well as the Ig-like domain and parF genes (but not traM, Figure 5d). rpUC4K-10 has thus most likely acquired the entire pGDT4 sequence located between the two ISYps3 transposons (Figure 5). Altogether, these results show that most pUC4K transfers generated a variety of genetic modifications that were systemat- ically accompanied by the transposition of the pGDT4-borne ISYps1 element. Replicative transposition of ISYps1 and formation of cointegrates ISYps1 belongs to the IS6 family, known to transpose through replicative transposition. This mode of transposition gives rise exclusively to replicon fusions (cointegrates), in which the donor and target replicons are separated by two IS copies in direct orientation. The cointegrate can be subsequently resolved by recombination between the two IS copies [17]. To determine whether ISYps1 transposes through this mechanism, this IS was cloned into the suicide mobilizable vector pSW23T and introduced into a replication-permissive E. coli strain, yielding v7249(pSWYps1.1) (Table 1). After mating of this donor strain with v4826, a non-replication permissive recA- recipient, v4826::pSWYps1.1 transconjugants resulting from pSWYps1.1 integration into the recipient chromosome were obtained with a frequency of 8.5(60.4)61026 (which corresponds to the frequen- cies of both conjugation and transposition, Table S5). Since the frequency of conjugation under these conditions was found to be 3.4(60.9)61023 (Table S5), the transposition frequency of ISYps1 is thus approximately 261023. The genomic DNA of eight independent v4826::pSWYps1.1 colonies were digested with HindIII (which cuts once in pSW23T and not in ISYps1), and hybridized with an ISYps1 probe. All eight clones harbored, as expected, two integrated copies of ISYps1 (Figure S2). Of note, all clones exhibited different hybridization profiles. To further determine whether association between ISYps1 and a conjugative plasmid allows cointegrate transfer, the IS was cloned on a non-mobilizable vector (pSW23) and introduced into an E. coli strain carrying the conjugative plasmid R388 (Table 1). After mating of the resulting pi3(R388, pSWYps1.2) donor strain with the v4826 recipient that cannot sustain pSWYps1.2 replication, v4826(R388::pSWYps1.2) transconjugants were independently selected on Cm (pSWYps1.2 tag) and Tmp (R388 tag) MH agar plates. CmR clones were found at a frequency of 9(63)61025 (Table S5) and were all TmpR, while in the absence of an ISYps1 carried on the pSW23, no CmR transconjugants were obtained (Table S5). This further demonstrates that Yps1 drives the formation of cointegrates that can be subsequently transferred by conjugation. Under these conditions, R388 was transferred at a frequency of 2.2(60.7)61021 (Table S5), indicating that transfer of these cointegrates occurs at high frequencies (<261025). To further characterize these events, the plasmid profiles of five independent R388::pSWYps1.2 cointegrates were analyzed after restriction with MfeI (10 sites in R388, one in pSWYps1.2). All five pSWYps1.2 insertions were in different locations on R388 (data not shown). The two MfeI junction fragments from one of these R388::pSWYps1.2 cointegrates were cloned into the EcoRI site of pUC18 and the precise cointegrate location was determined by sequencing. Transposition of pSWYps1.2 occurred in the orf5 cassette of the R388 integron [18] and led, as for the two pUC4K insertion events analyzed above, to an 8 bp duplication. The duplicated sequence (GATCCGAG) was different from the other two, further indicating the absence of a specific integration site. Our results thus demonstrate that ISYps1 is able to transpose into a variety of insertion sites by replicative transposition through Generalized DNA Transfer in Y. pseudotuberculosis cointegrate formation, mediating the transfer of potentially any piece of non-mobilizable DNA molecule. Discussion We have evidenced a mechanism of HGT that convey the conjugative transfer or virtually any piece of chromosomal or plasmid DNA in a natural isolate of Y. pseudotuberculosis. This mechanism shares some characteristics with those previously described, but has several novel and unique properties. GDT4 is not observed at temperatures $20uC and its efficiency increases as the temperature decreases. Although some conjugative plasmids have been previously shown to be self-transferable at 14uC but not at 37uC [19,20], to our knowledge no plasmid able to conjugate at 4uC has ever been described. Temperature- dependent plasmid transfer is primarily mediated by H-NS and Hha proteins, which can be both plasmid and/or chromosome encoded [20]. The pGDT4 sequence did not reveal any gene encoding such proteins, but it is known that chromosomally encoded Hha and YmoA (equivalent to H–NS) act as thermoreg- ulators in Y. enterocolitica [21]. These proteins (also encoded by the Y. pseudotuberculosis genome [8]) may thus modulate pGDT4 transfer at cold temperatures. Interestingly, H-NS is an integral part of bacterial stress response pathways and its function is known to be sensitive to changes in environmental conditions such as temperature [22,23]. A cold stress could thus be a signal for the bacteria to transfer their genetic material by GDT4. The low temperature and an iron-poor liquid environment may also induce changes in the bacterial membrane structure, as observed for the closely related organism Y. pestis, in which the transcription patterns of various genes encoding components of the bacterial membrane were modified during iron starvation [24] or growth at 10uC [25]. These modifications might facilitate the formation of pores through which chromosomal DNA could translocate. Indeed, large cell aggregates in which bacteria appeared to be connected by bridges were observed. Similar tight bacterial contacts, designated conjugative junctions [26] or conjugational junctions [27] have been observed during RP4 or F-mediated mating of E. coli, respectively. However, these physical properties do not seem to be pGDT4-mediated, as we also observed large bacterial aggregates and possibly intercellular channels with IP32953, a strain that does not harbor this plasmid (data not shown). These bacterial aggregates have some similarities with biofilms in which bacteria are also closely connected. Biofilm formation occurs under natural conditions in a variety of bacterial species [28], including Y. pseudotuberculosis [29]. It could thus be hypothesized that GDT4 may take place between bacteria residing within biofilms in their natural ecological niches. This observation also suggests that acquisition of new functions, including virulence factors, by Y. pseudotuberculosis takes place in the environment rather than in a mammalian host. Another characteristic feature of GDT4 is that transfer of chromosomal DNA occurs only in a liquid medium. Like other conjugative processes, GDT4 requires a pilus-like mating system and a mating channel to occur, as demonstrated by the fact that inactivation of the pGDT4-borne pilus apparatus abolished this mechanism. While some plasmids transfer better on plates, some others encoding long flexible pili allow DNA transfer efficiencies of the same magnitude in liquid and on solid media [30,31], and this applied to pGDT4. In contrast, the absence of transfer of chromosomal DNA on agar was unexpected. Also unexpected was the fact that the efficiency of transfer in broth was not affected by a strong agitation, as opposed to recent findings showing that a vigorous shaking negatively affected the transfer of several PLoS Genetics | www.plosgenetics.org 7 March 2012 | Volume 8 | Issue 3 | e1002529