Fig. 6

c-di-GMP does not regulate CRP gene transcription, protein stability or DNA binding. a Transcript levels for crp in V. cholerae with wild type (vector) or reduced c-di-GMP (pPDE) were measured by qRT-PCR. PDE gene expression was induced as described in the Methods. The data were normalized relative to the wild-type containing vector only, using rpoB as the reference gene. Shown are the means and standard deviations from at three independent samples. b CRP protein (23.6 kDa) levels in lysates of V. cholerae with wild type (vector) or reduced c-di-GMP (pPDE) were measured by western blot. V. cholerae Δcrp containing vector was included as a negative control. RNA Polymerase was detected on the same blot as a loading control. The images shown are from a representative of three independent experiments. Densitometry analyses were done by comparing the intensities of the GbpA bands to the intensities of the RNAP band in the same lane, then normalizing the value to that of wild type V. cholerae with vector. The fold change relative to the wild type is shown below each lane. c Using electrophoretic mobility shift assays, purified recombinant CRP was tested for the ability to bind and shift a DNA fragment encompassing the gbpA promoter in the presence or absence of cAMP and/or c-di-GMP. As a control, a non-specific DNA (indicated by “NS”) fragment was added to all binding reactions and was confirmed not to be shifted by CRP. *In the final lane, c-di-GMP was added in 10-fold excess of cAMP