The second messenger cyclic dimeric guanosine-monophosphate (c-di-GMP) is a central regulator of many different cellular activities in the bacterial domain and it plays an especially important role in the lifestyle transition of Vibrio cholerae between the marine environment and human infection. One of the primary effectors of this signal, the Vc2 c-di-GMP-binding riboswitch of V. cholerae, has been heavily studied in vitro, yet it remains poorly understood in vivo. Riboswitches have been traditionally characterized as cis-acting RNA elements that serve to regulate the gene expression of downstream coding sequences, but the relationship between the Vc2 element and its downstream gene, tfoY, is uncharacterized. In this work, we determine that tfoY is a vital component of the V. cholerae c-di-GMP program, specifically involved in motility, biofilm formation, and the direct genetic regulation of c-di-GMP metabolic enzymes. We also reveal that V. cholerae possesses both Vc2-dependent and Vc2-independent mechanisms for c-di-GMP regulation of tfoY expression. And finally, we present a novel paradigm for riboswitch and c-di-GMP gene regulation in which the stability and abundance of a small RNA is controlled by the ligand binding state of the Vc2 riboswitch aptamer domain.
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