Clostridium difficile is an anaerobic Gram-positive bacterium that causes intestinal infections with symptoms ranging from
mild diarrhea to fulminant colitis. Cyclic diguanosine monophosphate (c-di-GMP) is a bacterial second messenger that typically
regulates the switch from motile, free-living to sessile and multicellular behaviors in Gram-negative bacteria. Increased intracel-
lular c-di-GMP concentration in C. difficile was recently shown to reduce flagellar motility and to increase cell aggregation. In
this work, we investigated the role of the primary type IV pilus (T4P) locus in c-di-GMP-dependent cell aggregation. Inactivation
of two T4P genes, pilA1 (CD3513) and pilB1 (CD3512), abolished pilus formation and significantly reduced cell aggregation un-
der high c-di-GMP conditions. pilA1 is preceded by a putative c-di-GMP riboswitch, predicted to be transcriptionally active
upon c-di-GMP binding. Consistent with our prediction, high intracellular c-di-GMP concentration increased transcript levels
of T4P genes. In addition, single-round in vitro transcription assays confirmed that transcription downstream of the predicted
transcription terminator was dose dependent and specific to c-di-GMP binding to the riboswitch aptamer. These results support
a model in which T4P gene transcription is upregulated by c-di-GMP as a result of its binding to an upstream transcriptionally
activating riboswitch, promoting cell aggregation in C. difficile.Clostridium difficile is an anaerobic Gram-positive bacterium that causes intestinal infections with symptoms ranging from mild diarrhea to fulminant colitis. Cyclic diguanosine monophosphate (c-di-GMP) is a bacterial second messenger that typically regulates the switch from motile, free-living to sessile and multicellular behaviors in Gram-negative bacteria. Increased intracellular c-di-GMP concentration in C. difficile was recently shown to reduce flagellar motility and to increase cell aggregation. In this work, we investigated the role of the primary type IV pilus (T4P) locus in c-di-GMP-dependent cell aggregation. Inactivation of two T4P genes, pilA1 (CD3513) and pilB1 (CD3512), abolished pilus formation and significantly reduced cell aggregation un- der high c-di-GMP conditions. pilA1 is preceded by a putative c-di-GMP riboswitch, predicted to be transcriptionally active upon c-di-GMP binding. Consistent with our prediction, high intracellular c-di-GMP concentration increased transcript levels of T4P genes. In addition, single-round in vitro transcription assays confirmed that transcription downstream of the predicted transcription terminator was dose dependent and specific to c-di-GMP binding to the riboswitch aptamer. These results support a model in which T4P gene transcription is upregulated by c-di-GMP as a result of its binding to an upstream transcriptionally activating riboswitch, promoting cell aggregation in C. difficile
