Gc agar plates

H. pylori strains P12 and P12 [TEM-1–CagA] (Schindele et al., 2016 (link)) were grown on GC agar plates (Oxoid) supplemented with vitamin mix (1%) and horse serum (Life Technologies; 8%) (serum plates), and cultured for 16 to 60 h in a microaerobic atmosphere (85% N₂, 10% CO₂, 5% O₂) at 37°C. AGS cells were cultivated in RPMI (Gibco) supplemented with 10% FCS (heat-inactivated; Life Technologies). Murine L929 fibroblasts were cultivated in DMEM (Gibco) supplemented with 10% FCS, 2 mM l-glutamine (Gibco) and 1 mM sodium pyruvate (Gibco) at 37°C in a 5% CO₂ incubator.

H. pylori strains (see Table S2 in the supplemental material) were grown on GC agar plates (Oxoid) supplemented with vitamin mix (1%) and horse serum (8%) (serum plates) and cultured for 16 to 60 h in a microaerobic atmosphere (85% N₂, 10% CO₂, 5% O₂) at 37°C. For TEM assays, H. pylori wild-type strain P12 and defined P12 knockout mutants were used.

N. gonorrhoeae strain 1291 (a male gonococcal urethritis isolate, provided courtesy of M. A. Apicella) was grown at 37°C with 5% CO₂ on GC agar plates (Oxoid) supplemented with IsoVitaleX (Becton Dickinson) for ∼16 h. Escherichia coli strains DH5α and BL21 were grown in Luria-Bertani broth or agar at 37°C. Kanamycin, ampicillin, and spectinomycin were used as required at final concentration of 50 μg/ml, 100 μg/ml, and 100 μg/ml, respectively. Growth rate experiments were performed in GC broth supplemented with IsoVitaleX, as described previously (72 (link)).

H. pylori strains were grown on GC agar plates (Oxoid) supplemented with vitamin mix (1%) and horse serum (8%) (serum plates) or cholesterol (cholesterol plates) and cultured for 16 to 48 h in a microaerobic atmosphere (85% N₂, 10% CO₂, 5%O₂) at 37°C. E. coli strains were grown on Luria–Bertani (LB) agar plates supplemented with antibiotics, as appropriate. Plasmids were introduced into H. pylori by transformation as described previously [48 (link)], and transformants were selected on serum plates containing 6 mg/l chloramphenicol, 8 mg/l kanamycin, 10 mg/l erythromycin, or 250 mg/l streptomycin, as appropriate. For liquid culture plate-grown H. pylori were resuspended in PBS and added in an OD of 0.075 into 6-well plates prepared with 2 ml Brucella Broth and 8 μl cholesterol (Gibco) as previously published [49 (link)]. After 24h at 37°C in a 10% CO₂-atmosphere vitality was checked under the microscope before use. All cell lines were maintained at 37°C and 5% CO₂ with the appropriate media. Generally, cells were grown in 75 cm² tissue culture flasks (BD Falcon) and in 6-, 12- and 24-well plates (tissue culture treated cell culture clusters, Costar, Corning Inc.).

N. gonorrhoeae strain MS11 (GenBank accession number NC_022240.1) and the derivative strain N2009 ((36 (link)), P⁺, Opa⁻, PorB_IA, Cam^r, Erm^r) were grown on GC agar plates (Oxoid), supplemented with vitamine mix (1%) and, if applicable, kanamycin (40 mg/l) for 14–16 h at 37°C in a humidified 5% CO₂ atmosphere. For transformation of N. gonorrhoeae, piliated bacteria were selected by colony morphology (37 (link)) under a stereo microscope. 5 × 10⁶ bacteria were mixed with 10 ng polymerase chain reaction (PCR) products in 50 μl PPM medium (15 g Proteose peptone; 5 g sodium chloride; 0.5 g soluble starch; 1 g potassium dihydrogen phosphate; 4 g dipotassium hydrogen phosphate for 1 l; pH 7.2; 1% vitamin mix, 0.5% sodium hydrogen carbonate, 10 mM magnesium chloride, sterilized by filtration), and spotted onto a GC agar plate. After overnight incubation, the bacteria were resuspended in PPM medium and plated onto GC agar plates containing kanamycin for selection of transformants. For RNA extraction, GC were grown in PPM medium as described for transformation at an OD of 0.2 and grown at 180 revolutions per minute (rpm) at 37°C to an OD 0.4–0.5 (mid-logarithmic phase).

Four suspected Neisseria spp. isolates were grown on GC agar plates (Oxoid) with Kellogg’s [29 (link)] and 5 % Fe(NO₃)₃ supplements at 37 °C with 5 % CO₂. Control species for India ink staining, Escherichia coli and Bacillus subtilis, were grown on nutrient agar (Oxoid) and incubated at 37 °C. Blood agar (Oxoid) included 7 % defibrinated horse blood (Oxoid) to test for haemolytic activity. API NH strips (bioMérieux) to identify the species were used according to the manufacturer’s instructions. The Nitrate Reduction Test (Sigma) was performed according to manufacturer’s instructions to identify samples capable of reducing nitrates.