Experiments with microbes are a powerful tool for addressing general questions in evolutionary ecology. Microbial evolution is also interesting in its own right, and often clinically relevant. I have used experimental evolution of bacteria (Pseudomonas spp.) in controlled laboratory environments to investigate the role of environmental heterogeneity in the evolution of phenotypic diversity. Some of my results provide insight on general processes, while others are specific to bacteria. (1) I have shown that variation in resource supply affects the evolution of niche breadth in complex environments containing a range of available resources, leading to a peak in phenotypic diversity at intermediate levels. (2) I have found that resource availability also affects selection against redundant phenotypic characters, which is strongest when resources are scarce. (3) Using experiments with bacteria and their protozoan predators, I have found that selection for predator resistance varies with resource supply during a model adaptive radiation. (4) I have looked at the role of periodic bottlenecks in population size in the evolution of antibiotic-resistant bacteria. My results highlight the importance of biochemical constraints specific to different resistance mutations. (5) Finally, I have shown that bacterial adaptation to novel carbon substrates affects different growth parameters simultaneously, and that the same response is seen in environments that maintain different levels of phenotypic diversity. These findings emphasize the role of environmental heterogeneity in the evolution of phenotypic diversity, but also show how ecological and genetic factors can constrain adaptation to a given niche within a heterogeneous environment.

Acinetobacter baumannii is an emerging multiresistant pathogen increasingly known to cause infections in the immuno-compromised patients. Carbapenems and colistin are considered to be the last resorts in treatment of infections involving multidrug resistant strains of A. baumannii. Resistance to carbapenems is well known due to the presence of intrinsic carbapenemase gene blaOXA-51-like, which may be governed by insertion elements, or by acquired carbapenemases like blaOXA-23-like, blaOXA-58-like or blaOXA-40-like genes, most of which are frequently associated with insertion elements. The acquired carbapenemases can be integrated with the host chromosome making the bacterium strongly resistant to a range of antibiotics. Recent reports also suggest that the ubiquitous and intrinsic enzymes encoded by the blaOXA-51-like gene can be mobilized on a plasmid. In this thesis, the prevalence of antibiotic resistance was examined for 96 strains isolated from various parts of the world. The resistances to aminoglycosides, fluoroquinolones and cephalosporins were studied with a major focus on resistance to carbapenems. Section 1 shows the transposition of ISAba1 and its varied influence in controlling the blaOXA-51-like gene and the blaADC gene. It explains how ISAba1 being a strong factor in influencing antibiotic resistance genes contributes to the plasticity of the organism Section 2 is related with a novel insertion element ISAba125 controlling the blaADC gene and as an element providing high resistance to ceftazidime in comparison to ISAba1. Section 3 analyses the multi-drug resistant profile of strains isolated from Cochabamba, Bolivia. Besides the classification of carbapenem resistance for the clinical strains, the aminoglycoside resistance and ciprofloxacin mechanisms are examined in this project Section 4 relates with the pattern of resistance in strains isolated from the Aberdeen Royal Infirmary. It describes two novel variants of the blaOXA-51-like gene, namely blaOXA-216 and blaOXA-217 and also the acquisition of the blaOXA-23-like gene in two isolates from different years and deemed identical by their PFGE pattern. Section 5 describes the influence of ISAba825 in controlling the blaOXA-51-like gene and the blaOXA-58-like from clinical isolates Section 6 is related with the insertional inactivation of the blaOXA-132 gene and the carbapenem resistance caused by the activation of the blaOXA-58 gene in isolate Ab244 Section 7 describes the influence of insertion elements in strains having high ciprofloxacin resistance. This project is concerned with the role of efflux pump control system adeRS and how they influence the adeABC operon causing increased and decreased expression of the genes. Section 8 describes the multi drug resistant pattern of 36 strains each isolated from Europe and the United States In conclusion, there are various factors that influence the resistance profile of multidrug resistant A. baumannii isolates with insertion sequences such as ISAba1, ISAba2, ISAba3, ISAba825, IS1008, ISAba125, ISAba16 governing the expression or providing alternate mechanisms of resistance for the better fitness of the bacterium. Mutations in the genes identified in this study also have a crucial role in imparting resistance to this bacterium.

