The relentless evolution of antibiotic resistance in pathogens is one of the most pressing medical concerns of the 21st century. Antibiotic resistance and antibiotic drugs originated in environmental bacteria, where they have been integral to their evolution for millions of years. The application of antibiotics in medicine and agriculture has selected for mobilization and dissemination of resistance genes in pathogens. Understanding their evolution here will aid in combating their evolution in pathogens. This work expands the known mechanistic, functional, and genetic diversity of resistance (i.e. resistomes) in environmental bacteria. I systematically parse the extensively drug-resistant resistome of Paenibacillus sp. LC231, which was sampled from an underground ecosystem spatiotemporally isolated from the surface for over 4 Myr. Paenibacillus sp. LC231 was resistant to 26 of 40 drugs tested. Informatic annotation of resistance genes and functional genomes revealed 18 new resistance elements including five determinants without characterized homologs and three mechanisms not previously known to confer resistance. I investigated the resistome of Brevibacillus brevis VM4 to study the relationship between species diversity and resistance diversity in the Paenibacillaceae family, which includes Paenibacillus sp. LC231. I found that resistome diversity does not correlate with species diversity, consistent with horizontal transfer of resistance genes. In each of Paenibacillus sp. LC231 (MphI) and B. brevis VM4 (MphJ), I identified Mphs with unique substrate specifies. I identified the molecular determinants of substrate discrimination in MphI and in doing so, I developed a general strategy for understanding and predicting the functional evolution of resistance enzymes. Together, this work expands the known diversity of resistance that will enable better detection of resistance in pathogens. / Thesis / Doctor of Philosophy (PhD) / Infections caused by antibiotic resistant bacteria are a significant medical problem. Bacteria will always become resistant to antibiotic drugs. Understanding how resistance evolves is essential for increasing the effective lifetime of these drugs. Antibiotics have been naturally produced by bacteria for millions of years, which caused the spread of resistance in environmental bacteria. Medical and agricultural antibiotic use by humans caused resistance in environmental bacteria to transfer to pathogenic bacteria. My work expands the known causes of resistance in environmental bacteria so that we can better detect the causes of resistance in pathogens. In doing so, I demonstrate that multi-drug resistance is over 4 million years old and that environmental bacteria naturally transfer resistance genes. Furthermore, I develop a way to predict the evolution of new resistance functions by inferring their evolutionary histories.

Bacterial endosymbionts of eukaryotes are generally studied in terms of their benefit or detriment to their hosts. The constraints that the host's life history imposes on its endosymbionts are rarely considered, although bacterial genome content and size are influenced by both the biotic and abiotic factors in the environment. The host organism is the primary habitat of the endosymbiont. Thus, desecribing the environment a host provides its endosymbiont is essential for understanding the evolution of endosymbiotic bacteria. I propose a system to classify the endosymbiotic environment by three characteristics: 1) host life cycle 2) host metabolism, and 3) endosymbiont location relative to host tissues. Insect-bacterial mutualisms have been classified in terms of endosymbiont environment. The majority of insect-bacterial mutualisms currently studied involve monophagous, hemimetabolous hosts that provide a relatively constant endosymbiotic enviroment. A relatively constant environment may explain the extremely reduced genomes of their endosymbionts. In contrast, polyphagous, holometabolous hosts provide the most variable endosymbiotic environment. In this work, I examined the interactions between the polyphagous, holometabolous insect, Bactrocera oleae (Rossi), and the enteric gammaproteobacterium, Candidatus Erwinia dacicola, throughout host development. Candidatus Erwinia dacicola was found in the digestive system of all life stages of wild olive flies. PCR and microscopy demonstrated that Ca. Erwinia dacicola resided intracellularly in the gastric caeca of the larval midgut, but extracellularly in the lumen of the foregut and ovipositor diverticulum of adult flies. I document the widespread distribution and high frequency of Ca. Er. dacicola in ten populations of wild olive flies sampled in four countries (3 Old World and 1 New World). The relative abundance of the bacterium was highest in adults and less prevalent in the egg and pupal stages. Among adult flies, the bacterium was most common in ovipositing females. These results suggest that Ca. Er. dacicola is a persistent, autochthonous endosymbiont of the olive fly. Finally, mating initiation was examined to study the influence of Ca. Er. dacicola on mating between a laboratory and a wild population of olive flies from Israel. Behavioral differences between the two populations, not presence of the endosymbiont, explained mating initiation.

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.

