Bacteria are mediators of biogeochemical cycles and are in this way vital for maintaining life on earth. Their distribution, abundance and functioning are driven by environmental heterogeneity and dynamic change in abiotic and biotic factors. Both, freshwater lakes and oceans play central roles in the global carbon cycle and bacteria in these systems perform many services for the ecosystems, such as the transfer of organic carbon from primary producers to higher trophic levels. With estimated relative abundances up to 50% of the total bacterioplankton, the Alphaproteobacteria lineage SAR11 is the most abundant group of aquatic bacteria. It is globally distributed and can be partitioned into multiple sub-clades, one of which is exclusive to freshwaters. Until recently, the distribution, abundance and ecological role of this freshwater SAR11 named LD12 was unknown. The aim of the thesis was to study the drivers and mechanisms that influence the dynamics of aquatic bacterial communities in general and the SAR11 and LD12 groups in particular. The thesis consists of environmental surveys of a mesotrophic Lake Erken and the western Southern Ocean, an experiment and a data-mining exercise to reveal the phylogenetic structure of the SAR11 lineage on various temporal and spatial scales. The analysis of a long-term bacterioplankton community survey in lake Erken provided insights about the dynamics of the entire bacterial community and the LD12 population over an annual cycle. The results demonstrate that LD12 can be an equally abundant member of freshwater communities as marine SAR11 in oceans. LD12 featured strong seasonality and was positively coupled to environmental conditions indicative for an oligotrophic lifestyle. LD12 as well as other dominant lake bacterioplankton also maintained stable populations throughout spatial and temporal varying environments, but at high phylogenetic resolution, habitat preferences were revealed, particularly in response to oxygen concentrations. The later was not the case in LD12 as a single ribotype dominated. This is in stark contrast to the habitat partitioning with light availability, depth and water masses observed for marine SAR11 subclades in the Southern Ocean. The global data-mining corroborated that LD12 as a group was much less diverse than SAR11 furthermore, suggesting that the marine-freshwater barrier acted as a population bottleneck. My work shows that bacterial populations can respond in very different ways to environmental drivers, highlight the importance of highly resolved temporal and spatial scales as well as the need for high phylogenetic resolutions to target ecologically coherent populations.

<p>Microbial metagenomics is the exploration of taxonomical diversity of microbial communities in environmental habitats using large, exhaustive DNA sequence datasets. However, due to inherent limitations of sequencing technology and the complexity of environmental genomes, current analytical approaches do not reveal the existence of all microbes that may be present. In this study, a new classification approach is proposed based upon unique proteins that are specific for different clades of Alphaproteobacteria to predict the presence and absence of species from these groups of bacteria in published metagenomic datasets. In this work, 264 previously–identified, published conserved signature proteins (CSPs) characteristic of individual taxonomic clades of Alphaproteobacteria are used as probes to detect the presence of bacteria in metagenomic datasets. Although public genome sequence information has increased manifold since these CSPs were initially identified 6 years ago, results indicate that nearly all of these CSPs (259 of 265) are specific for their previously characterized clades. Furthermore, they are confirmed to be present in the newly–identified and sequenced members of these clades. In view of their specificity and predictive ability in different monophyletic clades of Alphaproteobacteria, the sequences of these CSPs provide reliable probes to determine the presence or absence of these Alphaproteobacteria in metagenomic datasets. In this work, CSPs are used to determine the presence of Alphaproteobacteria diversity in 10 published metagenomic datasets (bioreactor, compost, wastewater, activated sludge, groundwater, freshwater sediment, microbial mat, marine, hydrothermal vent and whale fall metagenomes), which cover diverse environment and ecosystems. It is indicated that the BLAST searches with these CSPs can be used to efficiently identify Alphaproteobacteria species in these metagenome dataset and substantial differences can be determined in the distribution and relative abundance of different Alphaproteobacteria species in the tested metagenome datasets. Thus the CSPs, which are specific for different microbial taxa, provide novel and powerful means for identification of microbes and for their taxonomic profiling in metagenomic datasets.</p> / Master of Science (MSc)

Le transfert latéral de séquences (LST) joue un rôle critique dans l'évolution des bactéries. Les alphaprotéobactéries (ALPs), avec leurs différentes tailles, leurs divers modes de vie et leurs "mobilome", constituent un modèle idéal pour étudier l'histoire évolutive des bactéries. Le "rhizome" représente la diversité des origines des gènes chez ces alphaprotéobactéries : ceci est notamment observable chez les Rickettsiales, appartenant aux ALPs, qui possèdent des génomes en mosaïque. Toutefois, les ALPs contribuent également aux contenus génomiques de différents génomes bactériens comme eucaryotes. De plus, plusieurs gènes codant pour les ribosomes chez les ALPs ont participé à la formation des mitochondries. En premier lieu, nous avons ici mis en place une approche pour mettre en évidence l'histoire évolutive des LSTs. Cette approche est basée sur l'établissement de profils phylétiques, suivi de la recherche de séquences homologues, la reconstruction phylogénétique et enfin la définition des séquences transferts basée sur l'utilisation d'un motif spécifique. Nous avons ainsi montré que 42 gènes des Rickettsiales sont issus de transferts provenant de différentes espèces des trois domaines de la vie. Cette approche est applicable à l'étude de LSTs pour un grand nombre de génomes d'intérêt. Deuxièmement, nous avons séquencé et annoté le génome d'Odyssella thessalonicensis, puis étudié l'histoire évolutive de la mitochondrie, de Candidatus Pelagibacter ubique (CPu), d'O. thessalonicensis et des alphaprotéobactéries. Nos résultats montrent que CPu provient probablement d'un ancêtre intracellulaire facultatif en commun avec les espèces de Rickettsiales. / Lateral sequence transfer (LST) plays a critical role in the bacterial evolu- tion. Alphaproteobacteria with different genomes in size, their diverse lifestyles and their "mobilome" are an ideal model for studying the evolutionary history of the bacteria. The different origins of genes of alphaproteobacteria species can be represented as a "rhizome". In constrast, the alphaproteobacteria contributed in the creation of different genomes such as bacteria, eukaryotes (the nematod, the insect). Moreover, many ribosomal genes of alphaproteobacteria have participated in the formation of the mitochondria. In the first part, we have done an approach to define LSTs. This approach is primarily based on the application of phylogenetic profiles, followed by the search for homologous sequences, then the phylogenetic reconstruction, and finally the definition of sequence transfer by a specific pattern. We found that 42 instances of transfers of Rickettsiales came from distantly related species of different domains of life (eukaryote, bacteria, archaea). We can apply this approach for studying LSTs of greater genomes of interest. In the second part, we sequenced and annotated the Odyssella thesalonicensis, then studied the evolutionary history of mitochondrion, Candidatus Pelagibacter ubique, O. thessalonicensis and alphaproteobacteria. Our results showed that Candidatus Pelagibacter ubique has probably originated from an ancestor facultative intracellular with Rickettsiales species.