Mycobacterium tuberculosis (Mtb), a pathogenic bacterium, is the causative agent in the vast majority of human tuberculosis (TB) cases. Nearly one-third of the world’s population has been affected by TB and annually two million deaths result from the disease. Because of the high cost of medication for a long term treatment with multiple drugs and the increase of multidrug-resistant Mtb strains, faster-acting drugs and more effective vaccines are urgently demanded. Several metabolic pathways of Mtb are attractive for identifying novel drug targets against TB. Hence, a high quality genome-scale metabolic network of Mtb (HQMtb) was reconstructed to investigate its whole metabolism and explore for new drug targets. The HQMtb metabolic network was constructed using an unbiased approach by extracting gene annotation information from various databases and consolidating the data with information from literature. The HQMtb consists of 686 genes, 607 intracellular reactions, 734 metabolites and 471 E.C. numbers, 27 of which are incomplete. The HQMtb was compared with two recently published Mtb metabolic models, GSMN-TB by Beste et al. and iNJ661 model by Jamshidi and Palsson. Due to the different reconstruction methods used, the three models have different characteristics. The 68 new genes and 80 new E.C. numbers were found only in the HQMtb and resulting in approximately 52 new metabolic reactions located in various metabolic pathways, for example biosynthesis of steroid, fatty acid metabolism, and TCA cycle. Through a comparison of HQMtb with a previously published human metabolic network (EHMN) in terms of protein signatures, 42 Mtb metabolic genes were proposed as new drug targets based on two criteria: (a) their protein functional sites do not match with any human protein functional sites; (b) they are essential genes. Interestingly, 13 of them are found in a list of current validated drug targets. Among all proposed drug targets, Rv0189c, Rv3001c and Rv3607c are of interest to be tested in the laboratory because they were also proposed as drug target candidates from two research groups using different methods.

Les enzymes lipolytiques sont des ester hydrolases impliquées dans le métabolisme lipidique. Leurs caractéristiques se sont révélées être des atouts dans de nombreuses applications industrielles. Chez les microalgues, l’isolement et la caractérisation de ces enzymes d’un point de vue structural et fonctionnel restent des domaines de recherche peu explorés à ce jour.Certaines espèces bénéficient pourtant de contenus en lipides intéressants, source de matière première pour les industries de l’agroalimentaire ou de l’énergie. Par exemple, l’acide docosahexaénoique (DHA), un acide gras polyinsaturé de la série des omégas 3, est reconnu pour ses propriétés en santé humaine. Parmi de nombreuses espèces, Isochrysis galbana, une microalgue unicellulaire appartenant à la classe des Prymnesiophycées est considérée comme une source possible de DHA. La présence d’acides gras libres a été montrée par l’analyse des lipides, suggérant la présence d’enzymes lipolytiques potentiellement intéressantes pour leur sélectivité et leur spécificité de substrat.L’analyse d’une banque de marqueurs de séquences exprimées a permis l’identification de séquences susceptibles de coder des enzymes lipolytiques. Les ARN messagers ont été extraits et les ADN complémentaires ont été clonés.Ce travail de thèse présente l’analyse et le clonage de deux gènes codant une ester hydrolase putative et une thioestérase putative, issues de la microalgue Isochrysis galbana.Les deux séquences codent des protéines de poids moléculaires de 35,41 kDa et de 42,31 kDa. Elles montrent 30 à 40 % d’identité et de similarité avec des hydrolases notamment des carboxylestérasesde différents organismes. Les séquences protéiques ont permis l’identification du pentapeptide consensus Gly-X-Ser-X-Gly caractéristique des enzymes lipolytiques et les acides aminés Ser/Asp/His de la triade catalytique.Les deux séquences codantes ont été clonées et exprimées dans la levure Saccharomyces cerevisiae et la bactérie Escherichia coli. Le clonage dans E. coli a permis d’identifier à la taille attendue une protéine par Western blot. En présence de cette protéine, la composition en acides gras des lipides de la bactérie a été modifiée. L’analyse CPG a notamment montré une augmentation des proportions en acides gras C16 :1 et C18 :1 par rapport au témoin. Ce résultat permet de caractériser l’activité thioestérase pour IgTeCe. / Lipolytic enzymes present in all known species play a key role in lipid metabolism and are involved in several industrial processes. They catalyse lipid hydrolysis and synthesis. Actually and particularly in microalgae, isolation and characterization of this type of enzyme remains an unexplored research area.The potential of the lipidic content of microalgae in food industry or energy field requires specific lipolytic enzymes. Docosahexaenoic acid (DHA), an 3 poly insaturated fatty acid (3 PUFA) is well known for its beneficial effects on human health. Among many species, Isochrysis galbana, a unicellular marine microalga belonging to the Prymnesiophyceae class, is considered as a potential alternative source of DHA.Lipid analysis of I. galbana shows free fatty acids and suggests the presence of lipolytic enzymes with potential interesting selectivities and substrate specificities. Analysis of incomplete expressed sequence tag (EST) listed in the EST bank of Isochrysis galbana, identified incomplete genes that encode lipolytic enzymes. Messenger RNAs were extracted, characterized and cloned.This work describes the analysis and cloning of two genes encoding a putative ester hydrolase and a putative thioesterase in marine microalgae Isochrysis galbana. Sequences encode two proteins with predicted molecular weights of approximately 35,41 kDa and 42,31 kDa. Slight similarity and identity (from 30 to 40 %) were observed between the gene sequence and various  fold hydrolase found in diverse phyla (including carboxylesterase).Sequences also included the consensus Gly-X-Ser-X-Gly and the catalytic triad Ser/Asp/His. To characterize the predicted enzymatic functions, an experimental procedure was introduced: coding sequences were cloned into expression vectors and expressed in Saccharomyces cerevisiae and in Escherichia coli.Western blot identification of recombinant enzyme shows a convenient protein production in bacteria. Furthermore, the expression of the protein in E. coli shifted the fatty acid composition predominantly towards C16:1 and C18:1 fatty acids. The enzyme called IgTeCe showed a thioesterase activity.

