Data Mining Using Hidden Markov Models (HMM2) to Detect Heterogeneities into Bacteria Genomes

PosterThe Streptococcus genus contains both pathogenic bacteria and bacteria used in the food-processing industry. We are developing a statistical segmentation method to identify heterogeneous sequences such as sequences acquired from recent horizontal transfer or genes weakly or strongly expressed. The method is based on second order Hidden Markov Models (HMM2). After an automatic unsupervised training, this method allows to demarcating some particular areas into a genome. After checking the efficiency of such models on various controls and on chimeric sequences generated in silico, we choose a HMM2 (3-mer, 5 states) to analyse the complete genome sequence of S. Thermophilus CNRZ1066 (1.8 Mb). More the 80 atypical segments were extracted and are currently analysed further

Data Mining Using Hidden Markov Models (HMM2) to Detect Heterogeneities into Bacteria Genomes

International audienceThe Streptococcus genus contains both pathogenic bacteria and bacteria used in the food-processing industry. We are developing a statistical segmentation method to identify heterogeneous sequences such as sequences acquired from recent horizontal transfer or genes weakly or strongly expressed. The method is based on second order Hidden Markov Models (HMM2). After an automatic unsupervised training, this method allows to demarcating some particular areas into a genome. After checking the efficiency of such models on various controls and on chimeric sequences generated in silico, we choose a HMM2 (3-mer, 5 states) to analyse the complete genome sequence of S. Thermophilus CNRZ1066 (1.8 Mb). More the 80 atypical segments were extracted and are currently analysed further

Using Markov Models to Mine Temporal and Spatial Data

Référence du projet ANR BIODIVAGRIM : ANR 07 BDIV 02Markov models represent a powerful way to approach the problem of mining time and spatial signals whose variability is not yet fully understood. In this chapter, we will present a general methodology to mine different kinds of temporal and spatial signals having contrasting properties: continuous or discrete with few or many modalities. This methodology is based on a high order Markov modelling as implemented in a free software: carottAge (Gnu GPL)Les modèles de Markov sont des modèles puissants pour analyser des signaux temporels et spatiaux dont la variabilité n'est pas entièrement comprise. Dans ce chapitre, nous présentons notre méthodologie pour fouiller différentes sortes de signaux ayant des propriétés différentes: signaux continus ou discrets, simples ou composites. Cette méthodologie s'appuie sur des modèles de Markov cachés du second-ordre tels qu'implantés dans la boîte à outils CarottAge (licence Gnu-GPL)

Multiple and Variable NHEJ-Like Genes Are Involved in Resistance to DNA Damage in Streptomyces ambofaciens

International audienceNon-homologous end-joining (NHEJ) is a double strand break (DSB) repair pathway which does not require any homologous template and can ligate two DNA ends together. The basic bacterial NHEJ machinery involves two partners: the Ku protein, a DNA end binding protein for DSB recognition and the multifunctional LigD protein composed a ligase, a nuclease and a polymerase domain, for end processing and ligation of the broken ends. In silico analyses performed in the 38 sequenced genomes of Streptomyces species revealed the existence of a large panel of NHEJ-like genes. Indeed, ku genes or ligD domain homologues are scattered throughout the genome in multiple copies and can be distinguished in two categories: the " core " NHEJ gene set constituted of conserved loci and the " variable " NHEJ gene set constituted of NHEJ-like genes present in only a part of the species. In Streptomyces ambofaciens ATCC23877, not only the deletion of " core " genes but also that of " variable " genes led to an increased sensitivity to DNA damage induced by electron beam irradiation. Multiple mutants of ku, ligase or polymerase encoding genes showed an aggravated phenotype compared to single mutants. Biochemical assays revealed the ability of Ku-like proteins to protect and to stimulate ligation of DNA ends. RT-qPCR and GFP fusion experiments suggested that ku-like genes show a growth phase dependent expression profile consistent with their involvement in DNA repair during spores formation and/or germination

Défense de Streptococcus thermophilus contre le stress oxydatif (existence d'un système de réponse, construction et sélection d'une collection de mutants, identification de gènes impliqués)

