FIG 2.

S. aureus contains a bona fide guanine riboswitch in the 5′-UTR of xpt. (a to d) Riboswitch transcriptional reporters were generated by fusing the xpt 5′-UTR riboswitch from B. subtilis (EK1) and S. aureus (EK2) to lacZ and integrated into the amyE locus of B. subtilis. Strains EK1 (a) and EK2 (b) were grown to log phase in Bacillus medium changing the concentration of exogenous guanine in a dose-response curve and assayed for reporter activity by Miller assay. (c and d) Strains EK1 (c) and EK2 (d) were grown to log phase in Bacillus medium or in the presence of 1 mM exogenous guanine, hypoxanthine, adenine, or xanthine and assayed for reporter activity by Miller assay. (e) S. aureus xpt-riboswitch reporter was integrated into the rmpF-isdB locus of S. aureus and grown to log phase in tryptic soy broth supplemented with 1 mM exogenous guanine, guanosine, hypoxanthine, xanthine, adenine, 2-aminopurine, 2′-deoxyguanosine, 6-thioguanine, and 2-acetamido-6-hydroxypurine, and reporter activity was measured. (f) Water-LOGSY NMR spectra of guanine (C and D) and adenine (A and B) in the presence (A and C) and absence (B and D) of purified riboswitch aptamer. The guanine signal at 7.95 ppm corresponds to the proton in position 8 of the purine ring. The change in sign upon the addition of riboswitch RNA to guanine indicates that the nucleotide is interacting (C and D). The adenine signals at 8.1 and 8.15 ppm correspond to the protons in position 2 and 8. There is no sign change upon the addition of riboswitch to adenine (A and B). The broad signals at 6.2 ppm (in guanine) and 6.8 ppm (in adenine) correspond to the NH₂ groups. These protons exchange with the bulk water, leading to a strong negative signal in all spectra that has no information for binding interaction. The signal at 0 ppm corresponds to the resonance of 2,2-dimethylsilapentane-5-sulfonic acid (DSS). Other signals visible in the riboswitch spectra stem from buffer impurities. Results are presented as means ± standard deviations (SD) (error bars) or means plus SD (n = 3) (a to e). Statistical analyses were performed using Student's t test. Values that are statistically significantly different from the value for the control (vehicle) are indicated by asterisks as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.