Extended Data Fig. 1.

(A) Schematic of undirected transformation of FA19 (parental CanB^19G) to identify causative factors for CO₂-dependence (B) Plating efficiency in the presence and absence of CO₂ following undirected transformation of gDNA from N. gonorrhoeae strain FA6140 into the N. gonorrhoeae lab strain FA19 (N = 10, 10, 9 from left to right from two independent experiments, error bars represent SEM). Significance (from left to right, p = 0.00018, p = 0.00028) determined by two-sided Mann-Whitney U test. (C) Putative reaction catalyzed by NGO2079/CanB and downstream metabolic products. (D) SNPs present in whole-genome sequencing of two transformants in (B). Arrow indicates the SNP identified in Figure 1. (E) (Top) Alphafold predicted homodimeric structures of the CanB^19E variant (teal) and the CanB^19G variant (green). (Bottom) Magnified Alphafold predicted structures overlaying the glutamate (maroon) and glycine (red) at position 19. (F) Proportion of sequenced isolates by year with the CanB^19G variant. *p<0.05, **p<0.01, ***p<0.001