Figure 4.

Identification of guanine analogs that inhibit cell growth and repress reporter gene expression. a) Growth inhibition by guanine analogs. Each guanine analog was added to B. subtilis cultures to a final concentration of 100 μM, and the relative growth compared to that observed with no added analog (−) was determined after incubation for 10 hr at 37°C. The average growth for three independent cultures is shown, wherein open and filled bars identify analogs that inhibit bacterial growth less than or greater than 50%, respectively. Error bars represent the standard deviation between three independent assays. b) Plots comparing the K_D and MIC values for three guanine analogs. The MIC values for G6, G7 and G15. MIC (GMM) and MIC (LB) values were established using the wild-type 1A1 B. subtilis strain. MIC (G6-resistant strain, GMM) values were established using a 1A1 B. subtilis variant that was generated to exhibit resistance to G6 (the variant carrying the near complete deletion of the terminator stem of the pbuE riboswitch, see Figure 5). MIC values for G7 and G15 in rich medium were not detected even at 1 mM, as represented by the > symbol. C–d) Plots of β-galactosidase expression for the wild-type and mutant (M1; see Methods) xpt-pbuX constructs normalized to the level of expression observed without added ligand. The xpt-pbuX guanine riboswitch was placed upstream of a lacZ reporter gene in a wild-type B. subtilis strain, and each compound was added to this strain at 75% of the MIC for the compound, or 200 μM in the case of guanine. The annotations (−) and gua designate no added ligand and guanine, respectively. A normalized value of one equals 45 Miller units for the wild-type xpt-pbuX strain and 27 Miller units for the mutant xpt-pbuX strain.