Les êtres vivants sont confrontés à des parasites qui diminuent leur fitness et se répandent dans la population. En réponse, les hôtes ont développé de nombreuses défenses immunitaires qui sont souvent mises en défaut par l'évolution des parasites. Ces défenses sont de plus souvent extrêmement diversifiées génétiquement. Quel est donc l'apport de la diversité génétique des défenses contre l'évolution des parasites ? Répondre à cette question expérimentalement nécessite un système biologique pour lequel on peut étudier la diversité génétique de l'hôte et l'évolution et la propagation du parasite. Les systèmes bactéries/phages sont de bons candidats pour une telle étude : leur manipulation au laboratoire est aisée, leurs cycles de vie sont rapides et ils ont de forts taux de mutation. La découverte récente de l'immunité CRISPR--Cas a ouvert de nombreuses possibilités : cette dernière a la propriété unique de générer dans le même fond génétique que l'hôte sensible de nombreux allèles de résistance. De plus, son mécanisme de fonctionnement reposant sur une interférence à ARN, la cible d'une résistance est très précisément connue ainsi que les possibilités de la contourner. Ce système permet donc l'étude expérimentale de l'impact de la diversité génétique sur la propagation et l'évolution des parasites, et sur la co-évolution antagoniste. Dans cette thèse, nous cherchons à 1) déterminer l'impact de la composition de la population d'hôtes sur la probabilité qu'une épidémie créée par un virus mutant ait lieu (émergence évolutive), 2) expliciter les causes de l'hétérogénéité de durabilité des résistances et 3) étudier la dynamique co-évolutive entre population génétiquement diversifiée d'hôtes et de parasites. Nous montrons que la composition de la population d'hôtes module fortement la probabilité d'émergence évolutive : une faible diversité génétique associé à un taux intermédiaire d'hôtes sensibles maximisant la probabilité d'émergence évolutive. Dans un second temps, nous montrons que l'immunité CRISPR génère des résistances dont la durabilité est hétérogène et cette hétérogénéité ne peut pas être expliquée par une hétérogénéité des fitness des mutants contournant CRISPR. Enfin, nous montrons que la diversité des résistances est maintenue à court terme par l'hétérogénéité des populations de parasites et que la dynamique co-évolutive est accélérée en présence d'une population génétiquement diverse. Enfin, nous proposons des pistes de recherche qu'il nous parait intéressant d'étudier dans le futur. / Living organisms face parasites which decrease their fitness and spread into their population. In response, hosts have evolved countless immune defenses that are often circumvented by parasite evolution. These defenses are usually extremely diverse. What is the impact of such genetic diversity on the protection against the evolution of parasites? Answering this question experimentally requires an experimental system in which host genetic diversity and parasite evolution and spreading can be monitored. Phages and bacteria systems are ideal candidates for such studies as their handling is easy in the lab, their life cycle is short and their mutation rates is high. The recent discovery of CRISPR--Cas immunity has opened many possibilities. Indeed, this immunity has the unique property to generate in the same genetic background as the sensitive host, numerous resistant alleles. In addition, it relies on an interference--RNA-like pathway, which results in the precise understanding of phage bypassing and in the ability to predict the targeted sequence. This system hence allows the experimental study of the impact of host genetic diversity on the epidemiology and the evolution of parasites and on antagonist coevolution. In this PhD, we 1) study how the host population composition impacts the probability of an epidemic created by an escape mutant (evolutionary emergence), 2) try to understand the causes of the heterogeneity in durability of resistances and 3) monitor the coevolution dynamic between genetically diverse populations. We show that the composition of the host population impacts the probability of evolutionary emergence: a low resistances diversity with an intermediate proportion of sensitive hosts maximises the probability of evolutionary emergence. Second, we show that CRISPR--Cas resistances are heterogeneous in their durability and this is not explained by the heterogeneity of escape mutants fitness. Third, we show that resistances diversity is conserved in a short term by parasites genetic diversity and that the coevolutionary dynamic is fastened by parasite intra-specific genetic diversity. Finally, we discuss research questions that we find interesting to develop in the near future.

Evolution is an ubiquitous feature of living systems. The genetic composition of a population changes in response to the primary evolutionary forces: mutation, selection and genetic drift. Organisms undergoing rapid adaptation acquire multiple mutations that are physically linked in the genome, so their fates are mutually dependent and selection only acts on these loci in their entirety. This aspect has been largely overlooked in the study of asexual or somatic evolution and plays a major role in the evolution of bacterial and viral infections and cancer. In this thesis, we put forward a theoretical description for a minimal model of evolutionary dynamics to identify driver mutations, which carry a large positive fitness effect, among passenger mutations that hitchhike on successful genomes. We examine the effect this mode of selection has on genomic patterns of variation to infer the location of driver mutations and estimate their selection coefficient from time series of mutation frequencies. We then present a probabilistic model to reconstruct genotypically distinct lineages in mixed cell populations from DNA sequencing. This method uses Hidden Markov Models for the deconvolution of genetically diverse populations and can be applied to clonal admixtures of genomes in any asexual population, from evolving pathogens to the somatic evolution of cancer. To understand the effects of selection on rapidly adapting populations, we constructed sequence ensembles in a recombinant library of budding yeast (S. cerevisiae). Using DNA sequencing, we characterised the directed evolution of these populations under selective inhibition of rate-limiting steps of the cell cycle. We observed recurrent patterns of adaptive mutations and characterised common mutational processes, but the spectrum of mutations at the molecular level remained stochastic. Finally, we investigated the effect of genetic variation on the fate of new mutations, which gives rise to complex evolutionary dynamics. We demonstrate that the fitness variance of the population can set a selective threshold on new mutations, setting a limit to the efficiency of selection. In summary, we combined statistical analyses of genomic sequences, mathematical models of evolutionary dynamics and experiments in molecular evolution to advance our understanding of rapid adaptation. Our results open new avenues in our understanding of population dynamics that can be translated to a range of biological systems.

