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

Epigenetically-Inherited Centromere and Neocentromere DNA Replicates Earliest in S-Phase

Eukaryotic centromeres are maintained at specific chromosomal sites over many generations. In the budding yeast Saccharomyces cerevisiae, centromeres are genetic elements defined by a DNA sequence that is both necessary and sufficient for function; whereas, in most other eukaryotes, centromeres are maintained by poorly characterized epigenetic mechanisms in which DNA has a less definitive role. Here we use the pathogenic yeast Candida albicans as a model organism to study the DNA replication properties of centromeric DNA. By determining the genome-wide replication timing program of the C. albicans genome, we discovered that each centromere is associated with a replication origin that is the first to fire on its respective chromosome. Importantly, epigenetic formation of new ectopic centromeres (neocentromeres) was accompanied by shifts in replication timing, such that a neocentromere became the first to replicate and became associated with origin recognition complex (ORC) components. Furthermore, changing the level of the centromere-specific histone H3 isoform led to a concomitant change in levels of ORC association with centromere regions, further supporting the idea that centromere proteins determine origin activity. Finally, analysis of centromere-associated DNA revealed a replication-dependent sequence pattern characteristic of constitutively active replication origins. This strand-biased pattern is conserved, together with centromere position, among related strains and species, in a manner independent of primary DNA sequence. Thus, inheritance of centromere position is correlated with a constitutively active origin of replication that fires at a distinct early time. We suggest a model in which the distinct timing of DNA replication serves as an epigenetic mechanism for the inheritance of centromere position

CAGO: A Software Tool for Dynamic Visual Comparison and Correlation Measurement of Genome Organization

CAGO (Comparative Analysis of Genome Organization) is developed to address two critical shortcomings of conventional genome atlas plotters: lack of dynamic exploratory functions and absence of signal analysis for genomic properties. With dynamic exploratory functions, users can directly manipulate chromosome tracks of a genome atlas and intuitively identify distinct genomic signals by visual comparison. Signal analysis of genomic properties can further detect inconspicuous patterns from noisy genomic properties and calculate correlations between genomic properties across various genomes. To implement dynamic exploratory functions, CAGO presents each genome atlas in Scalable Vector Graphics (SVG) format and allows users to interact with it using a SVG viewer through JavaScript. Signal analysis functions are implemented using R statistical software and a discrete wavelet transformation package waveslim. CAGO is not only a plotter for generating complex genome atlases, but also a platform for exploring genome atlases with dynamic exploratory functions for visual comparison and with signal analysis for comparing genomic properties across multiple organisms. The web-based application of CAGO, its source code, user guides, video demos, and live examples are publicly available and can be accessed at http://cbs.ym.edu.tw/cago

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>udp</i> in close relatives of <i>E.coli</i>.

<p>The detected sites are highlighted in the consensus of alignment. A) Alignment of the upstream regions. Green – CRP-boxes, magenta – CytR-boxes. B) SWAS and information content plots. Scores are plotted corresponding to the middle (4^th) position of a 8 bp window. Blue – O_CYTRD, magenta – O_CYTRP, red – averaged positional information content.</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

Alignment and SWAS plots of upstream regions of <i>tsx</i> in close relatives of <i>E. coli</i>.

<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