La structure spatiale de l'ADN est fortement affectée par le surenroulement. Lorsqu'il est surenroulé, l'ADN circulaire (ou linéaire et contraint topologiquement aux extrémités) se replie et forme des boucles appelées plectonèmes, rapprochant dans l'espace des régions éloignées du chromosome, perturbant ainsi de l réseau de régulation de la transcription de la cellule. Les chromosomes bactériens sont surenroulés négativement et ce surenroulement est connu pour jouer un rôle important dans la régulation de la transcription. Cependant, à cause de la nature globale de la contrainte topologique associée à l'ADN, les méthodes actuelles ne sont capables de simuler que de petites molécules (jusqu'à quelques milliers de paires de bases, KB). Cette thèse présente un nouvel algorithme utilisé pour réaliser des simulations de Monte-Carlo d'ADN surenroulé, basé sur une molécule. Cet algorithme a été utilisé pour réaliser des simulations de longues molécules (plusieurs dizaines de KB) et apporter un nouvel éclairage sur des questions encore débattues à propos de la structure de l'ADN surenroulé à cette échelle. Cette méthode permet d'étudier l'effet de la position des gènes le long de l'ADN sur leur co-localisation et leur co-régulation, et laisse entrevoir la possibilité de simuler le repliement d'un chromosome bactérien entier. / Superhelicity strongly affects the 3D structure of DNA. When supercoiled, circular DNA (or linear DNA with topolocically constrained ends) folds and forms loops called plectonemes, bringing some distant parts of the chromosme close to one another in space, thus perturbing the transcription regulation network of the cell. Bacterial chromosomes are negatively supercoiled and superhelicity is known to play a important role in the regulation of the transcription. However, due to the global nature of the topological constraint imposed to the DNA, current methods have only been able to simulate small moelcules (up to a few kilobasepairs, KB). This thesis presents a novel algorithm used to performed Monte-Carlo simulations of supercoiled DNA, featuring a local approach to the topological constraint via the computation of the twist of the molecule. Using this efficient algorithm, stimulations of long molecules (tens of KB) were performed and shed a new light on debated questions about the structure of supercoiled DNA at this scale. This method allows to study the effect of the position of genes along the DNA on their co-localisation and co-regulation, and to envision the possibility of simulating the folding of a whole bacterial chromosome.

No description available.

Advances in optical imaging techniques have allowed quantitative studies of many biological systems. This dissertation elaborates on our efforts in both developing novel imaging modalities based on detection of optical absorption and applying high-sensitivity fluorescence microscopy to the study of biology. / Chemistry and Chemical Biology

In this thesis, on the basis of the asymmetrical charge distribution of E. coli topoisomerase I, I developed a new rapid procedure to purify E. coli DNA topoismoerase I in the milligram range. The new procedure includes using both cation- and anion-exchange columns, i.e., SP-sepharose FF and Q-sepharose FF columns. E. coli topoisomerase I purified here is free of nuclease contamination. The kinetic constants of the DNA relaxation reaction of E. coli DNA topoisomerase I were determined as well. I also used isothermal titration calorimetry to investigate the energetics of DNA supercoiling by using the unwinding properties of DNA intercalators, ethidium and daunomycin. After comparing the enthalpy changes of these DNA intercalators binding to supercoiled and nicked or relaxed plasmid DNA pXXZ06, I determined the DNA supercoiling enthalpy is about 12 kcal/mol per turn of DNA supercoil, which is in good agreement with the previously published results.

