Generalized DNA Transfer in Y. pseudotuberculosis Figure 2. Conjugative properties of pGDT4. (A) Comparison of the efficiency of transfer of pGDT4 and the irp2K chromosomal locus. The donor 637-irp2K-ureBS(pGDT4T) and the recipient 637-NalR were co-incubated at 4uC in LB-aa’ and independent experiments were performed 4 to 5 times. (B) Efficiency of transfer of pGDT4 to various recipient strains. The donor 637-irp2K-ureBS(pGDT4T) was co-incubated at 4uC with either 637-NalR, 953- NalR or 637c-NalR. The results of two independent experiments were combined. (C) Efficiency of transfer of pGDT4 at various temperatures in liquid and on solid media. The experiments were performed twice independently. Shown are mean values of transfer frequencies (vertical bars) and standard error of the mean (sem, vertical lines). The detection limit was 10210. doi:10.1371/journal.pgen.1002529.g002 4) flanked by 34 bp inverted repeats and contains the ISYps3 transposase and resolvase, as well as ISYps2, an IS110 family member. None of the IS present on the Y. pseudotuberculosis IP32953 chromosome [8] were present on pGDT4 and conversely, no sequences homologous to the pGDT4-borne IS: ISYps1 (primer pair 727A/B), ISNCY (729A/B), ISYps2 (730A/B), ISL3 (731A/B) and ISYps3 (732A/B) were detected by PCR in the IP32637 genome (data not shown). The remaining genes included a large proportion of putative coding sequences of unknown functions, and a few loci with predictable functions, of which some shared homology with Y. pseudotuberculosis chromosomal genes (Table S4). The most remarkable example was the putative product of pGDT4_0059, which shared 41% amino acid identities with YPTB3789, a chromosomal Ig-like domain protein [16]. pGDT4–mediated generalized DNA transfer Since pGDT4 carries a large set of genes predicted to be involved in conjugative transfer, we wondered whether this pGDT4-specific mobility function mediates GDT4. To investigate this potential role, the Mpf function was inactivated by allelic exchange of a large portion of the pil region (from pilL to pilV) of pGDT4 with a Tmp cassette in 637(pUC4K). After co-incubation of the resulting strain 637(pUC4K, pGDT4Dpil) with the 953- Figure 3. Genetic map of pGDT4. doi:10.1371/journal.pgen.1002529.g003 PLoS Genetics | www.plosgenetics.org 5 March 2012 | Volume 8 | Issue 3 | e1002529