Genomic comparisons of S. meliloti, A. tumefaciens, and R. etli , and between Rhizobium leguminosarum bv viciae and Rhizobium etli , have shown that chromosomes are well conserved both in gene content and gene order, whereas plasmids presented few common regions and lacked synteny, except for some pairs of plasmids whose features indicate that they were part of the ancestral genome, and may be considered as secondary chromosomes [26, 27]. In R. etli, the symbiotic and self-transmissible plasmids are the less conserved replicons  with fewer collinear blocks .
In this paper we show that a conjugative plasmid from a bean nodulating S. fredii strain is formed by large segments of replicons found in strains belonging to different species from diverse geographic origins. These replicons include two plasmids of R. etli, and a S. fredii chromosome. In GR64, bean-nodulation is provided by pSfr64b. Although the phylogenetic relationship of the GR64 nifH gene shows that it is closely related to the R. etli gene (Figure 4), pSfr64b differs from the typical R. etli pSym in other features (see above).
We have previously reported that R. etli pRet42a is able to form a cointegrate with the pSym, and thus promote its conjugative transfer, and that in some cases (10% of the events), resolution of the cointegrate leads to the generation of recombinant plasmids containing segments of both pRet42a and the pSym . Also, the occurrence of frequent genomic rearrangements in rhizobial species has been amply documented [19, 20, 25, 28].
Integrating these data, we propose that the R. etli plasmids were transferred to a S. fredii strain and recombination events among the plasmids, the chromosome, and possibly another endogenous S. fredii plasmid, led to the generation of plasmids pSfr64a and pSfr64b. This would indicate that pSfr64a is an evolutionary "new" plasmid of chimeric origin, that was generated after R. etli strains arrived to Europe, following the discovery of America, when bean seeds coated with bacteria were most likely introduced to that continent . It is noteworthy that pSfr64a, in spite of carrying a large segment of chromosomal origin, would not be considered as a secondary chromosome, as it can be cured without affecting the saprophytic phenotype of the strain (data not shown). It is possible that such a plasmid is an "intermediate" in the formation of secondary chromosomes. Other plasmids with a structure similar to that of pSfr64a, have yet to be described. The finding of such a plasmid in a natural environment may be a living example of a pathway that allows shuffling of the repABC genes, which has been proposed as a strategy to explain the plasmid diversity of Rhizobium . Also, the fact that the repABC genes are located adjacent to the transfer region that is similar to that of pRet42a, and separate from the other sequences that are similar to the R. etli pSym, highlights the impact of evolutionary forces leading to this arrangement, which is highly conserved in many plasmids, and must have evolved in a relatively short time period.
Strain NGR234 was isolated in 1965 by M. J. Trinick, from Lablab purpureus nodules in Papua New Guinea . The complete genome of strain NGR234 has been sequenced . Very recently, the classification of NGR234 was changed from Rhizobium sp to Sinorhizobium fredii. However, no genomic sequence of a type strain of S. fredii is available at present. Genome analysis of other S. fredii strains, both, typical and bean-nodulating, would help to define if the sequence migrated to a plasmid in a S. fredii ancestor, or in a more recent event.
The segment containing sequences similar to the R. etli transmissible plasmid pRet42a includes the genes involved in conjugative transfer. Conjugative transfer of Agrobacterium tumefaciens pTi and other rhizobial plasmids is subject to quorum-sensing regulation [3, 4, 31]. In pRet42a, transcription of tra and trb genes is activated by the autoinducer TraI and the transcriptional regulators TraR and CinR. The repressor encoded by traM is not active . Plasmid pSfr64a contains similar regulatory genes, indicating that its transfer is probably regulated by quorum-sensing. Some differences, such as absence of cinR may account for specific responses to different host-related or environmental conditions. Preliminary data indicate the participation of new elements for the activation of the conjugative transfer of pSfr64a.
A comprehensive study of the regulatory mechanisms governing pSfr64a transfer will be addressed in the future.
We have shown that the pSym of GR64 is able to perform pSfr64a-dependent conjugative transfer. The process could be similar to what occurs in CFN42, where pRet42a forms a cointegrate with the pSym, allowing its transfer. Alternatively, pSfr64b mobilization could be induced in trans. The analysis of this process will be pursued in the future.
R. etli plasmid p42a was defined as self-transmissible because it may be transferred from diverse genomic backgrounds, such as Agrobacterium, containing no other plasmids [5, 32]. The conjugation experiments performed in this work, show that pRet42a transfer is significantly decreased in GR64 background, suggesting the presence of host-specific elements that interfere with the transfer function. Regarding pSfr64a, conjugation occurs at high frequency when the donor is the native strain. Transfer has not been determined from plasmid-less strains, so that the lack of transfer from R. etli background could be due to the presence of an inhibitor, or to the lack of a required factor, encoded in the chromosome or pSfr64b. These data suggest that a plasmid may be "sequestered" by a host, and imply that the plasmid needs to adjust the appropriate expression of conjugal transfer functions to the new host environment.