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

International audienceThe 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

Comparative Genomics of CytR, an Unusual Member of the LacI Family of Transcription Factors

CytR is a transcription regulator from the LacI family, present in some gamma-proteobacteria including Escherichia coli and known not only for its cellular role, control of transport and utilization of nucleosides, but for a number of unusual structural properties. The present study addressed three related problems: structure of CytR-binding sites and motifs, their evolutionary conservation, and identification of new members of the CytR regulon. While the majority of CytR-binding sites are imperfect inverted repeats situated between binding sites for another transcription factor, CRP, other architectures were observed, in particular, direct repeats. While the similarity between sites for different genes in one genome is rather low, and hence the consensus motif is weak, there is high conservation of orthologous sites in different genomes (mainly in the Enterobacteriales) arguing for the presence of specific CytR-DNA contacts. On larger evolutionary distances candidate CytR sites may migrate but the approximate distance between flanking CRP sites tends to be conserved, which demonstrates that the overall structure of the CRP-CytR-DNA complex is gene-specific. The analysis yielded candidate CytR-binding sites for orthologs of known regulon members in less studied genomes of the Enterobacteriales and Vibrionales and identified

Alignment and SWAS plots of upstream regions of <i>ycdZ</i> in distant Enterobacteriales.

<p>Notation as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044194#pone-0044194-g005" target="_blank">Fig. 5</a>.</p

Organization of upstream regions of five experimentally proven <i>E. coli</i> members of the CytR regulon.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044194#pone.0044194-Kallipolitis1" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044194#pone.0044194-TretyachenkoLadokhina1" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044194#pone.0044194-Pedersen3" target="_blank">[37]</a>. CytR-binding sites (O_CYTR) are highlighted in magenta, cores of CRP-binding sites (O_CRP) – in green. Numbers denote spacer lengths. Dots denote gaps in the alignment.</p

Sequence LOGOs of CytR-binding motifs, direct repeat type.

<p>Notation as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044194#pone-0044194-g003" target="_blank">Fig. 3</a>. A) O_CYTRD LOGO for <i>cytR</i> from 16 Enterobacteriales; B) O_CYTRD LOGO for <i>cytR</i> from 6 Vibrionales; C) O_CYTRP LOGO for <i>cdd</i> from 14 Enterobacteriales.</p

Sequence LOGOs of the CRP, CytR-distal, CytR-proximal operators.

<p>Horizontal axis: position in the binding site; vertical axis: informationcontent in bits. The height of each individual symbol reflects its prevalence at a given position, the height of each column is proportional to the positional information content in this position. A) O_CRP LOGO ; B1) O_CYTRD LOGO; B2) O_CYTRP LOGO.</p

Alignment and SWAS plots of upstream regions of <i>ycdZ</i> in the Vibrionales.

<p>Notation as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044194#pone-0044194-g005" target="_blank">Fig. 5</a>.</p

TMHMM predictions for YcdZ.

<p>Five transmembrane domains are predicted by TMHMM.</p

Alignment and SWAS plots of upstream regions of <i>cytR</i> in the Enterobacteriales.

<p>Notation as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044194#pone-0044194-g005" target="_blank">Fig. 5</a>.</p

The list of genomes with abbreviations.

*<p>Abbreviations in the left column were taken from KEGG database.</p>**<p>The accession number for the second chromosomes is in parentheses.</p>***<p>In the alignments, these genomes are denoted by the first two letters and a digit denoting the chromosome (1 or 2).</p