Variations in the relative amounts of ¹²C and ¹³C in microbial biomass can be used to infer the pathway(s) autotrophs use to fix and assimilate dissolved inorganic carbon. Discrimination against ¹³C by the enzymes catalyzing autotrophic carbon fixation is a major factor dictating the stable carbon isotopic composition (δ¹³C = {[¹³C/¹²Csample/¹³C/¹²Cstandard] - 1} X 1000) of biomass. Six different forms of ribulose 1,5-bisphosphate carboxylase/oxygenase or RubisCO (IA, IB, IC, ID, II, and III), the carboxylase of the Calvin-Benson-Bassham cycle (CBB), are utilized by algae and autotrophic bacteria that rely on the CBB cycle for carbon fixation. To date, isotope discrimination has been measured for form IA, IB, and II RubisCOs. Isotopic discrimination, expressed as ε values (={[¹²k/¹³k] - 1} X 1000; ¹²k and ¹³k = rates of ¹²C and ¹³C fixation) range from 18 to 29‰, explaining the variation in biomass δ¹³C values of autotrophs that utilize these enzymes. Isotope discrimination by form IC RubisCO has not been measured, despite the presence of this enzyme in many proteobacteria of ecological interest, including marine manganese-oxidizing bacteria, some nitrifying and nitrogen-fixing bacteria, and extremely metabolically versatile organisms such as Rhodobacter sphaeroides. The purpose of this work is to determine the e value for the form IC RubisCO enzyme from R. sphaeroides. Under standard conditions (pH 7.5 and 5 mM DIC), form IC RubisCO had an ε value of 29‰. Sampling the full phylogenetic breadth of RubisCO enzymes for isotopic discrimination makes it possible to constrain the range of δ¹³C values of organisms fixing carbon via the Calvin-Benson-Bassham cycle. These results are helpful for determining the degree to which CBB cycle carbon fixation contributes to primary and secondary productivity in microbially-dominated food webs.

Our understanding of microbial evolution is largely dependent on available genomic data of diverse organisms. Yet, genome-sequencing efforts have mostly ignored the diverse uncultivable majority in favor of cultivable and sociologically relevant organisms. In this thesis, I have applied and developed cultivation independent methods to explore microbial diversity and obtain genomic data in an unbiased manner. The obtained genomes were then used to study the evolution of mitochondria, Rickettsiales and Haloarchaea. Metagenomic binning of oceanic samples recovered draft genomes for thirteen novel Alphaproteobacteria-related lineages. Phylogenomics analyses utilizing the improved taxon sample suggested that mitochondria are not related to Rickettsiales but rather evolved from a proteobacterial lineage closely related to all sampled alphaproteobacteria. Single-cell genomics and metagenomics of lake and oceanic samples, respectively, identified previously unobserved Rickettsiales-related lineages. They branched early relative to characterized Rickettsiales and encoded flagellar genes, a feature once thought absent in this order. Flagella are most likely an ancestral feature, and were independently lost during Rickettsiales diversification. In addition, preliminary analyses suggest that ATP/ADP translocase, the marker for energy parasitism, was acquired after the acquisition of type IV secretion systems during the emergence of the Rickettsiales. Further exploration of the oceanic samples yielded the first draft genomes of Marine Group IV archaea, the closest known relatives of the Haloarchaea. The halophilic and generally aerobic Haloarchaea are thought to have evolved from an anaerobic methanogenic ancestor. The MG-IV genomes allowed us to study this enigmatic evolutionary transition. Preliminary ancestral reconstruction analyses suggest a gradual loss of methanogenesis and adaptation to an aerobic lifestyle, respectively. The thesis further presents a new amplicon sequencing method that captures near full-length 16S and 23S rRNA genes of environmental prokaryotes. The method exploits PacBio's long read technology and the frequent proximity of these genes in prokaryotic genomes. Compared to traditional partial 16S amplicon sequencing, our method classifies environmental lineages that are distantly related to reference taxa more confidently. In conclusion, this thesis provides new insights into the origins of mitochondria, Rickettsiales and Haloarchaea and illustrates the power of cultivation independent methods with respect to the study of microbial evolution.