The catabolite control protein CcpA is a pleiotropic regulator that mediates the global transcriptional response to rapidly catabolizable carbohydrates, like glucose in Gram-positive bacteria. By whole transcriptome analyses, we characterized glucose-dependent and CcpA-dependent gene regulation in Clostridium difficile. About 18% of all C. difficile genes are regulated by glucose, for which 50% depend on CcpA for regulation. The CcpA regulon comprises genes involved in sugar uptake, fermentation and amino acids metabolism, confirming the role of CcpA as a link between carbon and nitrogen pathways. Using combination of chromatin immunoprecipitation and genome sequence analysis, we detected 55 CcpA binding sites corresponding to ∼140 genes directly controlled by CcpA. We defined the C. difficile CcpA consensus binding site (creCD motif), that is, ‘RRGAAAANGTTTTCWW’. Binding of purified CcpA protein to 19 target creCD sites was demonstrated by electrophoretic mobility shift assay. CcpA also directly represses key factors in early steps of sporulation (Spo0A and SigF). Furthermore, the C. difficile toxin genes (tcdA and tcdB) and their regulators (tcdR and tcdC) are direct CcpA targets. Finally, CcpA controls a complex and extended regulatory network through the modulation of a large set of regulators.

The global regulator CymR represses the transcription of a large set of genes involved in cystine uptake and cysteine biosynthesis in Bacillus subtilis and Staphylococcus aureus. This repressor belongs to the widespread and poorly characterized Rrf2 family of regulators. The crystal structure of CymR from B. subtilis reveals a biologically active dimer, where each monomer folds into two tightly packed domains: a DNA‐binding domain, which houses a winged helix–turn–helix (wHTH) motif; and a long dimerization domain, which places the wHTH motifs at the extremes. This architecture explains how these small regulators can span 23–27‐bp DNA targets. The wHTH motif of CymR resembles those of the GntR superfamily of regulators, such as FadR and HutC. Superimposing the FadR wHTH motifs bound to their DNA fragments onto the wHTH motifs of the CymR dimer structure suggests that the DNA target and/or the protein must undergo some conformational changes upon binding. The CymR structure also hints at a possible location of the Fe–S centre associated with several Rrf2‐type regulators. Structured digital abstract http://www.uniprot.org/uniprot/O34527 http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0407 to http://www.uniprot.org/uniprot/O34527 by http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0114 (View Interaction http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8182482, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-8182474)