Figure 1. Transfer frequencies at various temperatures of three distantly located chromosomal loci. The donor 637-irp2K-ureBS- 5076T and recipient 637DHPI-NalR strains were co-incubated in LB-aa’ with agitation. Transfer frequency was calculated as the number of NalR (or KanR, SpeR, Tmp R ) and RifS transconjugants per RifR donor cells. Shown are mean values of transfer frequencies (vertical bars) and standard error of the mean (sem, vertical lines) of two independent experiments at each temperature. Mean transfer frequencies (6sem) at 4uC: 1.3(60.7)61028 for irp2K, 0.7(60.3)61028 for ureBS and 0.4(60.4)61028 for or5075T, and at 12uC: 0.03(60.02)61028 for the three loci. Transfer frequencies at temperatures $20uC were system- atically below the detection limit (10210). doi:10.1371/journal.pgen.1002529.g001 pGDT4 is transferable Plasmid analysis of transconjugants resulting from the co- incubation of the 637-irp2K-ureBS donor strain with the 637c-NalR recipient revealed that about half of them had acquired pGDT4 together with the chromosomal irp2 (8/20) or ureB (11/20) tagged region, thus indicating that pGDT4 is also transferable. To further study pGDT4 transfer capacity, the plasmid was labeled by allelic exchange of the pGDT4-4kb segment with a Tmp cassette. When the 637-irp2K-ureBS donor strain carrying the tagged pGDT4T was co-incubated with the 637-NalR recipient, transconjugants harboring pGDT4T were obtained with a frequency approximately 103 times higher than that of chromo- somal genes (Figure 2A and Table S3). The transfer frequency of pGDT4T increased 200 fold when 953-NalR instead of 637-NalR was used as a recipient (Figure 2B and Table S3), indicating that properties inherent to the recipient cells may influence their capacity to take up pGDT4. The difference in the ability of the two strains to acquire this plasmid could not be explained by a mechanism of surface exclusion, as the frequency of transfer of pGDT4T to IP32637 harboring or not harboring a resident pGDT4 was similar (Figure 2B and Table S3). Some unidentified intrinsic properties of the recipients such as a difference in their restriction/modification systems may be responsible for this difference. As observed with chromosomal DNA, no transfer of pGDT4T was detected when the bacteria were mated at temperatures $28uC (Figure 2C and Table S3). However, in contrast to chromosomal DNA [3], pGDT4T was transferable on nitrocellulose filters, at frequencies similar to those observed in a liquid medium (Table S3), but again, only at low temperature (Figure 2C). Therefore, transfer of pGDT4 is also temperature- dependent, but in contrast to chromosomal genes, it occurs at much higher frequencies, and both in liquid and on solid media. Since the presence of pGDT4 is required for transfer, we wondered whether its presence could confer GDT4 properties to a strain that is naturally unable to mediate chromosomal transfer. For this purpose, a 953-ureBS transconjugant that had acquired Generalized DNA Transfer in Y. pseudotuberculosis pGDT4 simultaneously with chromosomal genes was used as donor and co-incubated with a 637DHPI-RifR recipient. While the parental 953-NalR strain was unable to transfer chromosomal DNA, the 953-ureBS(pGDT4) transconjugant gained the capacity to retransfer the acquired ureBS locus, though with a frequency 10 times lower (1029) than that observed during the first transfer. These results further point at pGDT4 as a key element in the mechanism of chromosomal transfer. Sequence analysis of pGDT4 To determine whether pGDT4 could encode its own transfer machinery, the plasmid was sequenced (EMBL accession number FM178282). The schematic map of the 94,967 bp circular plasmid molecule is shown on Figure 3. Of the 102 predicted coding sequences (cds) identified on pGDT4, 74 had homologs in databases (Table S4). Four major functional groups of genes were delineated on pGDT4: 1) 2) 3) A DNA fragment of <44 kb (pGDT4_0086-0024) carried genes predicted to be involved in conjugative transfer. Overall, these genes showed the closest similarity with those of the 153 kb conjugative plasmid pADAP of Serratia entomophila [11] that encode proteins involved in mating pair formation (MPF), DNA transfer and post conjugative replication. The pGDT4 MPF belongs to the MPFI class, according to the most recent classification, for which R64 is the paradigm [12]. Interestingly, we found that in pGDT4, traX and traY are fused in a single traXY gene, while they are separated but adjacent in the related pADAP MPF operon. This is the first report of such organization, which seems to be functional as transfer of pGDT4 is occurring at reasonable rates. The classification of conjugative plasmids based on their encoded relaxase has recently emerged as powerful and meaningful in terms of plasmid physiology [13,14]. The pGDT4 MobA relaxase is likely encoded by pGDT4_0022. This putative relaxase and its closest homolog, the protein pADAP_128 from pADAP, both fall in the MOBP13 class [14]. Proteins encoded by its neighboring genes, pGDT4_0023 and 0024, likely corre- spond to the relaxosomal accessory proteins MobB and MobC that act together with MobA in this family. Usually, in this type of mobABC backbone, the oriT is located between mobC (pGDT4_0024) and mobB (pGDT4_0023), as demon- strated for instance in pADAP [11]. The second group of genes gathered loci involved in plasmid stabilization and replication such as anti-restriction and toxin-antitoxin genes (pGDT4_0028-0031), the parFG oper- on (pGDT4_0062/0063, Table S4), encoding a partition system of type I (Walker type ATPase), and a replication initiation protein of the IncFII_RepA family (pGDT4_0081, Table S4). This replication protein is highly related to the RepA protein encoded by the Yersinia enterocolitica plasmid pYVe8081 [15], but this plasmid is otherwise unrelated to pGDT4 and has, for example, a partition system from a different family (SopAB). The third group corresponded to mobile genetic elements, mostly transposases and insertion sequences (IS), which are commonly encountered on plasmids. pGDT4 carries one ISL3 transposon, one uncharacterized IS (ISNCY) with a transposase of the ISPlu15 subgroup belonging to the ISNCY orphans in the IS repository ISfinder, four copies (three complete and one truncated) of a new IS6 family member (ISYps1), and two copies of the transposon ISYps3 (Figure 3 and Table S4). The latter is a 7.3 kb Tn3-like transposon PLoS Genetics | www.plosgenetics.org 4 March 2012 | Volume 8 | Issue 3 | e1002529