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See detailDéfinition du régulon DasR chez Streptomyces coelicolor, un réservoir pour la découverte de nouveaux gènes essentiels à l'induction de la différenciation morphologique et la production de métabolites secondaires
Colson, Séverine ULg

Doctoral thesis (2011)

Ce travail vise à comprendre les mécanismes d’induction du développement chez les Streptomyces. Chez ce genre bactérien, le facteur de transcription DasR a été identifié comme le premier régulateur global ... [more ▼]

Ce travail vise à comprendre les mécanismes d’induction du développement chez les Streptomyces. Chez ce genre bactérien, le facteur de transcription DasR a été identifié comme le premier régulateur global à la charnière entre le métabolisme primaire et le métabolisme secondaire, capable de percevoir l'état nutritionnel de l'environnement et d'adapter en conséquence la réponse des différents processus associés au développement (Rigali et al., 2006 et Rigali et al., 2008). La première ambition de cette thèse de doctorat a été d'évaluer l'ampleur du régulon DasR chez S. coelicolor afin d'obtenir une liste - exhaustive et appuyée par des critère de fiabilité - des gènes ciblés par ce régulateur global. L'hypothèse à la base de ce travail de prédiction était qu'au sein du régulon DasR se trouvaient peut-être des gènes de fonction encore inconnue mais essentiels au développement. Comprendre le rôle de ces protéines pouvait déboucher sur l'élucidation de nouvelles voies d'induction du développement et donc peut-être de nouvelles voies à exploiter pour induire le réveil des gènes cryptiques. La première démarche visant à répondre à ce premier objectif nous a amené à constater les lacunes des programmes web de prédiction des régulons existant, notamment le manque de flexibilité au niveau des critères de recherche et au niveau de l'exploitation des résultats obtenus, ainsi que l'absence de moyen permettant d'estimer la fiabilité de la prédiction. C'est pourquoi, dans un premier temps, nous nous sommes lancés le défi de créer un nouvel outil de prédiction des régulons procaryotiques qui répondrait davantage aux attentes des biologistes. Ce programme, nommé PREDetector (Prokaryotic Regulatory Elements Detector), a été réalisé en collaboration avec le Professeur Louis Wehenkel (Department of Electrical Engineering and Computer Science, Université de Liège) (Hiard et al., 2007). Les caractéristiques et les possibilités offertes par PREDetector sont expliquées et illustrées dans le premier chapitre des résultats. Le second chapitre des résultats - ainsi que l'annexe de cette thèse - sont quant à eux dédiés à l'exploitation de ce programme et la caractérisation de l'ensemble des cibles de DasR prédites chez S. coelicolor. Ensuite, et toujours motivé par un souci de présenter un travail fiable pour d'ultérieures investigations, une comparaison des régulons DasR prédits chez différents streptomycètes et autres actinomycètes (prédictions et démarches détaillées dans l'annexe de thèse), vient appuyer notre définition du noyau dur du régulon DasR - conservon DasR - déduit pour l'organisme modèle S. coelicolor. Enfin, nous passons à l'étape d'utilisation de ces données bioinformatiques avec l'étude de gènes/protéines dont l'expression est dépendante de DasR dans le but de peut-être établir de nouvelles connexions entre le métabolisme primaire et le développement chez la souche modèle S. coelicolor. De manière intéressante, le régulon DasR de S. coelicolor comprend plusieurs gènes codant pour des systèmes de transport ABC de sucre. A l'instar du PTSGlcNAc, ces transporteurs situés à l'interface cellule/environnement peuvent aussi être les senseurs de molécules cruciales pour l'induction du développement. Après le PTSGlcNAc, nous nous sommes donc concentrés sur l'étude du premier transporteur ABC de sucre régulé par DasR - en termes de fiabilité des prédictions et de conservation inter-espèces/-genres -, c'est-à-dire, le système DasABC. Dans le troisième chapitre des résultats nous démontrerons ainsi que DasA est une protéine impliquée dans le processus de différenciation morphologique de S. coelicolor (Colson et al., 2008). [less ▲]

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See detailThe Chitobiose-Binding Protein, DasA, Acts as a Link between Chitin Utilization and Morphogenesis in Streptomyces Coelicolor
Colson, Séverine ULg; van Wezel, G. P.; Craig, Matthias ULg et al

in Microbiology (2008), 154(Pt 2), 373-82

Streptomycetes are mycelial soil bacteria that undergo a developmental programme that leads to sporulating aerial hyphae. As soil-dwelling bacteria, streptomycetes rely primarily on natural polymers such ... [more ▼]

