Since the gastric habitat of H. pylori is likely to be rich in DNA damaging agents, it will be of interest to study the roles of NER components in H. pylori

genetic diversification under in vivo conditions, e.g. in suitable animal models. Finally, the results show the functional versatility of apparently conserved housekeeping proteins such as the NER components, emphasizing the importance of comparative functional analyses in diverse organisms, such as other naturally competent and recombining bacteria. Methods Bacterial strains and culture conditions Bacterial strains used in this study are listed in Additional file 4: Table S1. H. pylori wild type strains 26695 [21] and J99 [38] were cultured from frozen stocks on blood agar plates (Blood agar base II, Oxoid, Wesel, Germany) learn more containing 10% horse blood and a mix of antibiotics (vancomycin [10 mg/l], polymyxin B [3.2 mg/l], amphotericin B [4 mg/l], and trimethoprim [5 mg/l]). The agar plates were kept in an incubator with 5% O2, 10% CO2 and 85% N2 at 37°C for 24–48 h. Mutant strains were cultivated on blood agar plates containing kanamycin (20 μg/ml), chloramphenicol (20 μg/ml), or both antibiotics as required. Liquid cultures were grown in brain heart infusion (BHI, Oxoid) medium with yeast extract

(2.5 g/l), 10% heat inactivated horse serum and an antibiotics cocktail (see above) in microaerobic atmosphere using air-tight jars (Oxoid) and Anaerocult® C gas generating bags (Merck). For the DNA cloning experiments, we used E. coli strains DH5α Anlotinib [39] and MC1061 [40]. These strains were grown in LB broth or on LB plates (Lennox L Broth, Invitrogen GmbH, Karlsruhe, Germany) supplemented with ampicillin (200 μg/ml), chloramphenicol (20 μg/ml) and/or kanamycin

(20 μg/ml) as required. DNA techniques All standard procedures (cloning, DNA amplification, see more purification and manipulation) were performed according to standard protocols [41]. Total genomic bacterial DNA was prepared using the QIAamp DNA Minikit (QIAGEN, Hilden, Germany). Large-scale purification of bacterial chromosomal DNA was Cytidine deaminase performed using QIAGEN Genomic-tip 100/G columns according to the manufacturer’s instructions. Plasmid DNA from E. coli strains was isolated using QIAGEN tip 100 columns. Insertion mutagenesis in H. pylori The construction of uvrA uvrB uvrC and uvrD mutants by natural transformation-mediated allelic exchange was performed as described previously [42]. A list of the oligonucleotides used for mutagenesis, including the introduced restriction sites is provided in Additional file 4: Table S2. Briefly, the target genes were amplified by PCR and cloned into pUC18. The resulting plasmids (Additional file 4: Table S3) were used for inverse PCR amplification.