and pRetCFN42 ORFs C59 wnt mouse is presented in Additional File 1. Figure 2 shows that the segment of pSfr64a shared with pRet42a has a high GC content, compared to the rest of the plasmid. This feature is also present in the similar pRet42a sequence. Figure 3 Colinearity between pSfr64a and other replicons. Dot matrix view of BLASTN comparisons of pSfr64a vs pRet42a, pRet42d and the chromosome

of NGR234. The ORFs similar to the pSym of CFN42 (pRet42d) include the repABC genes (Figure 2, Table 3). This is congruent with our finding that pSfr64a and pRet42d are incompatible (data not shown). The pSfr64a-pRet42d-shared ORFs are mainly involved in small Casein kinase 1 molecule metabolism (26 ORFs), and carbohydrate transport (13 ORFs). It is noteworthy that, in spite of the fact that pRet42d carries genes engaged in symbiotic functions, none

of these are present in pSfr64a. Within the region similar to pRet42d (ORFs 46 to 110), the colinearity is restricted to small segments, some of them in inverse orientation. (Figure 3, Additional file 1). The repABC genes (pSfr64a ORFs 164 to 166) were adjacent to the transfer region, separated from the other pRet42d genes. It has been amply documented that plasmid pRet42d is subject to frequent genomic rearrangements, due to the presence of reiterations and a high density of insertion sequences [16–20]. R. etli ORFs encoding transposon-related proteins located near to the sites where colinearity is disrupted are indicated in Figure 2 (purple arrows) and Additional File 1. For example, pSfr64a ORFs 122 to 146 are colinear with pRet42a ORFs 139 to 162. The adjacent ORF on pRet42a (ORF 138) encodes a transposon-related protein. It is possible that these sequences are related to the generation of rearrangements, causing the interruptions in colinearity. ORFs 114, 115, 116, 117, 118 and 121 show homology to ORFs encoded in another Rhizobium etli strain; IE4771 [21].