Streptomycetes are mycelial soil bacteria that undergo a developmental programme that leads to sporulating aerial hyphae. As soil-dwelling bacteria, streptomycetes rely primarily on natural polymers such as cellulose, xylan and chitin for the colonization of their environmental niche and therefore these polysaccharides may play a critical role in monitoring the global nutritional status of the environment. In this work we analysed the role of DasA, the sugar-binding component of the chitobiose ATP-binding cassette transport system, in informing the cell of environmental conditions, and its role in the onset of development and in ensuring correct sporulation. The chromosomal interruption of dasA resulted in a carbon-source-dependent vegetative arrest phenotype, and we identified a second DasR-dependent sugar transporter, in addition to the N-acetylglucosamine phosphotransferase system (PTS(GlcNAc)), that relates primary metabolism to development. Under conditions that allowed sporulation, highly aberrant spores with many prematurely produced germ tubes were observed. While GlcNAc locks streptomycetes in the vegetative state, a high extracellular concentration of the GlcNAc polymer chitin has no effect on development. The striking distinction is due to a difference in the transporters responsible for the import of GlcNAc, which enters via the PTS, and of chitin, which enters as the hydrolytic product chitobiose (GlcNAc(2)) through the DasABC transporter. A model explaining the role of these two essentially different transport systems in the control of development is provided. [less ▲]

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See detailPREDetector : Prokaryotic Regulatory Element Detector
Hiard, Samuel ULg; Rigali, Sébastien ULg; Colson, Séverine ULg et al

Poster (2007, November 12)

Background: In the post-genomic area, in silico predictions of regulatory networks are considered as a powerful approach to decipher and understand biological pathways within prokaryotic cells. The ... [more ▼]

Background: In the post-genomic area, in silico predictions of regulatory networks are considered as a powerful approach to decipher and understand biological pathways within prokaryotic cells. The emergence of position weight matrices based programs has facilitated the access to this approach. However, a tool that automatically estimates the reliability of the predictions and would allow users to extend predictions in genomic regions generally regarded with no regulatory functions was still highly demanded. Result: Here, we introduce PREDetector, a tool developed for predicting regulons of DNA-binding proteins in prokaryotic genomes that (i) automatically predicts, scores and positions potential binding sites and their respective target genes, (ii) includes the downstream co-regulated genes, (iii) extends the predictions to coding sequences and terminator regions, (iv) saves private matrices and allows predictions in other genomes, and (v) provides an easy way to estimate the reliability of the predictions. Conclusion: We present, with PREDetector, an accurate prokaryotic regulon prediction tool that maximally answers biologists’ requests. PREDetector can be downloaded freely at http://www.montefiore.ulg.ac.be/~hiard/predetectorfr.html [less ▲]

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See detailPREDetector: A new tool to identify regulatory elements in bacterial genomes
Hiard, Samuel ULg; Marée, Raphaël ULg; Colson, Séverine ULg et al

in Biochemical and Biophysical Research Communications (2007), 357(4), 861-864

In the post-genomic area, the prediction of transcription factor regulons by position weight matrix-based programmes is a powerful approach to decipher biological pathways and to modelize regulatory ... [more ▼]

In the post-genomic area, the prediction of transcription factor regulons by position weight matrix-based programmes is a powerful approach to decipher biological pathways and to modelize regulatory networks in bacteria. The main difficulty once a regulon prediction is available is to estimate its reliability prior to start expensive experimental validations and therefore trying to find a way how to identify true positive hits from an endless list of potential target genes of a regulatory protein. Here we introduce PREDetector (Prokaryotic Regulatory Elements Detector), a tool developed for predicting regulons of DNA-binding proteins in bacterial genomes that, beside the automatic prediction, scoring and positioning of potential binding sites and their respective target genes in annotated bacterial genomes, it also provides an easy way to estimate the thresholds where to find reliable possible new target genes. PREDetector can be downloaded freely at http://www.montefiore.ulg.ac.be/-hiard/PreDetector (c) 2007 Published by Elsevier Inc. [less ▲]

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See detailPREDetector : Prokaryotic Regulatory Element Detector
Hiard, Samuel ULg; Rigali, Sébastien ULg; Colson, Séverine ULg et al

Poster (2007, February 15)

Background: In the post-genomic area, in silico predictions of regulatory networks are considered as a powerful approach to decipher and understand biological pathways within prokaryotic cells. The ... [more ▼]

Background: In the post-genomic area, in silico predictions of regulatory networks are considered as a powerful approach to decipher and understand biological pathways within prokaryotic cells. The emergence of position weight matrices based programs has facilitated the access to this approach. However, a tool that automatically estimates the reliability of the predictions and would allow users to extend predictions in genomic regions generally regarded with no regulatory functions was still highly demanded. Result: Here, we introduce PREDetector, a tool developed for predicting regulons of DNA-binding proteins in prokaryotic genomes that (i) automatically predicts, scores and positions potential binding sites and their respective target genes, (ii) includes the downstream co-regulated genes, (iii) extends the predictions to coding sequences and terminator regions, (iv) saves private matrices and allows predictions in other genomes, and (v) provides an easy way to estimate the reliability of the predictions. Conclusion: We present, with PREDetector, an accurate prokaryotic regulon prediction tool that maximally answers biologists’ requests. PREDetector can be downloaded freely at http://www.montefiore.ulg.ac.be/~hiard/predetectorfr.html [less ▲]

