Mathematical modeling of evolutionary changes of oligonucleotide frequency patterns of bacterial genomes for genome-scale phylogenetic inferences

Modern phylogenetic studies from the advancement of next generation sequencing can benefit from an analysis of complete genome sequences of various microorganisms. Evolutionary inferences based on genome scale analysis were believed to be more accurate than gene-based ones. However, the computational complexity of current phylogenomic procedures and lack of reliable annotation and alignment free evolutionary models keep microbiologists from wider use of these opportunities. For example, the super-matrix approach of phylogenomics requires identification of clusters of orthologous genes in compared genomes followed by alignment of numerous sequences to proceed with reconciliation of multiple trees inferred by traditional phylogenetic tools. In fact, the approach potentially multiplies the problems of gene annotation and sequence alignment, not mentioning the computational difficulties and laboriousness of the methods. For this research, we identified that the alignment and annotation-free method based on comparison of oligonucleotide usage patterns (OUP) calculated for genome-scale DNA sequences allowed fast inferring of phylogenetic trees. These were also congruent with the corresponding whole genome supermatrix trees in terms of tree topology and branch lengths. Validation and benchmarking tests for OUP phylogenomics were done based on comparisons to current literature and artificially created sequences with known phylogeny. It was demonstrated that the OUP diversification between taxa was driven by global adjustments of codon usage to fit fluctuating tRNA concentrations that were well aligned to the species evolution. A web-based program to perform OUP-based phylogenomics was released on http://swphylo.bi.up.ac.za/. Applicability of the tool was proven for different taxa from species to family levels. Distinguishing between closely related taxonomic units may be enforced by providing the program with alignments of marker protein sequences, e.g. gyrA.Thesis (PhD)--University of Pretoria, 2018.BiochemistryPhDUnrestricte

A tale of two clades: genome evolution of oomycetes and fungi.

Some of the most ecologically-significant pathogens of plants, animals and marine life come from two groups of filamentous eukaryotes; the oomycetes and the fungi. Although similar in morphology and ecological niche, the two groups are only very-distantly related in terms of evolutionary history. The oomycetes are underresearched in evolutionary science, despite their historical and contemporary impact on food and environmental security. In contrast, fungi themselves are probably the most densely studied and sequenced group of organisms in evolutionary science outside of bacteria. This thesis is a collection of five published computational studies of the evolutionary biology of oomycetes and fungi. The first study is a systematic investigation of bacterial horizontal gene transfer into plant pathogenic oomycete species, which identifies 5 potential HGT events from prokaryotes into multiple oomycetes. The second study is a reconstruction of the evolutionary history of the oomycetes using wholegenome data from 37 species, which supports the larger groups within the oomycetes class but suggests that some exemplar oomycete genera are paraphyletic. Taking advantage of the abundance of genomics data available for all major fungal phyla, the third study reconstructs the evolutionary history of 84 fungal species using seven different phylogenomic techniques and critically evaluates each technique for accuracy, speed and other criteria. The fourth study looks at the pangenomes of four model fungal species, and compares the evolution of genomic variation, virulence and environmental adaptation within each species. The final study presents a refined iteration of the methodology used in the previous pangenome study as a self-contained software package and demonstrates the software’s capabilities through pangenome analysis and re-analysis of both model and non-model fungal species. Together, these studies cover a breadth of molecular evolution, comparative genomics, phylogenomics and pangenomics research for two similar, but evolutionarily-distinct groups of important microscopic eukaryotes

Bacterial niche adaptation at hydrothermal vents

At deep sea hydrothermal fields, a mixing gradient between hot reduced fluids and cold oxygenated sea water creates a number of micro-environments with different physico-chemical conditions in direct proximity to each other. Although many of the key microorganisms in these environments have been identified and described, a systematic understanding of their distribution across the mixing gradient and their niche partitioning is still missing. In my doctoral thesis, I investigated the interplay of geochemical settings, microbial community structures, and diversification of microorganisms in three collaborative studies of hydrothermal vent fields in the Atlantic and Pacific Oceans. Taken together, this thesis provides a detailed overview of the microbial community structures in hydrothermal vents. It deepens our understanding of niche differentiation of major marine sulfur oxidizers, and offers valuable insights into microbial diversification

Les génomes bactériens, une histoire de transferts de gènes, de recombinaison et de cladogénèse

