Generalized DNA Transfer in Y. pseudotuberculosis Figure 6. Model proposed for pUC4K mobilization based on the transposition mechanism of the IS6 family. Transposase-mediated replicon fusion of the two plasmid molecules generates a cointegrate carrying an additional copy of ISYps1 in the same orientation. Although only one type of cointegrate is represented here, different types of cointegrates mediated by each ISYps1 copy can be generated (indicated by dashed arrows). RecA-dependent homologous recombination between any two copies of ISYps1 present on the cointegrate will either regenerate the donor plasmid, leaving a single IS copy in the target pUC4K or create a rpUC4K containing a portion of pGDT4. Figure adapted from Mahillon J. and Chandler M. [17]. doi:10.1371/journal.pgen.1002529.g006 conjugative plasmids, including the F’ plasmid that encodes long and flexible pili [32]. Actually, growth in a liquid medium at low temperature could create the conditions optimal for GDT4. Indeed, this environment might be more favorable for the formation of tight bacterial aggregates and inter-cellular bridges through which long stretches of chromosomal DNA could transit. pGDT4 also triggered the conjugative transfer of the non- mobilizable plasmid pUC4K. Similarly, transfer of the non mobilizable plasmid pBR325 by an RP4::miniMu mobilizing plasmid was previously observed [33], but the mechanism underlying this genetic transfer was not characterized. cis- mobilization of non-mobilizable plasmid DNA can occur after integration of a conjugative plasmid into the genetic element to be transferred. Integration arises either by homologous recombina- tion between identical elements, often two copies of the same IS located on each DNA molecule (as for the Hfr formation in E. coli [34]), or through the formation of cointegrates mediated by specific transposons or ISs [35]. Integration of pGDT4 into pUC4K could not occur via homologous recombination as the two replicons do not share any common IS or identical DNA sequences. However, pGDT4 carries ISYps1, an IS which is predicted to belong to the IS6 family (http://www-is.biotoul.fr/is. html). ISYps1 is the second IS of the IS6-type identified in the genus Yersinia [36]. Members of this family have the capacity to create cointegrates by replicon fusion in the absence of a homologous IS on the target DNA [37]. Furthermore Tn3, which also moves by replicative transposition, has been found to mediate cis-mobilization of non mobilizable plasmids by this mechanism [35]. The fact that several rpUC4K plasmids obtained after pGDT4-mediated transfer carried a copy of ISYps1 argues for a role of this IS in pGDT4 integration into its target. We have demonstrated that ISYps1 is indeed transposing through replicative transposition. ISYps1 has a low specificity of recognition of the target sequence, as attested by our observation that the three insertion sites sequenced (two on pUC4K and one on R388) were different. This model also predicts the formation of cointegrates carrying two copies of the IS element, each flanking the sequence of the donor plasmid. We do have observed the formation of cointegrates between R388 and pSWYps1 flanked by the expected ISYps1 copies. A resolution step, which occurs through homolo- gous recombination between the two IS copies, is then required to separate the donor and target replicons, leaving a single IS copy in the target and restoring the donor plasmid. The rpUC4K-5 to -9 molecules that were found to carry one ISYps1 copy are most likely the results of such a resolution event. The presence on one recombinant plasmid (rpUC4K-10) of a portion of pGDT4 carrying ISYps1, ISYps2, ISYps3, ISNCY and ISL3 indicates that additional, complex rearrangements involving the Tn3-like transposon ISYps3 can also occur. Finally, the existence in some transconjugants of pUC4K plasmids with a size identical to that of the original molecule could be the result of the resolution of cointegrates containing pUC4K concatemers. GDT4 thus represents a remarkable illustration and validation of the model of Tn3-mediated transmission of non conjugative plasmids proposed by Crisona et al. in the 1980’s [35]. Following this model, the first step is the integration of pGDT4 into its target DNA by ISYps1-mediated replicon fusion during plasmid replication (Figure 6). As mentioned above, this generates a cointegrate which carries the two replicons separated on each side by an ISYps1 copy. This cointegrate then uses the conjugative machinery encoded by pGDT4 to promote its transfer to the recipient strain. The final step is the resolution of the cointegrate by homologous recombination between two ISYps1 copies or any other duplicated sequence present in the cointegrate. Since pGDT4 carries several ISYps1, the resolved molecules have different sizes and DNA composition. GDT4 is also able to mediate the translocation of chromosomal DNA, most likely by integration into the bacterial chromosome and transfer in a Hfr-like manner. The Hfr mechanism is one of the earliest and best described examples of chromosomal transfer PLoS Genetics | www.plosgenetics.org 8 March 2012 | Volume 8 | Issue 3 | e1002529