Cultivation and Genetic Manipulation of Helicobacter pylori

The H. pylori strains used in this study (Supplementary Table S1) were 26 695 (11 (link)) B128 (12 ,13 ) and X47-2AL (14 (link)). Strains were grown on Columbia agar plates supplemented with 7% horse blood and Dent selective supplement (Oxoid, Basingstoke, UK) for 24–48 h depending on the strain. Liquid cultures were performed in brain-heart infusion medium (Oxoid) supplemented with 10% fetal bovine serum and Dent. H. pylori plates and liquid cultures were incubated at 37°C under microaerobic conditions (10% CO₂, 6% O₂, 84% N₂) using an Anoxomat (MART microbiology) atmosphere generator. For liquid cultures, bacteria harvested from plates were inoculated at an optical density at 600 nm of 0.05 (OD₆₀₀ = 0.05) into 5 ml (tubes, shaking at 175 rpm) brain-heart infusion medium supplemented with 10% fetal bovine serum and Dent supplement. After 12–24 h, pre-cultures were diluted to an OD₆₀₀ of 0.05 into 25 ml (flasks, shaking at 125 rpm). Plasmids used for cloning were amplified in Escherichia coli strain JM109, which was grown in Luria–Bertani medium, supplemented either with kanamycin (50 μg.ml⁻¹) or chloramphenicol (30 μg.ml⁻¹). For H. pylori mutant selection and culture, antibiotics were used at the following final concentrations 20 μg.ml⁻¹ kanamycine (Sigma) and 8 μg.ml⁻¹ chloramphenicol (Sigma).