PCR was performed using the forward primer, 5′-ACGACAGGAAACCCTTTAGG-3′ and the reverse primer was 5′-AGCGTAATAAACAGGCACGC-3′. selleck chemicals It was also cloned into a pGEM-T easy vector (Promega). The imp/ostA and msbA genes were deleted by inverse PCR, and a chloramphenicol-resistant cassette (a gift from Dr. D. E. Taylor, University of Alberta) with its coding region (from the 1-bp start codon to the 624-bp stop codon)

was then cloned into the flanking regions to replace the full-length imp/ostA or msbA gene. This plasmid was natural transformed into the wild-type NTUH-S1 strain to generate click here deletion mutants. Chromosomal DNA of the transformants was checked by PCR with primers external and internal to the replacement site to verify the double-crossover event. Complementation of imp/ostA and msbA An imp/ostA complementation strain of NTUH-S1 was constructed as described previously [14]. The promoter site of msbA gene was predicted by using a tool available at the following website: http://​www.​fruitfly.​org/​seq_​tools/​promoter.​html. The msbA 3-Methyladenine clinical trial gene containing the predicted promoter region (upstream 73 bp) was obtained by PCR using the forward primer: 5′-CCAATCGCTTTAAGCTG-3′, and the reverse primer: 5′-TTAGCATTCTGTCAAACGCC-3′. Then the DNA fragment was cloned into the pGEM-T easy vector (Promega). The msbA gene with its promoter region was cut from the constructed pGEM-T easy vector and ligated

into the NruI site of the shuttle vector pHel3 (plasmid pHel3 was a gift from Dr. R. Haas, Max-Planck-Institute für Biologie, Tübingen, Germany). The constructed shuttle vector Amino acid was natural transformed into an msbA deletion mutant strain to generate the msbA complementation strain. Construction of the imp/ostA and msbA double

deletion mutant The gene encoding MsbA with its upstream 458-bp and downstream 474-bp flanking region was cloned into the pGEM-T easy vector as described above. A kanamycin-resistant gene aphA-3 from Campylobacter jejuni was then cloned between the flanking regions to replace the full length msbA gene. This plasmid was natural transformed into the wild-type NTUH-S1 strain to generate the deletion mutant. Chromosomal DNA of the transformants was checked by PCR with primers external and internal to the replacement site to verify the double-crossover event. Then, chromosomal DNA from msbA deletion mutant strain (Kmr) was natural transformed into the imp/ostA deletion mutant to obtain a double deletion mutant strain. It was also confirmed by PCR with primers external and internal to the msbA gene replacement site. Southern blotting Approximately 5 μg of genomic DNA from H. pylori NTUH-S1 and the mutants was digested by Hind III and incubated at 37°C overnight for complete digestion. The digoxigenin-labeled imp/ostA and msbA probes (primers were the same as those described for slot blot) was generated by PCR.