A principle challenge of modern biology is to understand how the human genome is organised and regulated within a nucleus. The field of chromatin biology has made significant progress in characterising how protein and DNA modifications reflect transcription and replication state. Recently our lab has shown that the human genome is organised into large domains of altered DNA helical twist, called DNA supercoiling domains, similar to the regulatory domains observed in prokaryotes. In my PhD I have analysed how the maintenance and distribution of DNA supercoiling relates to biological function in human cells. DNA supercoiling domains are set up and maintained by the balanced activity of RNA transcription and topoisomerase enzymes. RNA polymerase twists the DNA, over-winding in front of the polymerase and under-winding behind. In contrast topoisomerases relieve supercoiling from the genome by introducing transient nicks (topoisomerase I) or double strand breaks (topoisomerase II) into the double helix. Topoisomerase activity is critical for cell viability, but the distribution of topoisomerase I, IIα and IIβ in the human genome is not known. Using a chromatin immunoprecipitation (ChIP) approach I have shown that topoisomerases are enriched in large chromosomal domains, with distinct topoisomerase I and topoisomerase II domains. Topoisomerase I is correlated with RNA polymerase II, genes and underwound DNA, whereas topoisomerase IIα and IIβ are associated with each other and over-wound DNA. This indicates that different topoisomerase proteins operate in distinct regions of the genome and can be independently regulated depending on the genomic environment. Transcriptional regulation by DNA supercoiling is believed to occur through changes in gene promoter structure. To investigate DNA supercoiling my lab has developed biotinylated trimethylpsoralen (bTMP) as a DNA structure probe, which preferentially intercalates into under-wound DNA. Using bTMP in conjunction with microarrays my lab identified a transcription and topoisomerase dependent peak of under-wound DNA in a meta-analysis of several hundred genes (Naughton et al. (2013)). In a similar analysis, Kouzine et al. (2013) identified an under-wound promoter structure and proposed a model of topoisomerase distribution for the regulation of promoter DNA supercoiling. To better understand the role of supercoiling and topoisomerases at gene promoters, a much larger-scale analysis of these factors was required. I have analysed the distribution of bTMP at promoters genome wide, confirming a transcription and expression dependent distribution of DNA supercoils. DNA supercoiling is distinct at CpG island and non-CpG island promoters, and I present a model in which over-wound DNA limits transcription from both CpG island promoters and repressed genes. In addition, I have mapped by ChIP topoisomerase I and IIβ at gene promoters on chromosome 11 and identified a different distribution to that proposed by Kouzine et al. (2013), with topoisomerase I maintaining DNA supercoiling at highly expressed genes. This study provides the first comprehensive analysis of DNA supercoiling at promoters and identifies the relationship between supercoiling, topoisomerase distribution and gene expression. In addition to regulating transcription, DNA supercoiling and topoisomerases are important for genome stability. Several studies have suggested a link between DNA supercoiling and instability at common fragile sites (CFSs), which are normal structures in the genome that frequently break under replication stress and cancer. bTMP was used to measure DNA supercoiling across FRA3B and FRA16D CFSs, identifying a transition to a more over-wound DNA structure under conditions that induce chromosome fragility at these regions. Furthermore, topoisomerase I, IIα and IIβ showed a pronounced depletion in the vicinity of the FRA3B and FRA16D CFSs. This provides the first experimental evidence of a role for DNA supercoiling in fragile site formation.

No description available.

