as indicated on Figure S1. When the transfer of the irp2K locus was analyzed, the acquisition of the entire HPI by the recipient strain was further checked with primer pairs A10/144A and A9/143B (Figure S1 and Table S1). The frequency of DNA transfer was determined as the number of NalR (or KanR, SpeR, Tmp R ) RifS transconjugants per RifR donor cells. To determine whether free DNA molecules in the medium could mediate GDT4, the donor bacteria 637-irp2K-ureBS and the recipient 637DHPI-NalR were co- incubated in the presence of 100 U/ml of DNAse in the culture medium. The activity of the DNAse under these conditions was checked by adding 1 ug/ml of bacterial DNA to the culture medium and by observing that the added DNA was degraded. Transfer of pGDT4T Transfer of pGDT4T was studied after incubation of the donor (637-irp2K-ureBS(pGDT4T)) and various NalR recipient cells for four days at 4, 28 or 37uC in liquid or solid media. On solid medium, 26108 donor and recipient cells were mixed on a 0.45 mm nitrocellulose filter (Millipore) and at the end of the incubation period, the bacterial mixture was suspended in 1 ml of MH. Donor and recipient cells were quantified on MH-Rif and MH-Nal plates, respectively. Transconjugants having acquired pGDT4T were identified as NalR/Tmp R /RifS colonies. In each transfer experiment, 10 transconjugants were analyzed by PCR for the presence of pGDT4T with primer pair 358A/346B (Table S1). Finally, the pGDT4T transfer frequency was calculated as the number of NalR/TmpR/RifS transconjugants per donor cells. Sequencing of pGDT4 One transconjugant resulting from the co-incubation of the 637- irp2K-ureBS donor strain with the 637c-NalR recipient was used to obtain a plasmid extract which contained only pGDT4. Sequencing was performed using the whole genome shotgun strategy [54]. A 2– 3 kb insert library was generated by random mechanical shearing of pGDT4 DNA and cloning into pcDNA-2.1 (Invitrogen). Recom- binant plasmids were used as templates for cycle sequencing reactions consisting in 35 cycles (96uC for 30 s; 50uC for 15 s; 60uC for 4 min) in a thermocycler, using the Big dye terminator kit (V3.1, Applied Biosystems). Samples were precipitated and loaded onto a 96-lane capillary automatic 3700 DNA sequencer (Applied Biosystems). In an initial step, 1000 sequences from the library were assembled into 5 contigs using the Phred/Phrap/Consed software [55,56] (8-fold sequence coverage). Consed was used to predict links between contigs. PCR products amplified from the pGDT4 template were used to fill gaps and to re-sequence low quality regions using primers designed by Consed. Physical gaps were closed using combinatorial PCR. The correctness of the assembly was confirmed by ensuring that the deduced restriction map was identical to the one obtained experimentally. The traX and traY genes fusion into a single traXY gene was checked by re- sequencing this locus on the original pGDT4 DNA preparation. ISYps1, ISYps2 and ISYps3 designation were attributed by the ISfinder database (http://www-is.biotoul.fr/). The nucleotide sequence of pGDT4 has been submitted to the EMBL database under accession number FM178282. Details and properties of the different ISYps characterized in this work are accessible through the ISFinder web site. Electron microscopy Bacteria were negatively stained with 2% uranyl acetate onto glow discharged copper grids. The samples were observed in a Jeol 1200EXII and/or a JEM 1010 (Jeol) equipped with a Keenview camera (Eloise) at 80-kV accelerating voltage. Images were recorded with an Analysis Pro Software version 3.1 (Eloise). Generalized DNA Transfer in Y. pseudotuberculosis ISYps1 transposition assay The sequence corresponding to the ISYps1 copy carried on rpUC4K-6, flanked by its 113 bp upstream and 138 bp downstream regions, was amplified using primers 1039/1040 and cloned as an EcoRI-BamHI insert into the suicide mobilizable vector pSW23T [52], giving rise to pSWYps1.1. This plasmid was then introduced into E. coli v7249 [57], a strain allowing pSW23T replication and conjugative transfer. Conjugation between this donor strain and E. coli v4826 was performed as previously described [57], The frequency of conjugation–transposition frequency was calculated as the number of CmR transconjugants (v4826::pSWYps1.1) per total number of recipients (TcR). The conjugation frequency was established in parallel by conjugation from the same donor v7249(pSWYps1.1) to a v4826 pir+ derivative (obtained through transformation with plasmid pSU38Dpir which expresses pir [52]). The frequency of illegitimate recombination of the pSW23T which can lead to CmR transconjugants was established by conjugation between donor v7249(pSW23T) and v4826, and found to be 4.6(61.7)61028. Genomic DNA from 8 independent v4826::pSWYps1.1 colonies were extracted using QIAGEN Genomic Tips and buffer set, digested with HindIII, and hybridized with a probe internal to ISYps1 (generated by PCR amplification with primers 1041/1044 and labeled with a-32P dCTP, using the Random Primed labeling kit (Roche)). R388::pSWYps1.2 cointegrate formation assay The EcoRI-BamHI fragment carrying ISYps1 was transferred from pSWYps1.1 to the non-mobilizable version of pSW23 [52], giving rise to pSWYps1.2. This plasmid was then introduced into the E. coli pi3 pir+ strain that harbors the IncW conjugative plasmid R388, which does not carry any IS (GenBank BR000038), giving rise to pi3(R388, pSWYps1.2). Conjugation of this donor strain with v4826 yielded v4826(R388::pSWYps1.2). The fre- quency of cointegrate formation after mating was calculated as the number of CmR transconjugants per total number of recipients harboring R388 (TmpR). The ability of R388 to form transferable cointegrates with pSW23 in the absence of ISYps1 was assessed in the same conditions by replacing pSWYps1.2 by pSW23 in the pi3(R388) donor, and found to be inferior to 1029. Supporting Information Figure S1 Genetic map and location of primers used to insert antibiotic cassettes in the chromosomal irp2, ureB and or5076 genes. kan: kanamycin cassette, spe: spectinomycin cassette, tmp: trimethoprim cassette. (PDF) Figure S2 ISYps1 probing of 8 w4826::pSWYps1.1 transconju- gants after suicide conjugation of pSWYps1.1. (PDF) Table S1 Primers used in this study. The location of several of these primers is illustrated on Figure S1. Lower case letters indicate portions of spe (310B, 311A) or tmp (347B, 348A, 358B, 359A, 773B and 774A) sequences. (PDF) Table S2 Y. pseudotuberculosis and Y. pestis strains analyzed for the presence of pGDT4. These strains were taken from the collection of the Yersinia Research Unit (Institut Pasteur). +: amplification of a fragment of the expected size; 2: no amplification of a fragment of the expected size. A: Antiqua, M: Medievalis, O: Orientalis. (PDF) PLoS Genetics | www.plosgenetics.org 10 March 2012 | Volume 8 | Issue 3 | e1002529