Bacteria evolve during the colonization of human hosts, yet little is known about the selective pressures and evolutionary forces that shape this evolution. Illumination of these processes may inspire new therapeutic directions for combating bacterial infections and promoting healthy bacteria-host interactions. The advent of high-throughput sequencing has enabled the identification of mutations that occur within the human host, and various tools from computational and evolutionary biology can aid in creating biological understanding from these mutations. Chapter 1 describes recent progress in understanding within-patient bacterial adaption, focusing on insights made from genomic studies.

For many years, researchers have studied the social lives of bacteria to understand intra- and inter-species interactions. Cell-cell communication, also known as quorum sensing (QS), is used by bacteria to coordinate their behaviour in response to environmental conditions. The QS system in Streptococcus species is well known to regulate competence. Studies show that Streptococcus pneumoniae has two homologous QS systems: 1) the competence (Com) system that regulates competence; and 2) a bacteriocin-like peptide (Blp) system that regulates the production of bacteriocins. Both functions are widespread in the genus. In S. pneumoniae, the Blp QS system shares a common ancestor and has similar features to the Com QS system. However, the evolutionary relationship between these QS systems remains obscure. SUCRE methodology was developed to identify the QS homologous genes in the streptococcal species. SUCRE uses four complementary approaches: homology search, putative gene finding, regulon construction, and evolutionary analysis. The performance of SUCRE was assessed in comparison with other orthology detection methods. SUCRE is precise in identifying the QS homologous genes and has similar performance to OrthoMCL. The QS system structures are found to be conserved across the streptococcal species. A streptococcal species phylogeny was constructed from the ribosomal and tRNA synthetase gene families. Using the QS genes identified from SUCRE and the streptococcal species phylogeny, the study infers the evolution of the QS systems in Streptococcus species. The study shows that the QS systems evolved as a regulon unit. The paralogous relationship between each of the QS systems suggests that duplication has a huge influence on functional divergence of the QS systems in the genus. Although, horizontal gene transfer (HGT) is commonly found in bacteria, little evidence is found to support that the effect of HGT on the functional divergence of the QS systems in this genus. However, the QS regulon genes of the same QS system are found to be non- vertically transferred across species that signifies that the HGT event promotes the sequence variation between these genes.

Microbes are ubiquitous in virtually all habitats on Earth and affect human life in multiple ways, from the health-balancing role of the human microbiome, to the involvement of microbial communities in the global nitrogen and carbon cycles. The capacity of microbes to survive and grow in diverse environments relates directly to their ability to utilize available resources, be they from other microbes or from the environment itself. Hence, understanding how the environment shapes the metabolic functionality of individual microbes and complex communities constitutes an important area of research. In the first part of my thesis work, I explored how environmental nutrient composition and intracellular transcriptional regulation data can be integrated to provide insight into the temporal metabolic behavior of a bacterium through the use of genome-scale stoichiometric modeling approaches (Flux Balance Analysis). Thus I developed the method of Temporal Expression-based Analysis of Metabolism (TEAM), and applied it to Shewanella oneidensis, a bacterium studied for its important bioenergy and bioremediation applications. I found that TEAM improves on previous models' predictions of S. oneidensis metabolic fluxes, and recovers the overflow metabolism that has been seen experimentally. This study demonstrated the value of incorporating environmental context and transcriptional data for the prediction of time-dependent metabolic behavior. In the second part of my work, I extended the exploration of microbial metabolism from single species to complex communities in order to understand the robustness of metabolic functions. Specifically, I implemented novel mathematical analyses of metagenomic sequencing data to ask how functional stability of microbial communities could ensue despite large taxonomic variability. Upon representing in matrix form the metabolic capabilities of all genera found in 202 available metabolic ecosystem datasets, I compared the different communities with each other and with various randomized analogues. My results reveal new connections between the abundance of an organism in the community and the functions that it encodes. Furthermore, I found that genus abundances govern the metabolic robustness of a community more than the distribution of genetically encoded functions among the community members, suggesting that communities rely largely on ecological interactions to regulate their overall functionality.

