08 and 0.52. In addition, the alignments from these BLAST hits were deemed correct as judged by comparison to the multiple alignment Ruboxistaurin clinical trial presented in Figure 1. For each of the FliJ and HP0256 sequence groups, both Paircoil2 and PCOILS were run (for PCOILS, the multiple sequence alignment used to generate Figure 1 was used) . Allelic exchange mutagenesis Helicobacter DNA was isolated as previously described . Oligonucleotides were purchased from Eurofins MWG Operon (Germany). Oligonucleotides ML022FP/ML027RP (Table 4) were designed for the amplification of a 216 bp fragment containing the 3′ end
of HP0255 and the 5′ end of HP0256. Oligonucleotides ML028FP/ML023RP (Table 4) were designed for the amplification of a 245 bp fragment GW786034 mw at the 5′ end of HP0256. ML027RP and ML028FP had overlapping learn more sequences and included a BglII restriction site. The two amplicons were joined together by extension overlap PCR and the resulting DNA product was cloned into pUC18 (New England Biolabs, USA) following BamHI and EcoRI digestion. The resultant plasmid was cut with BglII and ligated with the chloramphenicol acetyl transferase (cat) gene which had been cut from the plasmid pRY109 . H. pylori cells were transformed with 1 μg of this plasmid for double-cross over gene disruption as previously described . Polymerase chain reactions (PCR) were
performed using 3 μM of each primer and 0.5 units per reaction of Vent Polymerase (New England Biolabs). Table 4 Oligonucleotide sequences used in this study. Primer Sequence (5′-3′) Gene Comments flgE-F GGCTAACGAGCGTGGATAAG flgE FP of flgE flgE-R GAGCGAGCGCTAAAGTCCTA flgE RP of flgE era-F AAGGCTAATGCGACCAGAAA hp0517 Arachidonate 15-lipoxygenase FP of era era-R GGAGCCCTGGTGTGTCTAAA hp0517 RP of era ML022FP CGGGATCCCGGGGCGAAAGATTGGAGATTT hp0256 Allelic exchange
mutagenesis ML027RP CCATCGTAGATCTGGGCTGC AGCGAATTTTTTCATAGAAAAATCG hp0256 Allelic exchange mutagenesis ML028FP GCAGCCCAGATCTACGATGGGCAATTAAAAAGCGCTCTAAGAAT hp0256 Allelic exchange mutagenesis ML023RP CGGAATTCCGTTACGCATGCAAGCCCTC hp0256 Allelic exchange mutagenesis HP0256-F2 TATAACAAGGAGTTACAACAATGAAAAAATTCGCTTCTGTG hp0256 FP of hp0256 HP0256-R GCGCGCATCGATTTACGCATGCAAGCCCTCTT hp0256 RP of hp0256 FLA-F2 GCGCGCGGATCCCATGCTCCTTTAAATTTTGC flaA FP of flaA promoter FLA-R TGTTGTAACTCCTTGTTATA flaA RP of flaA promoter minD-F TAATTTAGCGATCGGCTTGG minD FP of minD minF-R TCCATCACATCCACCACATC minD RP of minD hp0610-F ATAACGGCGTTCATTCTTGG hp0610 FP of hp0610 hp0610-R GCGGTTGTTATGCAAGGTTT hp0610 RP of hp0610 omp6-F GCCCGATTCTAAAGGGTTTC omp6 FP of omp6 omp6-R GGCCAAACTCTTTGGTGGTA omp6 RP of omp6 hpn-F ATGGCACACCATGAAGAACA hpn FP of hpn hpn-R GATGAGAGCTGTGGTGGTGA hpn RP of hpn HP0256-QF GCGCGCCCATGG AAAAATTCGCTTCTGTATTGG hp0256 FP of hp0256 HP0256-QR GCGCGCGGATCC TTACGCATGCAAGCCCTCTTT hp0256 RP of hp0256 FP, forward primer; RP, reverse primer.