Effect of mutations in the IR1 sequence on Crp binding and ahpC expression.
(A) Base substitution mutations of the conserved nucleotides within IR1. The consensus sequence of the Crp-binding sites is given at the bottom line. The mutated nucleotides are marked with the asterisks above the sequences. The numbers above the sequences indicate the positions of the mutated nucleotides relative to ahpC. (B) The 150-bp DNA fragments (9.3 ng, 100 fmol) containing the wild-type or mutated IR1 sequence (mutation 1 or 2) were incubated with various amounts of purified Crp in the presence of 100 µM cAMP. The amounts of Crp used are given above the lanes. The Crp-DNA reaction mixtures were subject to native PAGE. After electrophoresis, gels were stained with SYBR green EMSA gel staining solution. (C) Effect of the mutations within the IR1 sequence on the promoter activity of ahpC. The ahpC promoter activity was measured by determining β-galactosidase activity. M. smegmatis wild-type strains harboring pNCahpC, pNCM1, and pNCM2 were grown to an OD600 of 0.45 to 0.5 and treated with CHP or DMSO (the solvent for CHP stock solution: control). The cultures were further grown for 1 h. Cell-free crude extracts were used to measure β-galactosidase activity. All values are the means of two independent experiments. The error bars indicate the deviations from the means.