The frequency was calculated as number of transconjugants per donor; the range in the orders of magnitude obtained is shown. bNo transconjugants were detected under the detection level (<10-10). PstI EPZ5676 restriction profiles for the thirteen pA/C transconjugants selected for detailed Selleck BIBW2992 analysis (Table 4) showed that in some cases a distinct profile was generated in comparison with that of the wild-type YU39 pA/C transformed into DH5α (DH5α-pA/C). Examples of the plasmid (Figure 4A) and PstI restriction profiles
are shown (Figure 4B). Figure 4 Examples of pA/C transconjugants recovered in SO1 pSTV ::Km and DH5α. Panel A) shows the plasmid profiles of four different transconjugants in SO1 marked within dotted rectangles. The donor YU39 pA/C and the recipient SO1pSTV::Km strains are in the AZD5363 purchase first and last lanes, respectively. Within each dotted rectangle, in the first lane are the SO1 transconjugants; in the second and third lanes the DH5α transformants for the pA/C and pSTV of each transconjugant are shown. Panel B) displays examples of PstI restriction profiles of pA/C transconjugants of SO1
and DH5α compared with wild-type YU39 pA/C (DH5α-pA/C). In order to detect the presence of pX1 in the pA/C transconjugants, BamHI-NcoI restriction digests were performed, since these enzymes were used to analyze pX1. Most of the bands of the wild-type DH5α-pA/C were visible in selleck compound the restriction profiles of the transconjugants, but new bands were also evident (Figure 5). When hybridized with the complete pX1 as probe, positive signals in bands corresponding with the pX1 restriction profile were obtained in most
of the cases (Figure 5). SO1 transconjugant IA9 was negative for the pX1 hybridization, in agreement with the pX1 PCR screening; whereas the LT2 transconjugant IIIE9 produced hybridization signals, suggesting that this plasmid contained regions of pX1 not included in the PCR scheme (Figure 5 and Table 3). These results indicate that, with the exception of IIID8 and IIIE9, in most of the cases complete pX1 and pA/C formed co-integrates that were not resolved in the recipient strain. In any case, this finding indicates a type of cis-mobilization, in which the mobilized replicon is fused to a conjugative plasmid, which supplies both oriT and the tra functions . Figure 5 Representative restriction profiles for pA/C transconjugants.