The PCR products were subsequently digested with BglII (or PstI), and ligated with a kanamycin or chloramphenicol

resistance cassette (aphA-3 or cat; [43, 44] flanked by the compatible BamHI (or BglII) restriction sites. The direction of transcription of the antibiotic resistance genes (kanamycin [Km] and chloramphenicol [Cm]) was the same as that of the target gene to avoid possible polar effects. Plasmids containing the interrupted gene were used as suicide plasmids for natural transformations buy PS-341 of the H. pylori strain 26695. The successful chromosomal replacement of the target gene with the disrupted gene construct via allelic exchange (double crossover) was checked by PCR using suitable primer combinations. Functional complementation of mutants Functional complementation experiments for the uvrB and uvrC mutant strains were performed by inserting an intact copy of the

FG-4592 research buy target gene into the ureAB locus (Additional file 4: Table S3). To do so, the ORFs HP1114 and HP0821 were cloned in the pADC vector [45] downstream of the strong ureAB promoter, creating the plasmids pSUS2646 and pSUS2644 (Additional file 4: Table S2 and S3). Functional complementation of uvrA was performed by inserting an intact copy of the uvrA gene together with 400 bp of DNA upstream of the start codon containing the putative uvrA promoter into the rdxA locus. The ORF HP0705 plus the upstream region were cloned in the pCJ535 vector, creating the plasmid pSUS3009. These suicide plasmids were introduced via natural transformation into the single gene mutant strains 26695 uvrA, 26695 uvrB, and 26695 uvrC, and the transformants were selected on Km/Cm blood agar plates. The correct insertion of the complementing genes in the ureAB or rdxA locus was controlled by PCR and sequence analysis of the

insertion sites. In vitro transformation system of H. pylori, determination of mutation and recombination frequencies and import sizes The transformation system used to quantitate, in parallel, mutation and recombination rates as well as the length of the DNA fragments incorporated into the chromosome after recombination has been described previously [12]. Mutation rates obtained with this system have been shown to be in excellent agreement with fluctuation analysis [42]. From each experiment, at least 16 clones Aldol condensation were expanded in order to sequence a fragment (1663 bp) of the rpoB gene (see below). The experiments were reproduced three times for each H. pylori mutant strain. To determine the length of import events in the rpoB gene, a 2361 bp PCR fragment of rpoB was amplified with primers HPrpoB-1 and HPrpoB-6 as previously described [12] and Additional file 4: Table S2). This PCR product was used as template for the sequencing reactions with the primers HPrpoB-3, -4, -5, -6, -9w, and −10. The six sequences from each rifampicin resistant clone were assembled using the software Bionumerics V 4.