No description available.

Les communautés microbiennes de sédiments de subsurface ont été décrites jusqu’à 1922 mbsf (meters below the seafloor) et pourraient représenter 0,6% de la biomasse totale. Largement incultivées, ces communautés comprennent des groupes endémiques aux environnements de subsurface et des généralistes retrouvés dans des environnements contrastés, appartenant aux 3 domaines du vivant (Bacteria, Eukarya and Archaea). Bien que jouant un rôle majeur dans les grands cycles géochimiques, l’écologie microbienne des sédiments de subsurface reste peu connue. Les conditions hostiles de ces sédiments contrastent avec la présence d’activité et de viabilité microbiennes. Dans ce contexte, de nombreuses questions sur les modes de vie et les métabolismes des microorganismes enfouis demeurent. L’objectif de cette thèse était de mieux comprendre quelles stratégies adaptatives pouvaient être mises en place par les communautés microbiennes de subsurface et de caractériser leur potentiel physiologique. Pour cela, 3 approches ont été utilisées.(1) Une approche culturale a permis de décrire 2 nouvelles espèces bactériennes sédimentaires (Halomonas lionensis, ungénéraliste versatile, et Phaeobacter leonis, une bactérie marine typique). L’étude de la résistance aux conditions de subsurface de ces deux espèces et de la bactérie Sunxiuqinia faeciviva, isolée à 247 mbsf, a ensuite été étudiée. (2) Par une étude de génomique comparée et structurale, la plasticité physiologique de H. lionensis a été analysée. (3) Enfin, le potentiel fonctionnel de communautés microbiennes enfouies à 31 et 136 mbsf dans le bassin de Canterbury a été étudié, en analysant les 2métagénomes correspondants. Les résultats culturaux et génomiques montrent que H. lionensis et S. faeciviva résistent mieux aux stress de subsurface que P. leonis et, dans le cas de H. lionensis, ceci impliquerait des propriétés physiologiques variées pouvant expliquer le succès écologique du genre Halomonas. Les données de métagénomique indiquent que les diversités phylogénétique et fonctionnelle de subsurface du bassin de Canterbury sont distinctes de celles d’environnements de surface et suggèrent que des métabolismes comme la fermentation, la méthanogenèse ou la β-oxydation pourraient être importants. La présence de gènes d’importance écologique et évolutive a permis d’émettre des hypothèses sur les modes de vie de ces microorganismes et des évènements de recombinaison génomique de groupes toujours incultivés ont aussi pu être décrits / Microbial communities inhabiting marine subsurface sediments were described up to 1922 mbsf (meters below the sea floor) andcould represent 0.6% of the total biomass. This microbial diversity, remaining elusive to cultivation, comprises groups specific to subsurface environments and groups of generalists found in contrasted habitats, all belonging to the 3 domains of life (Bacteria,Eukarya and Archaea). Although playing a major role in global geochemical cycles, the microbial ecology of the subseafloor remains largely unknown. The hostile conditions of subsurface sediments contrast with the descriptions of microbial activity andviability in the subseafloor. In this context, many questions related to the microbial physiology and the lifestyles of buried communities remain to be answered. The objective of this thesis was to better understand which adaptive strategies could be deployed by subseafloor microbial communities and to characterize their physiological potential. In that aim, 3 approaches were used.(1) A cultural approach enabled describing 2 novel sedimentary bacterial species (Halomonas lionensis, a versatile generalist and Phaeobacter leonis a typical marine bacterium). The survival of these 2 species to subseafloor conditions and of the subsurface bacteria Sunxiuqinia faeciviva, isolated at 247 mbsf, was then studied. (2) Using a structural and comparative genomic approach, the physiological plasticity of H. lionensis was investigated. (3) Finally, the functional potential of the microbial communities buried at 31 and 136 mbsf in the Canterbury Basin was analyzed, by studying the 2 corresponding metagenomes. Cultural and genomics results showed that H. lionensis and S. faeciviva are more resistant to subsurface constrains than P. leonis and, in the case of H. lionensis, this may involve various physiological properties, maybe explaining thee cological success of the genus Halomonas. Metagenomic data showed that the functional and the phylogenetic diversity of the subseafloor are distinct from the ones from surface environments and highlighted the importance of metabolic pathways like fermentation, methanogenesis and β-oxidation. Genes of ecological and evolutionary interests enabled speculating about lifestyles of buried microorganisms and analyses of genomic fragments highlighted recombination events of still uncultivated microbial groups