Figure 4 Sketch drawing of the replicative transposition of Tn ces :: km into recipient chromosome and the strategy of hybridization. The transposase-mediated fusion of pTnkm and the target molecules generate a third copy of ISces. There are two theoretically possible results of transposition, depending on which ISces is duplicated. Three probes 1, 2, and 3, indicated

by dotted lines, represent an internal fragment of bla in cloning vector pUC18, ISces, and Km, respectively, were used for the survey of the transposition. The NdeI sites in kmRsmR transconjugants were indicated. No matter which ISces was duplicated, hybridization with probe 1 and 3, a 3.5 kb band and a 1.6 kb band is expected, respectively; with probe 2, besides the 1 kb and 3.5 kb expected bands, extra bands with variable sizes in each click here independent transconjugant are probably detected due to multi-transpositions. Although there is also a (remote) possibility for the duplication of the whole selleck kinase inhibitor Tnces::km element, the result will be similar except that more bands with probe 2 are expected. Figure 5 Southern blot hybridization analysis of the transconjugants of Tn ces :: km transposition in E. coli HB101. Two independent hybridizations

were performed. A: lane 1–4, independent KmRSmR transconjugants, lane 5, HB101, lane 6, JM109 (pTnkm); and B: lane 1–5, independent KmRSmR transconjugants, lane 6, HB101, lane 7, JM109 (pTnkm). Three probes of Km (a), ISces (b) and blapuc18 (c), respectively were used for hybridization as illustrated in Figure 4. To detect if the transposition of Tnces::Km displayed target site biases, the flanking sequences

of insertion Bumetanide sites of the transconjugants used in hybridization were determined by primer walking. For three transconjugants, it was found that Tnces::Km insertions occurred in three distinct sites on plasmid R388 and that an 8-bp direct repeat (DR) was produced after transposition (Table  2), which is a typical feature of IS6 family members (see the ISfinder database, http://​www-is.​biotoul.​fr) [34]. For the other six transconjugants, although repeated several times, it is difficult to get the flanking sequences of insertion sites by primer walking, probably due to sequence complexity caused by multiple transposition events of ISces. Table 2 DNA sequences flanking the insertion sites after random transposition of IS ces based transposon Tn ces :: km onto R388 Transconjugants Sequence of insertion sites (5’ to 3’) Tn02 GCCAACTTCCAAAGGAAAGAAGCCGCATAACC-ISces-GCATAACCTGCCCTCCCCCGCTCCGGCGGGGG Tn04 GAAGGCCAACGGTGGCGCCCAAGAAGGATTTC-ISces-AGGATTTCCGCGACACCGAGACCAATAGCGGAA Tn05 GAGCGGGCTTTTTTATCCCCGGAAGCCTGTGGA-ISces-CCTGTGGATAGAGGGTAGTTATCCACGTGAAAC The underlined sequences refer to the duplicated target sequences (DR). Discussion The taxonomy of B. cereus group has long been controversial, since many of the species are genetically heterogenous, with the exception of B.