Global transcriptional control by glucose and carbon regulator CcpA in Clostridium difficile

Laboratoire Pathogenèse des Bactéries Anaérobies, Département de Microbiologie Institut Pasteur, Université Paris Diderot, Sorbonne Paris Cité, Cellule Pasteur, 25 rue du Docteur Roux, Paris 75015, France, Institute for Information Transmission Problems, Russian Academy of Sciences, Moscow 127994, Russia and Sanford-Burnham Medical Research Institute, La Jolla, CA 92037, USA.
Nucleic Acids Research (Impact Factor: 9.11). 09/2012; 40(21). DOI: 10.1093/nar/gks864
Source: PubMed

ABSTRACT 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 (cre(CD) motif), that is, 'RRGAAAANGTTTTCWW'. Binding of purified CcpA protein to 19 target cre(CD) 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.

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    • "The LacI family contains a number of global regulators for central carbohydrate metabolic pathways, in particular the previously known regulators CcpA in Firmicutes and FruR in Enterobacteria. Here, we report a comparative genomics reconstruction of orthologous FruR regulons in γ-Proteobacteria, while the reconstructions of CcpA regulons in different lineages of Firmicutes has been reported previously (Ravcheev et al., 2011, 2013b; Antunes et al., 2012; Leyn et al., 2013) and is available in "
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    ABSTRACT: DNA-binding transcription factors (TFs) are essential components of transcriptional regulatory networks in Bacteria. LacI-family TFs (LacI-TFs) are broadly distributed among certain lineages of bacteria. The majority of characterized LacI-TFs sense sugar effectors and regulate carbohydrate utilization genes. The comparative genomics approaches enable in silico identification of TF-binding sites and regulon reconstruction. To study function and evolution of LacI-TFs, we performed genomics-based reconstruction and comparative analysis of their regulons. For over 1,300 LacI-TFs from over 270 bacterial genomes, we predicted their cognate DNA-binding motifs and identified target genes. Using the genome context and metabolic subsystem analyses of reconstructed regulons we tentatively assigned functional roles and predicted candidate effectors for 78% and 67% of the analyzed LacI-TFs, respectively. Nearly 90% of the studied LacI-TFs are local regulators of sugar utilization pathways, whereas the remaining 125 global regulators control large and diverse sets of metabolic genes. The global LacI-TFs include the previously known regulators CcpA in Firmicutes, FruR in Enterobacteria, and PurR in Gammaproteobacteria, and the three novel regulators, GluR, GapR, and PckR, that are predicted to control the central carbohydrate metabolism in three lineages of Alphaproteobacteria. Phylogenetic analysis of regulators combined with the reconstructed regulons provides a model of evolutionary diversification of LacI-TFs. The obtained genomic collection of in silico reconstructed regulons in Bacteria is available in the RegPrecise database ( It provides a framework for future structural and functional classification of the LacI protein family and identification of molecular determinants of the DNA and ligand specificity. The inferred regulons can be also used for functional gene annotation and reconstruction of sugar catabolic networks in diverse bacteria.
    Frontiers in Microbiology 06/2014; 5:294. DOI:10.3389/fmicb.2014.00294 · 3.99 Impact Factor
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    • "The increased abundance of dihydrodipicolinate synthase 2 (CD3223), involved in both lysine biosynthesis and in production of dipicolinate in spores – recognised as one of the organism's main virulence factors – suggests that cells under heat stress are more reliant upon fermentation and metabolism of amino acids, possibly due to lifting of carbon catabolite repression [69], although there is limited evidence in our data for such an effect upon either carbohydrate utilisation or amino acid fermentation pathways. The catabolite control protein CcpA (CD1064) is a pleiotropic regulator that via binding to well defined creCD sites upstream of some 18% of C. difficile genes, enables both positive and negative control of global transcription in response to carbohydrate availability [69]. Our microarray data suggest down regulation of CD1064 transcripts, implying that lifting of ccpA mediated transcriptional control could be important for survival and maintenance of metabolism under heat stress. "
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    ABSTRACT: Clostridium difficile is considered to be the most frequent cause of infectious bacterial diarrhoea in hospitals worldwide yet its adaptive ability remains relatively uncharacterised. Here, we used GeLC/MS and the exponentially modified protein abundance index (emPAI) calculation to determine proteomic changes in response to a clinically relevant heat stress. Reproducibility between both biological and technical replicates was good, and a 37°C proteome of 224 proteins was complemented by a 41°C proteome of 202 proteins at a 1% false discovery rate. Overall, 236 C. difficile proteins were identified and functionally categorised, of which 178 were available for comparative purposes. A total of 65 proteins (37%) were modulated by 1.5-fold or more at 41°C compared to 37°C and we noted changes in the majority of proteins associated with amino acid metabolism, including upregulation of the reductive branch of the leucine fermentation pathway. Motility was reduced at 41°C as evidenced by a 2.7 fold decrease in the flagellar filament protein, FliC, and a global increase in proteins associated with detoxification and adaptation to atypical conditions was observed, concomitant with decreases in proteins mediating transcriptional elongation and the initiation of protein synthesis. Trigger factor was down regulated by almost 5-fold. We propose that under heat stress, titration of the GroESL and dnaJK/grpE chaperones by misfolded proteins will, in the absence of trigger factor, prevent nascent chains from emerging efficiently from the ribosome causing translational stalling and also an increase in secretion. The current work has thus allowed development of a heat stress model for the key cellular processes of protein folding and export.
    PLoS ONE 02/2014; 9(2):e88960. DOI:10.1371/journal.pone.0088960 · 3.23 Impact Factor
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    • "In both organisms , carbon catabolite repression is thought to be responsible since glucose inhibition of toxin production is not observed in ccpA mutants that no longer produce the carbon catabolite repressor protein CcpA (Antunes et al., 2011; Mendez et al., 2012). Further support for the role of CcpA in mediating glucose-dependent inhibition of toxin production in C. difficile comes from the finding that the tcdA, tcdB and toxin regulator genes tcdR and tcdC are direct targets of the CcpA protein in vitro (Antunes et al., 2012). The observation that C. difficile ccpA mutants produce a reduced amount of toxin in comparison to the wild-type strain (Antunes et al., 2011) suggests that toxin regulation is complex and that unidentified regulators must also be involved in the CcpA-regulation cascade. "
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    ABSTRACT: The genus Clostridium comprises a large, heterogeneous group of obligate anaerobic, Gram-positive spore forming bacilli. Members of this genus are ubiquitous in the environment and although most species are considered saprophytic, several are pathogenic to both humans and animals. These bacteria cause a variety of diseases including neuroparalysis, gas gangrene, necrotic enteritis, food poisoning, toxic shock syndrome and pseudomembraneous colitis, which in most cases arise as a consequence of the production of potent exotoxins. Treatment options are often limited, underscoring the need for new treatment strategies and novel therapeutics. Understanding the fundamental mechanisms and signals that control toxin production in the pathogenic clostridia may lead to the identification of novel therapeutic targets that can be exploited in the development of new antimicrobial agents.
    Molecular Microbiology 02/2014; 91(2):221-31. DOI:10.1111/mmi.12469 · 4.42 Impact Factor
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