challenged LY2606368 purchase for a second round of conjugation using as recipients the original recipient strain (original) or DH5α. The frequency was calculated as number of transconjugants per donor; the range in the orders of magnitude obtained is shown. To address the extent of the CMY region transferred from pA/C to pX1 we used the PCR typing scheme developed in our previous studies (Figure 1A). Four of the pX1 transconjugants were positive for six of the seven genes present in the complete CMY region of pA/C (c. a. 12 kb), spanning from ISEcp1 to hypothetical protein 0093 (according to pSN254 annotation; GenBank:NC_009140); while the other four displayed a short version of the CMY region (c. a. 3 kb) including only ISEcp1, bla CMY-2, blc and sugE (Figure 1B and Figure 1C). Figure 1 Schematic diagram of the CMY regions of Typhimurium strain YU39 and pX1 :: CMY transconjugants. Panel A) shows a schematic diagram of the CYT387 CMY region in the pA/C plasmid of strain YU39 . Panel B) depicts a large CMY
region inserted into the intergenic region between 046 and 047 genes for IC2 transconjugant. Panel C) shows a short CMY region inserted into stbE gene for IIIC10 transconjugant. The PCR amplifications INCB28060 designed to assess the extension of the CMY regions are indicated by double arrowheads under the diagrams. The PCRs used to determine the pX1 CMY junctions are indicated by bars with circles. For the characterization of pX1 transconjugants IC2, ID1 and IIID2, that were negative for the 046-047 region, we used a combination of primers from the CMY region along with the primers for 046-047 to determine if this was the site of insertion (Figure 1B; PCRs H and I). We successfully
established that the IC2, ID1 and IIID2 transconjugants were positive for the CMY-046-047 junction (Table 3). Sequencing of these PCR products showed the exact insertion site for these pX1 transconjugants harboring a large CMY region. The schematic representation of the insertion of the CMY region into 046-047 in IC2 is presented in Figure 2A. Mapping according to the pOU1114 annotation revealed that the insertion site was in nucleotide 33,768. A repeat sequence of six nucleotides (TGAATA) flanking the CMY region was detected, corresponding to nucleotides 33,763 to 33,768 of pOU1114. We discovered that the hypothetical pheromone protein 0093 was truncated at nucleotide 4,168 removing 1,318 nucleotides of the complete ORF. Figure 2 Schematic representation for the insertion sites for the CMY region into the pX1 backbone. Panel A) depicts the insertion of the large CMY region into the intergenic region between 046 and 047 hypothetical proteins. Panel B) shows the insertion of the short CMY region into stbE. The numbers under the solid black arrows correspond to nucleotide numbers in the annotation of the reference pX1 pOU1114 (GenBank: DQ115387). The surrounding nucleotide sequences at the insertion points are shown.