In bacterial genomes, the frequent horizontal gene transfers (HGT) introduce genomic novelties that can promote the diversification of bacterial populations. In opposition, homologous recombination (HR) within populations homogenizes their genotypes, enforcing their cohesion. These processes of genetic exchange, and their patterns of occurrence among and within lineages, must have a great impact on bacterial cladogenesis. Beyond the pattern of exchanges actually occurring between bacteria, the traces of HR and HGT we observe in their genomes reflect what events were fixed throughout their history. This fixation process can be biased regarding the nature of genes or alleles that were introduced. Notably, natural selection can drive the fixation of transferred genes that bring new ecological adaptations. In addition, some mechanical biases in the recombination process itself may lead to the fixation of non-adaptive alleles. We aimed to characterize such adaptive and non-adaptive processes that are shaping bacterial genomes. To this end, several aspects of genome evolution, such as variations of their gene repertoires, of their architecture and of their nucleotide composition were examined in the light of their history of transfer and recombinationDans les génomes bactériens, les fréquents transferts horizontaux de gènes (HGT) introduisent des innovations génomiques qui peuvent entraîner la diversification des populations bactériennes. À l'inverse, la recombinaison homologue (RH) au sein des populations homogénéise leurs génotypes, et ainsi renforce leur cohésion. Ces processus d'échange génétique, et la fréquence à laquelle ils interviennent au sein et entre les populations, doivent avoir un grand impact sur la cladogénèse bactérienne. Au-delà de la configuration des échanges qui se sont réellement produits entre les bactéries, les traces de RH et de HGT que nous observons dans leurs génomes reflètent les événements qui ont été fixés tout au long de leur histoire. Ce processus de fixation peut être biaisé en ce qui concerne la nature des gènes ou allèles qui ont été introduits. La sélection naturelle peut notamment conduire à la fixation des gènes transférés qui apportent de nouvelles adaptations écologiques. En outre, des biais mécaniques dans le processus de recombinaison lui-même peuvent conduire à la fixation d'allèles non-adaptatifs. Nous avons cherché à caractériser certains de ces processus adaptatifs et non-adaptatifs qui façonnent les génomes bactériens. À cette fin, plusieurs aspects de l'évolution des génomes, comme les variations de leurs répertoires de gènes, de leur architecture et de leur composition en nucléotides ont été examinés à la lumière de leur histoire de transfert et de recombinaiso

Bacteriophages of Brevibacterium aurantiacum : diversity, host interactions, and impact in washed rind cheeses

Brevibacterium aurantiacum est l'un des principaux micro-organismes utilisés dans la production de fromages à croûte lavée dans le monde. L’utilisation de cette bactérie est dû à sa richesse métabolique, car elle produit des composés soufrés volatils, des pigments caroténoïdes et des enzymes lipolytiques et protéolytiques, qui sont nécessaires à la maturation d’une variété de fromages. Des souches de cette espèce bactérienne sont inoculées à la surface de fromages au cours de l'affinage et sont sensibles à des infections virales. Les bactériophages (phages), virus qui infectent les bactéries, sont omniprésents dans divers écosystèmes. Dans l'industrie laitière, ils sont reconnus pour perturber les procédés de production lors de l'infection de ferments lactiques, mais leur implication sur des fromages présentant des défauts de couleur et de saveur reste à démontrer. Ces anomalies de maturation de fromages à croûte lavée ont conduit à cette thèse. Le premier objectif de cette thèse de doctorat consistait à analyser le génome de la souche industrielle B. aurantiacum SMQ-1335 et qui est aussi sensible à des phages. Le deuxième objectif de la thèse visait à étudier les phages virulents infectant cette souche. D’ailleurs, cette étude rapporte la première description et caractérisation de phages infectant cette espèce bactérienne. Malgré la similitude entre ces phages, des répétitions en tandem d'ADN ont été identifiées dans des génomes viraux et une analyse approfondie a montré que ces segments d'ADN sont répandus parmi les phages. Le troisième objectif visait à étudier l'interaction phage-hôte via l’analyse du génome de souches mutantes insensibles aux phages. En étudiant ces souches mutantes, des gènes potentiellement nécessaires pour l'infection phagique ont été identifiés. Enfin, le quatrième et dernier objectif visait à évaluer l'impact des phages de B. aurantiacum dans la production de fromages à croûte lavée et ce, à l'aide des caillé modèles. À noter que le reclassement de la souche SMQ-1335, avant identifiée auparavant comme Brevibacterium linens, est décrit en annexe de cette thèse. Malgré des décennies d'études sur les phages laitiers, les phages de B. aurantiacum étaient encore inconnus. Mes travaux auront permis le développement d’un protocole reproductible pour isoler ces phages. Ces travaux ont également apporté de nouvelles connaissances sur les interactions phage-hôte et de leur impact dans les fromages affinés en surface.Brevibacterium aurantiacum is one of the key players in the production of washed rind cheeses produced worldwide. The importance of this bacterium to the dairy industry is due to its metabolic richness, as it produces volatile sulfur compounds, carotenoid pigments, and lipolytic and proteolytic enzymes, which play roles in the maturation of washed rind cheeses .As strains of this species are regularly inoculated on the cheese surface during ripening, there is a significant risk of viral attacks. Bacteriophage (phages), viruses that infect bacteria, are ubiquitous in the cheese environment. In the dairy industry, virulent phages have long been known to disrupt cheese processes by infecting lactic acid bacteria. The recent observations of color and flavor defects in washed rind cheeses suggested that phages may also infect strains of B. aurantiacum. These observations led to this thesis.The first objective of this PhD dissertation was to study the genomics of B. aurantiacum SMQ-1335, an industrial strain used in the production of washed rind cheeses. The second objective of the thesis was to study the diversity and biology of virulent phages infecting this industrial strain. This study was the first report of phages infecting B. aurantiacum. Despite the low diversity of the isolated B. aurantiacum phages, DNA tandem repeats were found in an intragenic region of the viral genomes and extended analysis showed that these DNA segments are widespread among phages. The third objective was to investigate phage-host interactions through the genome analyses of bacteriophage insensitive mutants, which were selected by challenging SMQ-1335 with phage AGM1. Host genes likely necessary for phage infection were identified and may explain why some of these mutants are phage-resistant. Finally, the fourth objective of this thesis evaluated the impact of virulent phages on the production of washed rind cheeses using model curds. Of note, the reclassification of the strain SMQ-1335, long believed to be Brevibacterium linens, is described in the annex of the thesis. Despite decades of studies on dairy phages, B. aurantiacum phages were still unknown. Here, a reproducible protocol to isolate these phages was developed, which may allow the isolation of new phages. This work also led to increased knowledge on phage-host interactions as well as on and their roles in surface-ripened cheeses

