Author Summary All living species have the capacity to evolve in order to adapt to new and often hostile conditions. Horizontal gene transfer is a major route for rapid bacterial evolution. Some clearly identified mobile genetic elements (plasmids, phages, etc.) are by essence exchanged between bacteria. However, the mechanisms generating the bacterial core genome diversity are much less understood. In this study we have characterized in Y. pseudotuberculosis, a natural bacterial pathogen causing mesenteric lymphadenitis and enteritis, a mechanism of horizontal gene exchange that conveys the transfer of virtually any piece of chromosomal or plasmid DNA to a new bacterial host. This generalized mechanism of DNA transfer is optimal when the bacteria encounter conditions that might resemble those they met in their natural ecological niches. We demonstrate that this transfer mechanism is extremely powerful, as the presence on a conjugative replicon of an insertion sequence having a low specificity of insertion and transposing through replicative transposition is sufficient to drive the horizontal transfer of virtually any piece of chromosomal or episomal DNA. As such, this mechanism is much less constrained than the classical Hfr mechanism described in laboratory E. coli and could be used by a wide variety of bacterial species for gene exchange and evolution. The aim of this work was to characterize the mechanisms underlying horizontal chromosomal gene transfer in Y. pseudotu- berculosis. We describe here a natural system of conjugative transfer, which may be used by a wide variety of bacterial species for gene exchanges, and which may represent a driving force for bacterial evolution. Results Generalized transfer of chromosomal and plasmid DNA in Y. pseudotuberculosis IP32637 Since we did not know whether the lateral transfer process previously observed was limited to the region encompassing the HPI or could involve any portion of the chromosome, two other loci (ureB and or5076) were labeled with a spectinomycin (Spe) and trimethoprim (Tmp) resistance cassette, respectively. These two genes were chosen because, based on the IP32953 sequence, they are predicted to be separated from each other and from the HPI (tagged with a kanamycin (Kan) cassette in the irp2 gene) by at least 1.5 Mb of chromosomal DNA (Figure S1). Moreover, the ureB gene, which is part of the urease locus, and or5076, encoding a putative toxin transporter [8] are not predicted to be involved in DNA transfer. After co-incubation of the donor 637-irp2K-ureBS- 5076T and recipient 637DHPI-NalR strains (Table 1) under conditions (4 days at 4uC in LB-aa’ with shaking) that we previously found to be optimal for HPI transfer [3], recipient strains having acquired either the irp2K (NalR, KanR, RifS), ureBS (NalR, SpeR, RifS) or or5076T (NalR, TmpR, RifS) antibiotic resistances were obtained. Acquisitions of the corresponding tagged loci were checked by PCR (Figure S1). Transfer frequencies were of the same magnitude for the three antibiotic- tagged loci (<1028, Figure 1). None of the transconjugants obtained had simultaneously acquired two of the antibiotic-tagged loci, indicating that the sizes of the chromosomal fragments transferred were inferior to 1.5 Mb. We previously showed that horizontal transfer of the HPI occurs only at low temperatures [3]. The same temperature dependency was observed for ureBS and or5076T: transfer of each of the three Generalized DNA Transfer in Y. pseudotuberculosis antibiotic-tagged loci was detected only when the donor and recipient strains were co-incubated at temperatures below 20uC (Figure 1), and was more efficient at 4uC than at 12uC ($13 fold higher), as previously observed for irp2K. Therefore, distantly located chromosomal loci can be transferred with similar efficiencies and temperature regulations. Whether this transfer mechanism could also mediate horizontal transmission of episomal molecules was addressed by introducing the non-conjugative and non-mobilizable plasmid pUC4K (KanR) into the donor 637-RifR. Using the defined optimal growth conditions, transfer of pUC4K from the 637(pUC4K) to the recipient 637DHPI-NalR was obtained and confirmed by PCR with primers 210A/210B (Table S1). This transfer occurred at a frequency of 2.3 (60.4)61027, which is at least 10 times higher than that of chromosomal loci. Therefore, the process of DNA transfer is not limited to chromosomal DNA but can also involve plasmid molecules. Altogether our results demonstrate the existence of a mecha- nism that potentially allows transfer of any chromosomal or episomal DNA molecule at low temperature. This mechanism was thus named GDT4 (for Generalized DNA Transfer at 4uC). IP32637 harbors a plasmid involved in GDT4 The capacity of other Y. pseudotuberculosis strains to mediate GDT4 was studied by tagging the IP32953 and IP32777 strains with both a Kan and Spe cassettes inserted into the irp2 and ureB loci, respectively (Table 1). When these two recombinant strains were used as donors, no IP32637 transconjugants having acquired either irp2K or ureBS were obtained, indicating that GDT4 is not a property common to the entire Y. pseudotuberculosis species. Strain IP32637 has the peculiarity of harboring an extra high molecular weight ($100 kb) plasmid [9]. The role of this additional plasmid in chromosomal transfer was assessed by comparing GDT4 in IP32637 and its IP32637c plasmid-cured derivative [9]. Two tagged donor strains, 637c-irp2K and 637c- ureBS (Table 1), were generated and co-incubated with the 953- NalR recipient. No transconjugants were obtained, indicating a role of this plasmid in DNA transfer. The high molecular weight plasmid was thus designated pGDT4. pGDT4 does not appear to be ubiquitous in the species Y. pseudotuberculosis as the genome sequences of IP32953 and of other Y. pseudotuberculosis strains available in databases did not evidence the presence of this plasmid. To get an insight into the frequency of pGDT4 carriage in this species, a 4 kb HindIII fragment of this episome, designated pGDT4.seq was cloned into pUC18, sequenced, and used to design primers (358A/B) for PCR screening. The analysis of a panel of 39 Y. pseudotuberculosis strains of serotypes I to V (Table S2) for the presence of the pGDT4 sequence identified two isolates (IP32699 and IP30215) that gave a PCR product of the expected size (Table S2). Both strains contained high molecular weight episomes whose HindIII- digestion patterns yielded some restriction fragments with a size similar to those of pGDT4, but the overall profiles of the three episomes were different (data not shown). Therefore, the plasmids found in IP32699 and IP30215 probably share some regions with pGDT4, but they are not identical to this plasmid. Since Yersinia pestis is a recent descent of Y. pseudotuberculosis [10], we also screened by PCR a panel of 51 strains of Y. pestis belonging to the three classical biovars (Antiqua, Medievalis and Orientalis) for the presence of the pGDT4-borne sequence. None of the strains tested yielded an amplification product (Table S2), suggesting the absence of vertical or horizontal transmission of pGDT4 to Y. pestis. PLoS Genetics | www.plosgenetics.org 2 March 2012 | Volume 8 | Issue 3 | e1002529