Transformation and Growth of N. gonorrhoeae

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).