Streptococcus thermophilus est un organisme anaérobie aérotolérant. Un système de défense inductible contre le peroxyde d'hydrogène a pu être mis en évidence, et participe à l'aérotolérance de la souche CNRZ368. Afin d'identifier des gènes impliqués dans la défense contre le stress oxydatif, des mutants obtenus par mutagenèse insertionnelle ont été sélectionnés pour leur sensibilité ou résistance à un tel stress. L'analyse moléculaire des mutants ainsi sélectionnés révèle que les loci interrompus peuvent être classés en 4 catégories : ceux portant des gènes présentant un lien potentiel avec la défense contre le stress oxydatif, ceux potentiellement impliqués dans la synthèse de la paroi, ceux potentiellement impliqués dans le métabolisme général, ceux portant des gènes de fonction inconnue. Deux mutants, interrompus dans des gènes homologues de rodA et pbp2b sont affectés dans leur forme cellulaire, confirmant que les gènes affectés chez ces deux mutants seraient les homologues fonctionnels des gènes rodA et pbp2b, impliqués dans l'élongation de la paroi latérale. Deux autres mutants sont affectés dans deux ORF distinctes mais localisées au même locus. Cette région contient au moins 4 ORF dont les produits hypothétiques sont homologues de protéines impliquées dans la maturation des centres Fe-S. Une analyse transcriptionnelle permet de montrer leur cotranscription. Une mutagenèse dirigée sur l'une des 4 ORF (nifU-like) permet d'impliquer celle-ci directement dans la défense contre le stress oxydatif. Parallèlement, une étude du phénotype colonial de la souche CNRZ368 a permis d'identifier l'existence de variants spontanés apparaissant à une fréquence proche du pourcent. La détection de ces phénotypes est conditionnelle à la présence d'oxygène. De plus, ces clones variants sont fréquemment résistants à un stress oxydatif, suggérant l'existence d'un lien (bien que non strict) entre ces variants et le niveau de résistance contre le stress oxydatif.Streptococcus thermophilus is an anaerobic aerotolerant organism. A hydrogen peroxide defence system had been shown to be inducible in the CNRZ368 strain, and may take part in its aerotolerance . In order to identify genes involved in S. thermophilus defence against oxidative stress, clones from a mutant collection obtained by insertional mutagenesis, had been selected for their sensitivity or resistance to an oxidative stress. Molecular analysis of mutants selected under these conditions showed that their disruption sites can be classified in 4 categories: those that carried genes potentially linked to oxidative stress defence, those potentially involved in cell wall biosynthesis, those potentially involved in general metabolism, those that carried genes of unknown function. Two mutants, disrupted in genes homologous to rodA et pbp2b were affected in their cell shape, confirming that these genes were probably functional homologues of rodA and pbp2b genes involved in elongation of lateral cell wall. Two other mutants were affected into two distinct ORF but localised in the same locus. This region contained at least 4 ORF whose hypothetical translation products are homologous to proteins involved in Fe-S centers maturation. A transcriptional analysis showed their cotranscription. Directed mutagenesis affecting one of the 4 ORF (nifU-like) allowed to demonstrate its direct involvement in oxidative stress defence.Moreover, study of CNRZ368 colonial phenotype revealed the existence of spontaneous variants that appeared with a frequency of almost one percent. Detection of variant phenotypes was conditional to oxygen presence during growth. In addition, variant clones often displayed a better resistance to oxidative stress than did the wild-type strain, suggesting a link (although it is not a strict link) between these variants and the resistance against oxidative stress level.NANCY1-SCD Sciences & Techniques (545782101) / SudocSudocFranceF

Complete Genome Sequence of Streptomyces ambofaciens DSM 40697, a Paradigm for Genome Plasticity Studies.

International audienceThe sequence of Streptomyces ambofaciens DSM 40697 was completely determined. The genome consists of an 8.1-Mbp linear chromosome with terminal inverted repeats of 210 kb. Genomic islands were identified, one of which corresponds to a new putative integrative and conjugative element (ICE) called pSAM3

Effects of rodA and pbp2b Disruption on Cell Morphology and Oxidative Stress Response of Streptococcus thermophilus CNRZ368

