coli was demonstrated. Further study is needed to look for appropriate genetic tools to analysis the transposition of Tnces in Bacillus spp. and the dynamics of other MGEs flanking the ces gene clusters. Methods Mocetinostat molecular weight strains and plasmids Emetic strains used in this study are listed in Table 1. A non cereulide-producing B. cereus isolate CER071 was used as negative control. E. coli DH5α and JM109 were
used as PXD101 bacterial hosts in electroporation experiments. Plasmid R388 (Trimethoprim resistant) , a conjugative plasmid devoid of transposon, was used for transposition assay. E. coli was routinely cultivated at 37°C in Luria-Bertani (LB) media. B. cereus group strains were grown at 30°C. Antibiotics were used at the following concentrations: Kanamycin (Km), 50 μg/ml; Ampicilin (Amp),
50 μg/ml and Trimethoprim (Tp), 50 μg/ml. Insertion site determination of the cereulide gene cluster and Tnces::Km Regions flanking the cereulide gene cluster sites of the emetic B. cereus isolates and the target site and flanking sequences of the composite transposon were obtained by the method of genome walking (Takara genome walking kit), using the primer walking sets listed in Table 3. All the sequences obtained by this method were validated by PCR and subsequent sequencing. Table 3 Primers used in this study Primers Target Sequences (5’ → 3’) EmF cesB GACAAGAGAAATTTCTACGAGCAAGTACAAT EmR GCAGCCTTCCAATTACTCCTTCTGCCACAGT 14 F pXO1-14 GGTAAAGAGTGCGGAAAATGA 14R AATACGCCAACGCCAACTTA Vildagliptin AZD9291 supplier 45 F pXO1-45 TGCAGCTCGTAATCCACAG 45R TGCTAATGATAAAACGCCTGG 50 F pXO1-50 TTCGTACAGATGAAACACAGG 50R GTGCCTCAAGATGAACCTTC 55 F pXO1-55 GATAGAGACTGCTCTTGGGAA 55R GGTCTTAGCCATGAGAGTAAAAACA 58 F pXO1-58 TGTGATGGACCTTTGTATTAATTTGT 58R ATACCCCGCATGGAGCTTAG ISF_SacI ISces GCAGAGCTCGGTTCTGGTGCAAAAACTTCAGGACA ISR_XbaI GCATCTAGAGGTTCTGGTGCAAAAAGATAATAAAG ISF_HindIII ISces GCAAAGCTTGGTTCTGGTGCAAAAACTTCAGGACA ISR_BamHI GCAGGATCCGGTTCTGGTGCAAAAAGATAATAAAG KmF_XbaI Km TCATCTAGATAAACCCAGCGAACCATTTG KmR_BamHI TCAGGATCCTCTAGGTACTAAAACAATTCATCCAG ISF3 ISces
TCTGGTGCAAAAACTTCAGG ISR3 AAGTCGCATACGACCAGGTA kmF3 Km GAGGATGAGGAGGCAGATTG KmR3 CGGCCAGATCGTTATTCAGT APF1 bla TTTGCCTTCCTGTTTTTGCT APR1 TTGCCGGGAAGCTAGAGTAA ISL-SP1 CTTCATCCTCTTCGTCTTGGTAGC ISL-SP2 GGTTCGCTGGGTTTATCTAGAGGT ISL-SP3 GACAGACTGGTCCCGTAAATCAAC ISR-SP1 ATATCGGGGAAGAACAGTATGTCG ISR-SP2 GTACCTAGAGGATCCGGTTCTGGT ISR-SP3 GACAGACTGGTCCCGTAAATCAAC IS-LR CTTTCGAATCAACAGCACGA CesD-SP1 GGCCTATTGTATAATGACAACG CesD-SP2 GGTGTATTATTTATCTTCGCCTG CesD-SP3 GGTATTTTAGGGGCGAAGGTTC MH-SP1 CACTCTTGCGTTTTTGCGTATC MH-SP2 AAACAATGAGCCCACCCCGAAA MH-SP3 CGCTTTTCCACATTCTTTACGG DNA manipulation and plasmid construction Plasmid and genomic DNA were isolated using Plasmid Mini-Midi kits and Bacterial genome extraction kit (QIAGEN), respectively.