Computer simulations provide a potentially powerful complement to conventional experimental techniques in elucidating the structures, dynamics and interactions of macromolecules. In this thesis, I present three applications of computer simulations to investigate important biomolecules with sizes ranging from two-residue peptides, to proteins, and to whole chromosome structures. First, I describe the results of 441 independent explicit-solvent molecular dynamics (MD) simulations of all possible two-residue peptides that contain the 20 standard amino acids with neutral and protonated histidine. 3JHNHα coupling constants and δHα chemical shifts calculated from the MD simulations correlated quite well with recently published experimental measurements for a corresponding set of two-residue peptides. Neighboring residue effects (NREs) on the average 3JHNHα and δHα values of adjacent residues were also reasonably well reproduced. The intrinsic conformational preferences of each residue, and their NREs on the conformational preferences of adjacent residues, were analyzed. Finally, these NREs were compared with corresponding effects observed in a coil library and the average β-turn preferences of all residue types were determined. Second, I compare the abilities of three derivatives of the Amber ff99SB force field to reproduce a recent report of 3JHNHα scalar coupling constants for hundreds of two-residue peptides. All-atom MD simulations of 256 two-residue peptides were performed and the results showed that a recently-developed force field (RSFF2) produced a dramatic improvement in the agreement with experimental 3JHNHα coupling constants. I further show that RSFF2 also improved modestly agreement with experimental 3JHNHα coupling constants of five model proteins. However, an analysis of NREs on the 3JHNHα coupling constants of the two-residue peptides indicated little difference between the force fields’ abilities to reproduce experimental NREs. I speculate that this might indicate limitations in the force fields’ descriptions of nonbonded interactions between adjacent side chains or with terminal capping groups. Finally, coarse-grained (CG) models and multi-scale modeling methods are used to develop structural models of entire E. coli chromosomes confined within the experimentally-determined volume of the nucleoid. The final resolution of the chromosome structures built here was one-nucleotide-per-bead (1 NTB), which represents a significant increase in resolution relative to previously published CG chromosome models, in which one bead corresponds to hundreds or even thousands of basepairs. Based on the high-resolution final 1 NTB structures, important physical properties such as major and minor groove widths, distributions of local DNA bending angles, and topological parameters (Linking Number (Lk), Twist (Tw) and Writhe (Wr)) were accurately computed and compared with experimental measurements or predictions from a worm-like chain (WLC) model. All these analyses indicated that the chromosome models built in this study are reasonable at a microscopic level. This chromosome model provides a significant step toward the goal of building a whole-cell model of a bacterial cell.

Transcription by RNA polymerase can induce the formation of hypernegatively supercoiled DNA both in vivo and in vitro. This phenomenon has been explained by a “twin-supercoiled-domain” model of transcription where a positively supercoiled domain is generated ahead of the RNA polymerase and a negatively supercoiled domain behind it. In E. coli cells, transcription-induced topological change of chromosomal DNA is expected to actively remodel chromosomal structure and greatly influence DNA transactions such as transcription, DNA replication, and recombination. In this study, an IPTG-inducible, two-plasmid system was established to study transcription-coupled DNA supercoiling (TCDS) in E. coli topA strains. By performing topology assays, biological studies, and RT-PCR experiments, TCDS in E. coli topA strains was found to be dependent on promoter strength. Expression of a membrane-insertion protein was not needed for strong promoters, although co-transcriptional synthesis of a polypeptide may be required. More importantly, it was demonstrated that the expression of a membrane-insertion tet gene was not sufficient for the production of hypernegatively supercoiled DNA. These phenomenon can be explained by the “twin-supercoiled-domain” model of transcription where the friction force applied to E. coli RNA polymerase plays a critical role in the generation of hypernegatively supercoiled DNA. Additionally, in order to explore whether TCDS is able to greatly influence a coupled DNA transaction, such as activating a divergently-coupled promoter, an in vivo system was set up to study TCDS and its effects on the supercoiling-sensitive leu-500 promoter. The leu-500 mutation is a single A-to-G point mutation in the -10 region of the promoter controlling the leu operon, and the AT to GC mutation is expected to increase the energy barrier for the formation of a functional transcription open complex. Using luciferase assays and RT-PCR experiments, it was demonstrated that transient TCDS, “confined” within promoter regions, is responsible for activation of the coupled transcription initiation of the leu-500 promoter. Taken together, these results demonstrate that transcription is a major chromosomal remodeling force in E. coli cells.

