FIG 2.

CRP directly affects frwC transcription. (a) qRT-PCR. The mRNA levels of frwC were compared between the Δcrp and WT strains. A standard curve was prepared for each RNA preparation with the 16S rRNA gene. The relative fold expression of frwC in the Δcrp mutant was determined by the 2^−ΔΔCt method and compared with that in the WT. The error bars indicate standard deviations. (b) lacZ fusion. A promoter-proximal region of frwC was cloned into the lacZ transcriptional-fusion vector pHRP309. The frwC-lacZ fusion plasmid was transformed into the WT and Δcrp strains, and frwC promoter activities were determined. (c) EMSAs for binding of CRP with the promoter of frwC. The radioactively labeled P_frwC DNA fragment was incubated with increasing amounts of purified His₆-CRP (lanes 1 to 4: 0, 1.3, 2.5, and 5.1 pmol, respectively) and subjected to native 4% polyacrylamide gel electrophoresis. The band of free DNA disappeared with increasing amounts of His₆-CRP, resulting in a retarded DNA band with decreased mobility, which presumably represented the DNA-CRP complex.