Diversité des archées et implication de la composante procaryote dans le cycle biogéochimique du méthane en milieu aquatique continental (études taxonomiques et fonctionnelles dans la colonne d'eau et les sédiments anoxiques du lac Pavin)

Le méthane, un des principaux gaz à effet de serre, est majoritairement produit et consommé par l'activité métabolique de microorganismes affiliés aux domaines des Archaea et des Bacteria. Afin d appréhender le cycle biogéochimique du méthane, il est essentiel d identifier l ensemble des acteurs impliqués dans ce dernier ainsi que les facteurs environnementaux modulant leurs activités. Les lacs d eau douce constituent une source importante de méthane, car, dans ces écosystèmes, les conditions environnementales favorisent la méthanogenèse au détriment d autres processus terminaux de la dégradation anaérobie de la matière organique. Au cours de cette thèse, les études sur les communautés impliquées dans le cycle biogéochimique du méthane ont été conduites dans la colonne d eau et les sédiments anoxiques du Lac Pavin (Auvergne), unique lac méromictique de France. Cet écosystème a été choisi comme site d'étude en raison des fortes concentrations en méthane présentes dans sa couche d'eau profonde qui contrastent avec les faibles émissions de ce gaz vers l'atmosphère. Ces observations géochimiques suggèrent une intense activité de production et de consommation du méthane, offrant un cadre pertinent pour l étude des communautés ciblées. Les approches moléculaires visant à caractériser la structure spatiale, la composition, les zones d'activité et les facteurs (ascendants et descendants) potentiellement impliqués dans la régulation des communautés de méthanogènes et de méthanotrophes ont été, au cours de ce travail, systématiquement associées à des approches culturales et microcalorimétriques afin d acquérir des données sur la physiologie des microorganismes impliqués dans le cycle du méthane. Les résultats obtenus mettent en évidence que les communautés de méthanogènes sont distribuées sur l ensemble de la colonne d eau anoxique et dans la strate superficielle des sédiments profonds. Ce groupe métabolique, essentiellement représenté par des espèces affiliées aux Methanosaetaceae et aux Methanoregulaceae, est particulièrement actif dans la zone benthique qui constituerait la source principale de méthane dans cet écosystème. Une nouvelle espèce méthanogène, Methanobacterium lacus, a été isolée de ces sédiments et décrite, et vient enrichir le faible nombre d'espèces méthanogènes isolées à ce jour à partir des lacs d'eau douce. L'étude écophysiologique de cette souche suggère que la température pourrait en partie expliquer la faible représentativité des Methanobacteriales dans cet écosystème. Une partie du méthane semble être directement consommée dans la zone anoxique (pélagique et benthique). L existence de ce processus d oxydation anaérobie, soutenu par les approches microcalorimétriques, pourrait être, dans les sédiments profonds, sous la dépendance de lignées candidates archéennes dont la physiologie reste encore énigmatique. Le remplacement progressif des méthanogènes par 2 lignées candidates d'archaea (MBG-D et MCG) le long du profil sédimentaire suggère qu'elle se développe dans des niche contrastées. La régulation putative des communautés archéennes par les virus a été analysée. Cette étude est la première à rapporter la présence de particules virales de type "archaeovirus" dans un environnement non-extrême (en termes de température, pH et salinité) ainsi que des particules virales pouvant représentées de nouvelles familles de virus. Une activité virale intense est suggérée dans ces sédiments par le nombre important de cellules infectées, comparativement à d'autres sédiments, et par le changement concomitant de la structure de la communauté virale et procaryotique avec la profondeur. Bien qu une partie du méthane soit probablement oxydée en anaérobiose, la consommation de ce métabolite est principalement dépendante de l activité de méthanotrophes aérobies dominées par des espèces affiliées au genre Methylobacter, un des principaux genres de méthanotrophes rencontré en milieu d eau douce. (...)Methane, a major greenhouse gas, is produced and consumed