L’entérobactérie Dickeya dadantii est responsable de la maladie de la pourriture molle sur de nombreux hôtes végétaux. Ce symptôme est essentiellement dû à la production d’un arsenal d’enzymes qui dégradent la pectine, ciment des parois des cellules végétales. Parmi ces enzymes, les pectate lyases (Pels) ont un rôle majeur dans le pouvoir pathogène en raison de leur capacité àreproduire, sous forme purifiée, le symptôme de la pourriture molle. La synthèse des Pels est soumise à un contrôle très fin qui fait intervenir différents régulateurs agissant de manière intégrée via un réseau de régulation. De nombreuses conditions environnementales modulent la synthèse des Pels via l’action de ces régulateurs. La température est un facteur qui agit sur leur synthèse et pour lequel les mécanismes moléculaires restaient non élucidés. Lors de cette étude, nous avons montré que le régulateur PecT, un répresseur du réseau de régulation, intervient dans la thermorégulation de la synthèse des Pels. PecT s’est avéré être également impliqué dans la thermorégulation de deux autres fonctions de virulence : la mobilité et la synthèse desexopolysaccharides de surface. La quantification des transcrits des gènes de ces 3 fonctions de virulence a permis de montrer que l’action de PecT dans ce contrôle a lieu au niveau transcriptionnel. Le mécanisme moléculaire de la thermorégulation exercée par PecT a été étudié plus en détail sur les gènes pel. Des résultats obtenus in vivo ont montré que la fixation de PecT sur les régionsrégulatrices des gènes pel est plus efficace quand la température augmente. La croissance de D. dadantii à hautes températures induit un relâchement de l’ADN. De manière remarquable, un relâchement artificiel de l’ADN par un traitement inhibant la gyrase entraine une augmentation de la fixation de PecT sur les gènes pel même pour des cellules cultivées à basses températures. De plus, la délétion de PecT se traduit par une augmentation de la capacité de D. dadantii à induire la pourriture molle à hautes températures. Ainsi la topologie de l’ADN et PecT agissent de manière concertée pour moduler la synthèse des Pels en fonction de la température.L’ensemble de ces données apporte une preuve supplémentaire de l’importance de la dynamique structurale de la chromatine dans l’ajustement de la physiologie bactérienne en réponse aux variations des conditions environnementales. / Bacteria are colonizers of various environments and host organisms, and they are often subjected to drastic temperature variations. Dickeya dadantii is a Gram-negative pathogen infecting a wide range of plant species. Soft rot, the visible symptom, is mainly due to the production of pectate lyases (Pels) that can destroy the plant cell walls. Production of Pels is controlled by a complex regulation system and responds tovarious stimuli, such as presence of pectin, plant extracts, growth phase, temperature or iron concentration. Although many studies have been carried out, the mechanisms of control of Pels production by temperature have not yet been elucidated. In bacteria, thermoregulation acting at the level of transcription initiation occurs usually both via transcription factors and DNA topology. We show that PecT, a previously identified repressor, is involved in the thermoregulation of the pel gene expression. Using in vivo Chromatin ImmunoPrecipitation (ChIP) coupled to quantitative RT-PCR(qRT-PCR), we reveal that PecT binding to the pel gene promoters is modulated by temperature. By manipulating the DNA topology in vivo, we further show that DNA supercoiling state is involved in the thermoregulation of pel gene expression by PecT. In addition, we show that the development of the pathogenicity of the pecT mutant according to changes in temperature is different from that of the parental strain. This report presents a new example of how plant pathogenic bacteria use transcription factor and DNA topology to adjust synthesis of virulence factors in response to temperature variation.

