The mechanistic role of the small bacterial second messenger cyclic di-GMP (c-di-GMP) in transcription initiation has remained unclear. In Vibrio cholerae, the causative agent of the disease cholera, VpsR is the master Enhancer Binding Protein (EBP) that binds c-di-GMP to increase biofilm gene expression at the biofilm biogenesis promoter, PvpsL, in vivo. Unlike typical EBPs that activate RNA polymerase (RNAP) containing the alternate sigma (sigma factor, sigma54, VpsR has several different features: 1) it lacks conserved residues needed to bind to sigma54 and hydrolyze ATP; 2) it retains a highly conserved aspartic acid (D59) residue, which is typically phosphorylated; and 3) it activates PvpsL in the absence of sigma54 in vivo. These features all suggest a different unknown mechanism of transcription activation.To address this mechanism, we have established an in vitro system and show for the first time that c-di-GMP is sufficient to directly activate transcription with another activator at PvpsL. Unlike other regulators which use c-di-GMP to promote oligomerization and/or increase DNA binding affinity, the presence of c-di-GMP neither affects VpsR oligomerization nor significantly changes the affinity of VpsR for PvpsL DNA. Instead, KMnO4 and DNase I footprinting reveal that the PvpsL/sigma70-RNAP/VpsR/c-di-GMP complex forms the open transcription bubble and adopts a different conformation from that formed by PvpsL/sigma70-RNAP with or without c-di-GMP or VpsR. To investigate the role of the D59 residue, we have characterized the phosphodefective variant D59A and the phosphomimetic D59E. While both variants dimerize and bind DNA with Kd(app)s similar to that of wildtype (WT), D59E activates transcription and forms the open transcription bubble while D59A yields basal transcription. DNase I footprints of the transcription complex made with D59E resemble those made with WT, while footprints with D59A resemble those of RNAP alone. We have also developed a method to denature and renature VpsR (VpsRREN). In the absence of the high-energy phosphate donor, acetyl phosphate, VpsRREN now resembles D59A. Addition of acetyl phosphate results in a VpsRREN that behaves like the previously purified WT VpsR and D59E in in vitro transcriptions, electrophoretic mobility shift assays (EMSAs), DNase I and KMnO4 footprinting.Lastly, we have also explored whether VpsR has additional regulatory gene expression roles. We have identified three new promoters that are regulated by VpsR and c-di-GMP in vitro: rbmA, rbmF, and vpsU. Interestingly, like PvpsL, the regulated genes of each promoter represent the first gene of their gene cluster or operon. Similar to PvpsL, binding of c-di-GMP does not alter protein-DNA contacts at these promoters in the absence of sigma70-RNAP. In vitro transcriptions require both c-di-GMP and VpsR, and the positions of the VpsR binding sites at these promoters reveal that VpsR can utilize both Class I and Class II activation to upregulate gene expression.In conclusion, VpsR represents a novel c-di-GMP dependent transcription regulator. Not only does it use c-di-GMP to promote open complex formation, but our data also suggests that phosphorylation is simultaneously required for that process. As the master regulator of biofilm formation, VpsR directly activates a set of vps and rbm promoters using different mechanisms of transcription activation. Understanding these mechanisms not only provide a new paradigm in c-di-GMP-dependent transcription activation and elucidate mechanistic processes that regulate biofilm formation, but also provide the foundation needed for the development of novel chemical inhibitors against V. cholerae and biofilm-based nocosomial infections
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