The RNA Domain Vc1 Regulates Downstream Gene Expression in Response to Cyclic Diguanylate in Vibrio cholerae

Abstract

In many bacterial species, including the aquatic bacterium and human pathogen Vibrio cholerae, the second messenger cyclic diguanylate (c-di-GMP) modulates processes such as biofilm formation, motility, and virulence factor production. By interacting with various effectors, c-di-GMP regulates gene expression or protein function. One type of c-di-GMP receptor is the class I riboswitch, representatives of which have been shown to bind c-di-GMP in vitro. Herein, we examined the in vitro and in vivo function of the putative class I riboswitch in Vibrio cholerae, Vc1, which lies upstream of the gene encoding GbpA, a colonization factor that contributes to attachment of V. cholerae to environmental and host surfaces containing N-acetylglucosamine moieties. We provide evidence that Vc1 RNA interacts directly with c-di-GMP in vitro, and that nucleotides conserved among this class of riboswitch are important for binding. Yet the mutation of these conserved residues individually in the V. cholerae chromosome inconsistently affects the expression of gbpA and production of the GbpA protein. By isolating the regulatory function of Vc1, we show that the Vc1 element positively regulates downstream gene expression in response to c-di-GMP. Together these data suggest that the Vc1 element responds to c-di-GMP in vivo. Positive regulation of gbpA expression by c-di-GMP via Vc1 may influence the ability of V. cholerae to associate with chitin in the aquatic environment and the host intestinal environment.

Cyclic di-GMP Regulates TfoY in Vibrio cholerae To Control Motility by both Transcriptional and Posttranscriptional Mechanisms

“…As a result, its physiological role in V. cholerae c-di-GMP regulation remains undefined. A related class I riboswitch in V. cholerae, termed Vc1, was recently shown to regulate the surface binding protein GbpA, functioning as an on switch in response to c-di-GMP (20).…”

Cyclic Diguanylate Regulates Virulence Factor Genes via Multiple Riboswitches in Clostridium difficile

“…In addition, the presence of a large set of genes regulated (both positively and negatively) by c-di-GMP riboswitches provides the opportunity to examine the differences in responsiveness of this class of c-di-GMP receptor to changes in intracellular c-di-GMP. For example, differences between riboswitches of the same type may result in changes to ligand affinity, resulting in responses to different threshold concentrations of c-di-GMP ( 20 , 39 ).…”

“…Initial transcriptional analyses indicated that class II riboswitches are uniformly “on” switches, while all but one class I riboswitch are “off” switches in response to c-di-GMP in C. difficile . However, there are multiple examples of ligands also affecting the activity of the promoter upstream of their cognate riboswitch ( 39 , 40 , 48 , 49 ). For example, in vivo studies of class I c-di-GMP riboswitches in Vibrio cholerae , Vc1 and Vc2, showed that c-di-GMP regulates the expression the downstream genes gbpA and tfoY at two levels: by controlling transcription initiation via the upstream promoters and via the riboswitches ( 39 , 40 ).…”

“…However, there are multiple examples of ligands also affecting the activity of the promoter upstream of their cognate riboswitch ( 39 , 40 , 48 , 49 ). For example, in vivo studies of class I c-di-GMP riboswitches in Vibrio cholerae , Vc1 and Vc2, showed that c-di-GMP regulates the expression the downstream genes gbpA and tfoY at two levels: by controlling transcription initiation via the upstream promoters and via the riboswitches ( 39 , 40 ). Moreover, the two control mechanisms acted in opposing directions ( 39 , 49 ).…”

“…For example, in vivo studies of class I c-di-GMP riboswitches in Vibrio cholerae , Vc1 and Vc2, showed that c-di-GMP regulates the expression the downstream genes gbpA and tfoY at two levels: by controlling transcription initiation via the upstream promoters and via the riboswitches ( 39 , 40 ). Moreover, the two control mechanisms acted in opposing directions ( 39 , 49 ). Using reporter fusions to the regions upstream of the functional c-di-GMP riboswitches in C. difficile , we found that c-di-GMP controls expression of CD630_19900 via the promoter.…”