Les bactéries pathogènes coordonnent de manière très stricte l’expression de leurs facteurs de virulence en fonction de leur état métabolique, des conditions externes et de l’état de l’hôte. La topologie du chromosome bactérien est également modulée par les conditions environnementales et l’état métabolique des cellules. Le surenroulement de l’ADN, est considéré désormais comme un élément clé de la régulation de l’expression génique. L’objectif de cette thèse est d’identifier les acteurs essentiels de la réponse aux conditions rencontrées durant l’infection chez la bactérie phytopathogène Dickeya dadantii, responsable de la pourriture molle d’une large gamme d’hôtes. Les symptômes de pourriture molle sont principalement associés à la synthèse d'enzymes extracellulaires, en particulier les pectinases qui vont dégrader la paroi des cellules végétales. Cependant une colonisation efficace de la plante requiert de nombreux facteurs additionnels. L’analyse du transcriptome de D. dadantii dans 32 conditions de culture proches de celles rencontrées lors du cycle infectieux a révélé qu’en moyenne 63% des gènes sont exprimés dans chacune des conditions testées alors que 82% des gènes sont exprimés dans au moins une des conditions analysées. Deux facteurs modifient profondément l’expression génique: la phase de croissance et la nature des stress appliqués. L’analyse des gènes différentiellement exprimés a permis d’établir des signatures transcriptionnelles et fonctionnelles spécifiques de chaque stress et d’identifier de nouveaux régulateurs potentiellement impliqués dans la survie aux stress. L’adaptation au stress acide étant peu connue chez les pathogènes de plante, la régulation de quelques gènes spécifiquement induits en stress acide a été approfondie. Ces études ont révélé que le régulateur OmpR est un élèment clé de la réponse au stress acide chez Dickeya. Afin d’établir un lien entre réponse aux stress et impact de la topologie de l’ADN et des NAPs, les profils transcriptionnels obtenus lors des stress ont été comparés à ceux obtenus dans des conditions de relaxation artificielle de l’ADN et chez des mutants fis ou hns. La distribution chromosomique des GDE a révélé des profils cohérents de gènes activés ou réprimés lors des variations de conditions environnementales quelque soit le milieu de culture utilisé et l’existence de patchs d’expression qui illustre l’organisation en domaines du chromosome bactérien.L’expression des gènes au sein des domaines est dépendante de leur propriété thermodynamique, de leur préférence vis à vis du surenroulement, et de leur réponse aux NAPs. Ainsi, Dickeya tire partie des variations topologiques de l’ADN au cours de l’infection pour coordonner son programme de virulence. Ces résultats illustrent la complexité des processus utilisés par D. dadantii pour s’adapter aux conditions de l’infection et coloniser ses hôtes. / Pathogenic bacteria strictly coordinate the expression of their virulence factors according to their metabolic states, external conditions and the host environment. The topology of the bacterial chromosome is also modulated by environmental conditions and the metabolic state of the cells. The DNA supercoiling is now considered as a key factor in the regulation of gene expression. The objective of this thesis is to provide a comprehensive picture of the Dickeya infection process by integrated analyses of gene expression patterns obtained under various stress conditions encountered by this pathogen in the course of infection. Dickeya dadantii is a plant pathogenic bacterium that causes soft-rot disease in a wide range of plant species. Soft rot symptoms are mainly associated with the synthesis of extracellular enzymes, particularly pectinases which degrade the plant cell wall. However, an effective colonization of the plant requires a number of additional factors. The transcriptome analysis of D. dadantii, grown in a suite of thirty-two different growth conditions similar to those conditions encountered during the infection cycle revealed that an average of 63% of genes was expressed in each individual condition, while 82% of genes are expressed in at least one of the analyzed conditions. Two factors profoundly alter gene expression: the growth phase and the nature of applied stress. Analysis of differentially expressed genes in this work has established specific transcriptional and functional signatures of each stress and proposed new regulators potentially involved in survival under stress conditions. In this way, we obtained the apparent « temporal map » of the bacterial responses to sequential stress conditions encountered during the infection. The chromosomal distribution of DEG revealed coherent patches of genes activated or repressed during changes in environmental conditions and highlighted a rational organization of the DEG in the chromosomal space. Gene expression within the chromosomal domains is dependent on primary sequence organisation, DNA thermodynamic stability, supercoil dynamics, and binding effects of two abundant nucleoid associated proteins, FIS and H-NS. Therefore, Dickeya takes advantage of DNA topological variations during the infection to coordinate its virulence program. These results illustrate the complexity of mechanisms used by D. dadantii to adapt to stress conditions and colonize its hosts.