Fluorometer

The Helicobacter hepaticus strain Hh-2 (ATCC 51448)^{59 (link)} was purchased from the Leibniz Institute DSMZ – German Collection of Microorganisms and Cell Cultures (DSM No.22909) and cultivated at the Max von Pettenkofer-Institute, LMU Munich. Bacteria from cryo stock were resuspended in Brain Heart Infusion (BHI) medium and put on blood agar plates (Columbia agar with 5% sheep blood, BD, Cat: 4354005). Plates were incubated in a chamber with anaerobic conditions (83% N₂, 10% CO₂, 7% H₂) for 4 days at 37°C. A subculture was cultivated further on in BHI medium with 3% sheep serum in a culture flask in the chamber with anaerobic conditions for additional 4 days at 37°C.
For Hh lysate (HhL) preparation, bacterial cells were harvested and washed 2–3 times with PBS. Cell pellets were resuspended in PBS and lyzed by sonification with the Sonifier 150 Cell Disruptor (Branson) 6 times for 3 min at level 3 on ice. Lyzed cells were centrifuged at 20,000 x g for 30 min at 4°C and the supernatant was mixed with a protease inhibitor (cOmplete ULTRA Tablets, Roche, Sigma-Aldrich, Cat: 05892953001). Protein concentration was determined using the Qubit Protein Assay Kit and Fluorometer (Invitrogen), according to the manufacturer’s instructions and the lysate was stored at −20°C until use for immunoblot or ELISA.

Genomic DNA extraction and purification were performed using Qiagen Genomic-tip 20/G kit (Qiagen, Germany). Previous to sequencing, Qubit Fluorometer and the double-stranded DNA (dsDNA) broad-range (BR) assay kit (Invitrogen, Waltham, MA, USA) was used to determine the concentration of the DNA. Sequencing libraries were prepared using Nextera XT DNA library preparation kit (Illumina, Inc., San Diego, CA, USA) and short reads paired-end sequencing of the S. Dublin strains was performed with MiSeq instrument (Illumina, Inc., San Diego, CA, USA) following the supplier’s instructions.