a, Enzyme names are shown above reaction arrows. RibA is a GTP cyclohydrolase II, RibB is a (3S)-3,4-dihydroxy-2-butanone 4-phasphate synthase. RibDG is a bifunctional enzyme encoding 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-phosphate deaminase and 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5′-phosphate reductase activity. RibH is a lumazine synthase. RibE is a riboflavin synthase. RibFC is another bifunctional enzyme encoding flaviokinase and FAD synthetase activity. Note, one molecule of GTP and two molecules of ribulose 5-phosphate are required to make one molecule of riboflavin. I, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′ phosphate; II, 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5′-phosphate; III, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5′-phosphate; IV, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione; V, (3S)-3,4-dihydroxy-2-butanone 4-phosphate; VI, 6,6-dimethyl-8-D-ribityllumazine. Note two molecules of VI dismutate (step 7) to give IV and riboflavin. Note also that the phosphatase converting III to IV (step 4) is not known. Adapted from Pedrolli et al.25. b, HPLC-based quantitative analysis of riboflavin, FMN, and FAD levels in E. coli strain MB5746 following genetic inactivation of riboflavin biosynthesis (ΔribA or ΔribB) or ribocil drug treatment. Wild-type strain MB5746 and isogenic ΔribA or ΔribB strains were grown overnight in CAMHB media containing 10 µM riboflavin, washed in CAMHB media lacking riboflavin, diluted (1:50) and grown for an additional 20 h before harvesting cells and HPLC analysis of cell lysates as described in the Methods. In parallel, overnight cultures of MB5746 grown in CAMHB were diluted (1:50) and treated with DMSO as a control or 20 μM ribocil, grown for an additional 20 h and analysed as above (middle panel). Data are presented as the mean of two technical repeats and are representative of two independent experiments. Riboflavin, FMN and FAD depletion levels are listed as a percentage relative to wild-type controls (right panel). c, HPLC-based quantitative analysis of riboflavin levels in 19 independent E. coli ribocilR mutant isolates versus the isogenic parent strain, MB5746. Overnight cultures of each strain were diluted 1:50 in CAMHB and treated with 10 µM ribocil or mock treated (1% DMSO). After growth at 37 °C with agitation for 20 h, cell lysates were prepared and riboflavin levels were quantitated as described in the Methods. Data are the mean of two technical repeats (error bars indicate range) and is representative of two independent experiments.