Mark-recapture methods use repeated captures of individually identifiable animals to provide estimates of properties of populations. Different models allow estimates to be obtained for population size and rates of processes governing population dynamics. State-space models consist of two linked processes evolving simultaneously over time. The state process models the evolution of the true, but unknown, states of the population. The observation process relates observations on the population to these true states. Mark-recapture models specified within a state-space framework allow population dynamics models to be embedded in inference ensuring that estimated changes in the population are consistent with assumptions regarding the biology of the modelled population. This overcomes a limitation of current mark-recapture methods. Two alternative approaches are considered. The "conditional" approach conditions on known numbers of animals possessing capture history patterns including capture in the current time period. An animal's capture history determines its state; consequently, capture parameters appear in the state process rather than the observation process. There is no observation error in the model. Uncertainty occurs only through the numbers of animals not captured in the current time period. An "unconditional" approach is considered in which the capture histories are regarded as observations. Consequently, capture histories do not influence an animal's state and capture probability parameters appear in the observation process. Capture histories are considered a random realization of the stochastic observation process. This is more consistent with traditional mark-recapture methods. Development and implementation of particle filtering techniques for fitting these models under each approach are discussed. Simulation studies show reasonable performance for the unconditional approach and highlight problems with the conditional approach. Strengths and limitations of each approach are outlined, with reference to Soay sheep data analysis, and suggestions are presented for future analyses.

Les éléments génétiques transposables (ET) sont des séquences d’ADN capables de se déplacer dans tous lesgénomes sous certaines conditions. Les ET ont des structures et des modes de transposition qui lesdifférencient en plusieurs groupes. Les éléments de la famille mariner sont ubiquistes ; lorsqu’ils sont actifs, ilsproduisent une transposase qui coupe l’élément et l’insère dans un autre locus. Des fragments d’éléments detype mariner (MLE) ont été précédemment identifiés chez la diatomée marine Amphora acutiuscula et ils semblaientactifs en condition de stress thermiques. Les diatomées sont caractérisées par une paroi siliceuse ornementée et,dans le milieu marin, elles jouent un rôle primordial dans les chaînes alimentaires.Dans le présent travail, par des méthodes de biologie moléculaire et de bioinformatique, nous avons recherché et caractérisé des MLE complets, et précisé leur activité en conditions de chocs thermiques et métalliques (cuivre et zinc). L’analyse des séquences amplifiées a mis en évidence la présence de MLE particuliers chez cette diatomée qui code une transposase ayant une triade catalytique DD43D jamais décrite jusqu’à présent.L’analyse phylogénétique place les MLE de diatomées dans une sousfamille différente mais proche de celle desMLE de plantes. Parmi les nombreuses copies de MLEprésentes dans le génome d’A. acutiuscula, certaines sont exprimées lorsque la diatomée est placée pendant 2 à 5 heures à unetempérature inférieure à sa température de culture. Par contre, l’expression des MLE n’a pas été mise en évidence chez cette espèce soumise aux stress métalliques appliqués. / Transposable elements (TE) are DNA sequences able to move in all genomes depending on conditions. TE have different structures and transposition mechanisms. Tc1-mariner elements are ubiquist ; when they are active, they produce a transposase which cuts and inserts the element into another locus. Fragments of mariner-like elements (MLE) hadpreviously been identified in the marine diatom Amphora acutiuscula and they seemed active under thermal stress. Diatoms are characterized by a siliceous ornamented cell wall and, in the ocean, they play a major role in trophic networks.In this work, with biomolecular and bioinformatic methods, we have searched for and characterized full length MLE, and precised their activity under thermal and metal (copper and zinc) stresses. The analyse of the DNA sequences obtained highlighted that MLE in diatoms are particular and that they encode a transposase which has a DD43D catalytic triad neverso far depicted. The phylogenetic analyse arranged diatom MLE in a subfamily different and close to that of plant MLE. Among the numerous MLE copies present in the genome of A.acutiuscula, some were expressed when the microalga was put at a lower temperature than the culture temperature for 2 to 5 hours. Conversely, metal stresses we applied did notinduce MLE expression in this species.