Abstract
3',5'-Cyclic diguanylic acid (c-di-GMP) is a bacterial second messenger molecule that is a key global regulator in Vibrio cholerae, 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 V. cholerae 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 tfoY 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 tfoY 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 V. choleraeIMPORTANCE The bacterial pathogen Vibrio cholerae 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 V. cholerae motility from a dense to a dispersive state. Our results suggest that the c-di-GMP signaling network of V. cholerae can exist in at least three distinct states to regulate biofilm formation and motility.