bNo transconjugants were detected under the detection level (<10-

bNo transconjugants were detected under the detection level (<10-10). cNumber of transconjugants analyzed. dNumber of transconjugants positive for the repA/C find more PCR marker. eNumber of transconjugants positive for the oriX1 PCR marker. We calculated that the transposition and co-integration events occurred within YU39 at frequencies between

10-6 and 10-9, based on the difference between the conjugation frequency of pA/C + pX1 and pX1::CMY transconjugants (10-7 and 10-10; Table 2 and Table 4) compared with that of pX1ydgA::Tn5 (10-1; Table 5). It is worth noting that these conjugation experiments involving a DH5α donor carrying pA/C and pX1 produced the same results observed as when the YU39 wild-type strain was used as donor, indicating that the interaction between these plasmids did not require additional elements from the YU39 genome. pColE1-like was preferentially

trans-mobilized along with pA/C To determine the genetic identity of the 5 kb plasmid the band was purified, digested and cloned. The sequences from the cloned fragments showed homology to the replication and mob genes of ColE1 plasmids, indicating that the 5 kb was a ColE1-like plasmid (pColE1-like). PCR screening using specific primers to amplify the pColE1-like mobA region (Additional file 3: Table S1) showed that YU39 and all the transconjugants displaying the 5 kb band were positive. The mobA PCR product was employed as a probe to hybridize YU39 and transconjugants Selleckchem Sotrastaurin plasmid profiles. These hybridizations confirmed

the identity of the 5 kb band and, in addition, showed that the pColE1-like was not involved in the formation of pA/C + X1 co-integrates or medroxyprogesterone pX1::CMY. The pColE1-like was mobilized in trans with all the DH5α pA/C + X1, with most of the SO1 pA/C transconjugants and with a few pX1::CMY transconjugants (Table 2), indicating stable co-existence with pA/C and pX1, and with pSTV when present. The YU39 pX1 is selleck chemical closely related to other E. coli and Salmonella pX1 The nucleotide sequences for the six regions selected for the pX1 PCR screening showed that the YU39 pX1 was highly similar to other pX1 plasmids. In a recent study, Johnson et al. proposed the use of the taxC sequence as a genetic marker to compare IncX plasmids [19]. The phylogenetic inference obtained by the comparison of the taxC partial sequence of the YU39 pX1 with those of IncX plasmids showed that it was closely related to other E. coli and Salmonella IncX1 plasmids (Figure 6). Similar phylogenetic reconstructions were observed for the other five YU39 pX1 sequences (data not shown). Figure 6 Genetic relationships of YU39 pX1 and other IncX plasmids. The dendrogram was constructed using the Maximum Likelihood method based on the HKY + G model with 500 bootstrap replicates.

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