VpsR and cyclic di-GMP together drive transcription initiation to activate biofilm formation in Vibrio cholerae

The small molecule cyclic di-GMP (c-di-GMP) is known to affect bacterial gene expression in myriad ways. In Vibrio cholerae in vivo, the presence of c-di-GMP together with the response regulator VpsR results in transcription from P(vpsL), a promoter of biofilm biosynthesis genes. VpsR shares homology with enhancer binding proteins that activate σ54-RNA polymerase (RNAP), but it lacks conserved residues needed to bind to σ54-RNAP and to hydrolyze adenosine triphosphate, and P(vpsL) transcription does not require σ54 in vivo. Consequently, the mechanism of this activation has not been clear. Using an in vitro transcription system, we demonstrate activation of P(vspL) in the presence of VpsR, c-di-GMP and σ70-RNAP. c-di-GMP does not significantly change the affinity of VpsR for P(vpsL) DNA or the DNase I footprint of VpsR on the DNA, and it is not required for VpsR to dimerize. However, DNase I and KMnO(4) footprints reveal that the σ70-RNAP/VpsR/c-di-GMP complex on P(vpsL) adopts a different conformation from that formed by σ70-RNAP alone, with c-di-GMP or with VpsR. Our results suggest that c-di-GMP is required for VpsR to generate the specific protein–DNA architecture needed for activated transcription, a previously unrecognized role for c-di-GMP in gene expression.