and is mediated by the F plasmid of E. coli [38–40]. F integrates stably into the E. coli chromosome through homologous recom- bination between IS copies present on both the F plasmid and the bacterial chromosome [38–42] to create Hfr strains, with transfer origins located at different chromosomal loci [43,44]. In contrast to the classical Hfr mechanism, integration of pGDT4 into the chromosome probably occurs, as in pUC4K, via the ISYps1- mediated replication fusion mechanism. At least three pieces of evidence support this hypothesis: (i) no IS element is shared by pGDT4 and the IP32637 chromosome, in contrast to what is expected for the Hfr mechanism, (ii) in Y. pseudotuberculosis, three distantly located chromosomal loci (irp2K, ureBS and or5076T) were transferred with similar frequencies, and (iii) in all eight E. coli transconjugants analyzed, pSWYps1 was inserted at different sites on the chromosome. ISYps1 thus appears to have a very low specificity of recognition, allowing its insertion at multiple sites on bacterial plasmids and chromosomes. After mobilization of the chromosomal fragment adjacent to the pGDT4 integration site and transfer to a recipient strain, following the Hfr-type transfer model, homologous recombination between the incoming DNA and the chromosome is expected to take place, leaving no trace of pGDT4 in the chromosome of the transconjugant. Our previous observation that RecA activity is necessary in the recipient, but not in the donor strain for chromosomal transfer [3], and the results of the present study showing that pGDT4 is absent from some transconjugants that have acquired chromosomal genes further support this model of horizontal transfer. Our study thus validates the model proposed by Willets et al. in the 1980’s for the mobilization of the E. coli chromosome via the formation of a cointegrate with the R68.45 plasmid during IS21 transposition [45]. Such cointegrate formations were widely used at that time to establish the genetic map of various bacterial species (see for instance [46,47]). Most importantly our results show, without the need for heterologous plasmids like RP4 or R68.45, that this type of chromosomal conjugative transfer may occur under natural conditions in wild type bacterial pathogens carrying endogenous plasmids. The capacity of wild type bacteria to naturally transfer large pieces of chromosomal DNA following the typical Hfr mechanism of homologous recombination between identical IS copies on the chromosome and the plasmids has been documented in a variety of bacteria, including extremophiles [48], Gram-positive cocci [49], and actinomycetes [50]. What we describe here is certainly a less constrained and more powerful mechanism, as it only requires the presence of an IS of the IS6 family on a conjugative replicon to generate cointegrates able to drive the horizontal transfer of any piece of DNA (chromosomal or episomal). It is remarkable that a high density of IS is commonly observed on plasmids. For instance the Shigella plasmid pWR100 carries 93 copies of complete or truncated IS belonging to 21 different types [51]. Thus, more than being IS depository, this location may reflect the broad selective advantage brought by plasmid/IS associations as a chromosomal transfer device. Such a ‘genetic symbiosis’, offers a means for the natural transfer of large blocks of genes conferring new metabolic properties or virulence functions. According to our model, GDT4 does not leave any signature in the recipient genome in most instances, and therefore its contribution to the numerous horizontal gene exchanges that shape bacterial genomes can hardly be quantified. However, according to the ISfinder database, approximately 5% of the known IS belong to the IS6 and Tn3 families, which use a replicative transposition mecha- nism. As they are found in all bacterial and archaeal phyla, the mechanism we describe here might be responsible for a substantial fraction of gene exchanges occurring among bacterial species. Generalized DNA Transfer in Y. pseudotuberculosis Remarkably, this mechanism of DNA transfer was optimal when the bacteria were grown under conditions (low temperatures, iron poor medium, biofilm-like bacterial aggregates) that might be close to those met by these microorganisms in their normal ecological niches. This natural GDT mechanism may thus play a significant role in bacterial evolution, genetic polymorphism, pathogenesis and adaptation to new environmental conditions. Methods Bacterial strains and growth conditions Bacterial strains used in this study are listed in Table 1 and Table S1. Wild type strains were taken from the collection of the Yersinia Research Unit (Institut Pasteur). Bacteria were grown in LB (Luria Bertani) or MH (Mueller Hinton) medium for 24 h at 28uC (Yersinia) or 37uC (E. coli) with agitation, or for 48 h on LB or MH agar plates. When necessary, kanamycin (Kan: 100 mg ml21), rifampicin (Rif: 100 mg ml21), nalidixic acid (Nal: 25 mg ml21), spectinomycin (Spe: 50 mg ml21), tetracycline (Tc: 15 mg ml21), chloramphenicol (Cm: 25 mg ml21), trimethoprim (Tmp: 20 mg ml21), thymidine (dT: 0.3 mM) or the iron chelator aa’- dipyridyl (0.2 mM, Sigma) were added to the medium. Mutagenesis of chromosomal or plasmid genes Spe (aadA) or Tmp (dfr) non-polar cassettes were PCR-amplified using primers described in Table S1, and pSW25 [52] or pGP704N- dfr [3] as templates, respectively. All allelic exchanges of chromosomal or plasmid genes by an antibiotic resistance cassette were done following the LFHR-PCR procedure [53]. The Spe and Tmp cassettes were introduced into the chromosomal ureB and or5076 genes, respectively, using primers that amplify upstream and downstream fragments of ureB and or5076, as shown on Figure S1 and Table S1. To label pGDT4, the plasmid was digested with HindIII and a 4 kb fragment (pGDT4-4kb) was purified and cloned into pUC18. Approximately 600 bp of each extremity of the cloned fragment were sequenced. These sequences were then used to design primers (358A/B and 359A/B, Table S1) that served for allelic exchange between the Tmp cassette and the target region of pGDT4 in strain 637-irp2K-ureBS. Correct insertion of the Tmp cassette was confirmed by PCR using primer pair 358A/359B. Mutagenesis of the pil region was done by replacing the pGDT4 region extending from pilL (pGDT4_0086) to pilV (pGDT4_0097) by a Tmp cassette, using primer pairs 773A/B and 774A/B (Table S1). The various antibiotic-tagged derivatives cured of pKOBEG- sacB were selected on sucrose plates. Growth conditions for DNA transfer and determination of the transfer frequency Optimal conditions for chromosomal DNA transfer in Y. pseudotuberculosis have been previously described [3]. Briefly, the donor strain (usually RifR) harboring chromosomal loci labeled with antibiotic cassettes and the recipient strain (usually NalR) were grown overnight in LB at 28uC with agitation. Equal amounts (56106) of donor and recipient cells were mixed in 25 ml of LB-aa’ and grown at 4uC with mild rotary agitation (80 rpm) for 4 days. Donor and recipient bacteria were quantified on Rif and Nal plates, respectively, and transconjugants were selected on Nal plates containing the appropriate antibiotic. To ensure that the colonies were not spontaneous NalR mutants of the RifR recipient strain, the Rif susceptibility of the transconjugants was systematically checked. For every single DNA transfer experiment, 10 to 20 transconjugant colonies were analyzed by PCR for the acquisition of the corresponding antibiotic-tagged locus with primer pairs 233B/166, 92A/322B and 348B/346A (Table S1) PLoS Genetics | www.plosgenetics.org 9 March 2012 | Volume 8 | Issue 3 | e1002529