Figure 4. Electron microscopy of IP32637 grown at 46C in LB with agitation. White arrows point at bridge-like structures. doi:10.1371/journal.pgen.1002529.g004 NalR recipient, no transconjugants having acquired pUC4K were obtained, indicating that the pilus-encoding region of pGDT4 is required for generalized DNA transfer. As the Mpf region is predicted to encode a conjugative machinery, GDT4 most likely occurs by a mechanism of conjugation. To rule out other possible mechanisms of transfer, DNAse was added to the medium during the co-incubation period. The transfer frequency of the irp2 locus from the donor 637-irp2K-ureBS to the recipient 637DHPI-NalR was not affected, arguing against an acquisition of naked DNA through a transformation process. Cell-free filtrates of the supernatant of the donor strain incubated with the recipient strain did not allow DNA transfer, suggesting the absence of transferable DNA released from the bacteria but protected from the action of a DNAse (inside phage particles or membrane vesicles). These results argue against a transfer of DNA by transformation or transduction and further point at conjugation as the most likely mechanism. However, since this conjugative process was observed in liquid medium under agitation, we wondered whether a strong shaking of the culture would disrupt the pilus-mediated interac- tions between bacterial cells, and therefore decrease the transfer frequency. Surprisingly, when we increased the agitation of the medium containing the donor and recipient cells to 130 rpm (which was vigorous under our experimental conditions), the frequency of transfer of the irp2 locus was not affected (0.961028). Electron microscopy analysis of IP32637 cells grown under conditions optimal for GDT4 did not reveal any pilus structures on the bacterial surface. In contrast, tightly aggregated bacilli that seemed to be connected by ‘‘bridges’’ were observed (Figure 4). IS–mediated DNA mobilization We noted that after pUC4K transfer, the plasmid sizes of pUC4K in 10 different transconjugants were variable. This was confirmed after digestion of the 10 plasmids with NdeI, an enzyme that has a single restriction site in pUC4K. Three plasmids (rpUC4K-1 to -3) had the expected pUC4K size, while the seven others (rpUC4K-4 to -10) had a size superior to that of the original molecule (data not shown), indicating that various types of rearrangements had occurred during plasmid transfer. Remark- ably, a search for the potential transposition of pGDT4-borne IS (ISYps1, ISL3, ISYps2, ISNCY or ISYps3) on rpUC4K molecules by Generalized DNA Transfer in Y. pseudotuberculosis Figure 5. Schematic representation of seven recombinant pUC4K molecules recovered from transconjugants. Ten trans- conjugants resulting from the co incubation of 637(pUC4K) and 637- NalR were analyzed. The donor plasmid pGDT4 harbors two copies of ISYps1 in direct orientation and one copy in opposite direction, two copies of ISYps3 transposon including ISYps3 and ISYps2 transposases, one ISL3 and one ISNCY. ISYps1u is a truncated copy of ISYps1. Stars indicate XhoI restriction sites. Genes used to search by PCR for the presence of inter-ISYps1 regions are boxed. doi:10.1371/journal.pgen.1002529.g005 PCR (primers described in Table S1) showed that all seven larger size recombinant plasmids (rpUC4K-4 to -10) harbored ISYps1. rpUC4K-5 to -9 had a size compatible with the acquisition of a single ISYps1 copy. Digestion with XhoI, an enzyme that cuts once in pUC4K and once in ISYps1, yielded two restriction fragments, thus confirming the presence of a single ISYps1 copy. However, two distinct restriction profiles were observed, one for rpUC4K-5 and -8 and one for rpUC4K-6, -7 and -9 (data not shown), indicating the occurrence of different genetic rearrangements. Sequencing of the regions encompassing the ISYps1 insertion site in one recombinant plasmid of each group (rpUC4K-5 and rpUC4K-6) demonstrated that the IS was inserted at two different sites, approximately 100 bp apart, and in opposite orientation (Figure 5a and 5b). The insertion generated an 8 bp duplication of the target sequence: AAAATAGG in rpUC4K-5 and TATTT- GAA in rpUC4K-6. rpUC4K-4 had a size superior to that of the above five plasmids. XhoI digestion revealed the presence of two ISYps1 copies on this plasmid (Figure 5c). To determine whether the region located between these two ISYps1 copies on rpUC4K-4 corresponded to a portion of pGDT4, a PCR amplification of three pGDT4 genes (the Ig-like domain, parF and traM), each located between two different ISYps1 copies on pGDT4 (Figure 5) was performed. No positive signal was detected, suggesting that the region located between the two ISYps1 is a duplicated portion of pUC4K (Figure 5c). The rpUC4K-10 plasmid was different from all others since the PCR analysis showed that it carries all five pGDT4-borne IS, as PLoS Genetics | www.plosgenetics.org 6 March 2012 | Volume 8 | Issue 3 | e1002529