Protease peptone

E. coli strains were grown in lysogeny broth (LB) with shaking at 250 rpm or supplemented with agar for growth on plates. All strains are shown in Supplementary Table 3. Strains were incubated at 37 °C unless otherwise mentioned. The antibiotics ampicillin (100 μg/ml), spectinomycin (50 μg/ml), kanamycin (50 μg/ml), or tetracycline (10 μg/ml) were added when needed. MacConkey agar was purchased from BD Diagnostic, Difco MacConkey Agar Base (281810). Simple MacConkey (SM) agar media composition for 300 ml was: Peptone from soybean meat (70178-100 G Sigma Aldrich, 5.1 g), Protease Peptone (P0431-250G Sigma Aldrich, 0.9 g), NaCl (Sigma Aldrich, 1.5 g), and Agar (Sigma Aldrich, 4.05 g).
The crp gene was deleted in Escherichia coli K-12 MG1655 cyaA::cat Δfnr and in Escherichia coli K-12 MG1655 cyaA::cat Δfnr cmkA216E by lambda-red recombineering with pSIM19 as previously described^{43 (link),44 (link)} and integration of a tetA PCR product with homology ends to the crp locus. The tetA PCR product was generated with oligo #3734 and #3735 (Supplementary Table 4). With the same method, cmk, cytR, and rpoH were deleted in K-12 MG1655 cyaA::cat Δfnr Δcrp. The tetA PCR products were performed with oligos #4010/#4011, #5249/ #5250 and #5290/#5391.

Gonococcal (GC) broth was prepared by dissolving 3.75 g of protease peptone (Sigma-Aldrich), 0.25 g of potassium phosphate monobasic (Sigma-Aldrich), 1 g of potassium phosphate dibasic (Sigma-Aldrich) and 1.25 g of sodium chloride (Sigma-Aldrich) into 250 mL of distilled water. The GC broth medium was sterilised by autoclaving at 121 °C for 15 min, cooled to 50 °C before adding 250 µl of the iron and 2.5 mL of the glucose supplement aseptically. GC agar was prepared by dissolving 9 g of GC agar base (CM0367, Oxoid) in 250 mL of distilled water, sterilised by autoclaving, and cooled before adding 250 μl of the iron and 2.5 mL of the glucose supplement.