Insertional mutagenesis was used to isolate clones from Streptococcus thermophilus CNRZ368 that were modified in their abilities to tolerate oxidative stress. During this process, two menadione-sensitive clones (6G4 and 18C3) were found to display abnormal cell morphologies and distorted chain topologies and were further studied. Molecular characterization of both 6G4 and 18C3 mutants indicated that they were disrupted in open reading frames homologous to rodA and pbp2b, respectively. Both genes encoded proteins in Escherichia coli that were described as being implicated in peptidoglycan synthesis during the process of cell elongation and to function in determining the rod shape of the cell. This work reports a possible connection between peptidoglycan biosynthesis and oxidative stress defense in S. thermophilus CNRZ368

Subtelomeres are fast-evolving regions of the Streptomyces linear chromosome

International audienceStreptomyces possess a large linear chromosome (6–12Mb) consisting of a conserved central region flanked by variable armscovering several megabases. In order to study the evolution of the chromosome across evolutionary times, a representativepanel of Streptomyces strains and species (125) whose chromosomes are completely sequenced and assembled was selected.The pan-genome of the genus was modelled and shown to be open with a core-genome reaching 1018 genes. The evolutionof Streptomyces chromosome was analysed by carrying out pairwise comparisons, and by monitoring indexes measuring theconservation of genes (presence/absence) and their synteny along the chromosome. Using the phylogenetic depth offered bythe chosen panel, it was possible to infer that within the central region of the chromosome, the core-genes form a highly conserved organization, which can reveal the existence of an ancestral chromosomal skeleton. Conversely, the chromosomal arms, enriched in variable genes evolved faster than the central region under the combined effect of rearrangements and addition of new information from horizontal gene transfer. The genes hosted in these regions may be localized there because of the adaptive advantage that their rapid evolution may confer. We speculate that (i) within a bacterial population, the variability of these genes may contribute to the establishment of social characters by the production of ‘public goods’ (ii) at the evolutionary scale, this variability contributes to the diversification of the genetic pool of the bacteria

Characterisation of Streptococcus thermophilus CNRZ368 oxidative stress-resistant mutants: involvement of a potential Rgg-like transcriptional regulator

International audienceEight mutants of Streptococcus thermophilus CNRZ368 presenting a menadione-resistant phenotype were selected and the locus mutated in each mutant was identified. Among these clones, 5 were disrupted in a gene of unknown function, 2 were impaired in genes involved in cellular metabolism and the last one (the mutant 15H7) was disrupted in rgg encoding a putative transcriptional regulator. To determine if rgg could be a key regulator of the superoxide defence of S. thermophilus, the 15H7 mutant was further characterised. The results from our work indicate that the rgg gene, mutated in the 15H7 genome, is a pseudogene composed of 2 ORFs (rggA and rggB) that are potentially translated in two different frames. Moreover, transcription analysis demonstrated the existence of a transcript containing rggA, rggB and orf2. Construction of $\Delta$rggA, $\Delta$rggB and orf2 mutants and their phenotypic analysis confirmed the involvement of rggA and rggB in the oxidative stress response. The question of the role of the pseudogene rgg is still open.Caractérisation de mutants résistants au stress oxydant chez Streptococcus thermophilus CNZR368 : implication d'un régulateur transcriptionnel putatif de type Rgg. Huit mutants de Streptococcus thermophilus CNRZ368, présentant un phénotype de résistance à la ménadione ont été sélectionnés et le locus muté a été identifié pour chacun d'eux. Parmi ces clones, 5 sont interrompus dans un gène de fonction inconnue, 2 sont mutés dans des gènes impliqués dans le métabolisme cellulaire et un autre (15H7) est interrompu dans le gène rgg codant potentiellement un régulateur transcriptionnel. Afin de déterminer si rgg est un régulateur majeur de la défense contre le stress superoxyde le mutant 15H7 a été plus amplement étudié. Les résultats de notre travail indiquent que le gène rgg, muté dans le génome de 15H7, est un pseudogène composé de deux ORF (rggA and rggB) potentiellement traduites dans deux phases de lecture différentes. De plus, l'analyse transcriptionelle démontre l'existence d'un transcrit englobant rggA, rggB et orf2. La construction des mutants $\Delta$rggA, $\Delta$rggB et orf2 ainsi que l'analyse de leur phénotype confirme l'implication de rggA et rggB dans la réponse contre le stress oxydant. La question concernant le rôle du pseudogène rgg est toujours ouverte