3',5'-Cyclic diguanylic acid (c-di-GMP) is a bacterial second messenger molecule that is a key global regulator in , but the molecular mechanisms by which this molecule regulates downstream phenotypes have not been fully characterized. One such regulatory factor that may respond to c-di-GMP is the Vc2 c-di-GMP-binding riboswitch that is hypothesized to control the expression of the downstream putative transcription factor TfoY. Although much is known about the physical and structural properties of the Vc2 riboswitch aptamer, the nature of its expression and function in has not been investigated. Here, we show that Vc2 functions as an off switch to inhibit TfoY production at intermediate and high concentrations of c-di-GMP. At low c-di-GMP concentrations, TfoY production is induced to stimulate dispersive motility. We also observed increased transcription of at high intracellular concentrations of c-di-GMP, but this induction is independent of the Vc2 riboswitch and occurs via transcriptional control of promoters upstream of by the previously identified c-di-GMP dependent transcription factor VpsR. Our results show that TfoY is induced by c-di-GMP at both low and high intracellular concentrations of c-di-GMP via posttranscriptional and transcriptional mechanisms, respectively. This regulation contributes to the formation of three distinct c-di-GMP signaling states in The bacterial pathogen must transition between life in aquatic environmental reservoirs and life in the gastrointestinal tract. Biofilm formation and bacterial motility, and their control by the second messenger molecule c-di-GMP, play integral roles in this adaptation. Here, we define the third major mechanism by which c-di-GMP controls bacterial motility. This pathway utilizes a noncoding RNA element known as a riboswitch that, when bound to c-di-GMP, inhibits the expression of the transcription factor TfoY. TfoY production switches motility from a dense to a dispersive state. Our results suggest that the c-di-GMP signaling network of can exist in at least three distinct states to regulate biofilm formation and motility.

Cyclic di-GMP (c-di-GMP) is a bacterial second messenger molecule that is important in the biology of Vibrio cholerae, but the molecular mechanisms by which this molecule regulates downstream phenotypes have not been fully characterized. We have previously shown that the Vc2 c-di-GMP-binding riboswitch, encoded upstream of the gene tfoY, functions as an off switch in response to c-di-GMP. However, the mechanism by which c-di-GMP controls expression of tfoY has not been fully elucidated. During our studies of this mechanism, we determined that c-di-GMP binding to Vc2 also controls the abundance and stability of upstream noncoding RNAs with 3′ ends located immediately downstream of the Vc2 riboswitch. Our results suggest these putative small RNAs (sRNAs) are not generated by transcriptional termination but rather by preventing degradation of the upstream untranslated RNA when c-di-GMP is bound to Vc2. IMPORTANCE Riboswitches are typically RNA elements located in the 5′ untranslated region of mRNAs. They are highly structured and specifically recognize and respond to a given chemical cue to alter transcription termination or translation initiation. In this work, we report a novel mechanism of riboswitch-mediated gene regulation in Vibrio cholerae whereby a 3′ riboswitch, named Vc2, controls the stability of upstream untranslated RNA upon binding to its cognate ligand, the second messenger cyclic di-GMP, leading to the accumulation of previously undescribed putative sRNAs. We further demonstrate that binding of the ligand to the riboswitch prevents RNA degradation. As binding of riboswitches to their ligands often produces compactly structured RNA, we hypothesize this mechanism of gene regulation is widespread.

SUMMARYCyclic di-GMP (c-di-GMP) is a bacterial second messenger molecule that is important in the biology of Vibrio cholerae, but the molecular mechanisms by which this molecule regulates downstream phenotypes have not been fully characterized. We have previously shown that the Vc2 c-di-GMP-binding riboswitch, encoded upstream of the gene tfoY, functions as an off-switch in response to c-di-GMP. However, the mechanism by which c-di-GMP controls expression of tfoY has not been fully elucidated. During our studies of this mechanism, we determined that c-di-GMP binding to Vc2 also controls the abundance and stability of upstream non-coding small RNAs (sRNA) with 3’-ends located immediately downstream of the Vc2 riboswitch. Our results suggest these sRNAs are not generated by transcriptional termination but rather by preventing degradation of the upstream untranslated RNA when c-di-GMP is bound to Vc2.IMPORTANCERiboswitches are typically RNA elements located in the 5’ untranslated region of mRNAs. They are highly structured and specifically recognize and respond to a given chemical cue to alter transcription termination or the translation initiation. In this work, we report a novel mechanism of riboswitch mediated gene regulation in Vibrio cholerae whereby a 3’ riboswitch, named Vc2, controls the stability of upstream untranslated RNA upon binding to its cognate ligand, the second messenger cyclic di-GMP, leading to the accumulation of previously undescribed sRNAs. We further demonstrate that binding of the ligand to the riboswitch prevents RNA degradation. As binding of riboswitches to their ligands often produces compactly structure RNA, we hypothesize this mechanism of gene regulation could be widespread.