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See detailConserved cis-acting elements upstream of genes composing the chitinolytic system of streptomycetes are DasR-responsive elements
Colson, Séverine ULg; Stephan, J.; Hertrich, T. et al

in Journal of Molecular Microbiology and Biotechnology (2007), 12(1-2), 60-66

For soil-dwelling bacteria that usually live in a carbon-rich and nitrogen-poor environment, the ability to utilize chitin -the second most abundant polysaccharide on earth is a decisive evolving ... [more ▼]

For soil-dwelling bacteria that usually live in a carbon-rich and nitrogen-poor environment, the ability to utilize chitin -the second most abundant polysaccharide on earth is a decisive evolving advantage as it is a source for both elements. Streptomycetes are high-GC Gram-positive soil bacteria that are equipped with a broad arsenal of chitinase-degrading genes. These genes are induced when the streptomycetes sense the presence of chitooligosaccharides. Their expression is repressed as soon as more readily assimilated carbon sources become available. This includes for example glucose or N-acetylglucosamine, the monomer subunit of chitin. Historically, the first cis-acting elements involved in carbon regulation in streptomycetes were found more than a decade ago upstream of chitinase genes, but the transcriptional regulator had so far remained undiscovered. In this work, we show that these cis-acting elements consist of inverted repeats with multiple occurrences and are bound by the HutC/GntR type regulator DasR. We have therefore designated these sites as DasR-responsive elements (dre). DasR, which is also the repressor of the genes for the N-acetylglucosamine-specific phosphotransferase transport system, should therefore play a critical role in sensing the balance between the monomeric and polymeric forms of N-acetylglucosamine. Copyright (c) 2007 S. Karger AG, Basel. [less ▲]

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See detailThe sugar phosphotransferase system of Streptomyces coelicolor is regulated by the GntR-family regulator DasR and links N-acetylglucosamine metabolism to the control of development
Rigali, Sébastien ULg; Nothaft, H.; Noens, E. E. E. et al

in Molecular Microbiology (2006), 61(5), 1237-1251

Members of the soil-dwelling, sporulating prokaryotic genus Streptomyces are indispensable for the recycling of the most abundant polysaccharides on earth (cellulose and chitin), and produce a wide range ... [more ▼]

Members of the soil-dwelling, sporulating prokaryotic genus Streptomyces are indispensable for the recycling of the most abundant polysaccharides on earth (cellulose and chitin), and produce a wide range of antibiotics and industrial enzymes. How do these organisms sense the nutritional state of the environment, and what controls the signal for the switch to antibiotic production and morphological development? Here we show that high extracellular concentrations of N-acetylglucosamine, the monomer of chitin, prevent Streptomyces coelicolor progressing beyond the vegetative state, and that this effect is absent in a mutant defective of N-acetylglucosamine transport. We provide evidence that the signal is transmitted through the GntR-family regulator DasR, which controls the N-acetylglucosamine regulon, including the pts genes ptsH, ptsI and crr needed for uptake of N-acetylglucosamine. Deletion of dasR or the pts genes resulted in a bald phenotype. Binding of DasR to its target genes is abolished by glucosamine 6-phosphate, a central molecule in N-acetylglucosamine metabolism. Extracellular complementation experiments with many bld mutants showed that the dasR mutant is arrested at an early stage of the developmental programme, and does not fit in the previously described bld signalling cascade. Thus, for the first time we are able to directly link carbon (and nitrogen) metabolism to development, highlighting a novel type of metabolic regulator, which senses the nutritional state of the habitat, maintaining vegetative growth until changing circumstances trigger the switch to sporulation. Our work, and the model it suggests, provide new leads towards understanding how microorganisms time developmental commitment. [less ▲]

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See detailPreDetector : Prokaryotic Regulatory Element Detector
Hiard, Samuel ULg; Rigali, Sébastien ULg; Colson, Séverine ULg et al

Poster (2006, May 17)

PreDetector is a stand-alone software, written in java. Its final aim is to predict regulatory sites for prokaryotic species. It comprises two functionalities. The first one is very similar to Target ... [more ▼]

PreDetector is a stand-alone software, written in java. Its final aim is to predict regulatory sites for prokaryotic species. It comprises two functionalities. The first one is very similar to Target Explorer1. From a set of sequences identified as potential target sites, PreDetector creates a consensus sequence and computes its scoring matrix. This sequence and matrix can be saved on a file and, then, be used to find along a selected genome the sequences that are close enough to the consensus sequence. To this end, a score is attributed to each locus in the genome according to the similarity measure defined by the matrix. The output of this functionality is filtered with a cut-off score and then directly used as input by the second one. The second functionality starts by fetching the gene positions of the selected species from the NCBI server. The loci having above cut-off score are then classified into four classes, allowing multiple classes for one element. This gives the biologists a better view of his discovered sequences. [less ▲]

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