mainly by the metabolic activity of microorganisms affiliated to the domains Archaea and Bacteria. In order to understand the biogeochemical cycling of methane, it is essential to identify all the biological actors involved, as well as environmental factors modulating their activity. Freshwater lakes are a major source of methane because environmental conditions occurring in these ecosystems favor methanogenesis over other terminal processes of anaerobic degradation of organic matter. In this thesis, studies of communities involved in the biogeochemical cycling of methane were carried out in the water column and anoxic sediment of Lake Pavin (Auvergne), the unique French meromictic lake. This ecosystem has been selected as study site due to the high concentrations of methane in its deep water layer which contrast with the very low emission of this gas in the atmosphere. These geochemical observations suggest an intense activity of production and consumption of methane, providing an appropriate framework for studying the communities involved. Molecular approaches to characterize the spatial structure, composition, activity areas and factors (bottom-up and top-down) potentially involved in the regulation of methanogens and methanotrophs were, in this work, systematically associated to cultural and microcalorimetric approaches to acquire data on the physiology of microorganisms involved in the methane cycle. The results show that methanogens are distributed throughout the permanent anoxic water column (monimolimnion) and mainly in the superficial layer of the sediment situated under the monimolimnion. This metabolic group, mainly represented by species affiliated to Methanosaetaceae and Methanoregulaceae, is particularly active in the benthic zone which would be the main source of methane in this ecosystem. A new species of methanogen, Methanobacterium lacus, was isolated from these sediments and described. It enhances to the small number of methanogenic species isolated to date from freshwater lakes. The ecophysiological study of this strain suggests that the temperature could partly explain the low representation of Methanobacteriales in this ecosystem. A part of the methane appears to be directly consumed in the anoxic zone (pelagic and benthic). The existence of this process of anaerobic oxidation, supported by microcalorimetric approaches, could be in deep sediments, dependent on archaeal candidate lineages whose physiology remains enigmatic. The gradual replacement of methanogens by two archaeal candidate lineages (MBG-D and MCG) along the sedimentary profile suggests that they live in contrasted niche. The putative regulation of the archaeal communities by virus was analyzed. This study has reported the first observations of archaeovirus-like particles in a non-extreme environment (in term of temperature, pH and salinity) and virus-like particles which might represent new viral families. An intense viral activity in these sediments is suggested by i) the important number of visibly infected cells and ii) the concomitant change of the viral and prokaryotic communities with depth. While a fraction of methane is probably oxidized anaerobically, the consumption of this metabolite is mainly dependent on the activity of aerobic methanotrophs dominated by species affiliated to the genus Methylobacter, one of the main types of methanotrophs found in freshwater environments.These methanotrophs have a large area of activity, extending around both sides of the red/ox interface in the water column. This wide distribution may partly explain the low quantity of methane released by the Lake Pavin. (...)CLERMONT FD-Bib.électronique (631139902) / SudocSudocFranceF

Modelling the Seasonal Growth of the Brown Seaweed Fucus Vesiculosus in the Kiel Outdoor Benthocosms

Warming and acidification of the oceans as a consequence of increasing CO2-concentrations occur globally. In mesocosm experiments, the single and combined impact of elevated seawater temperature and pCO2 (1,100 ppm) on the brown alga Fucus vesiculosus together with its ssociated community (epiphytes and mesograzers) was studied in four consecutive experiments (from April 2013 to April 2014). Based on these experiments, a numerical boxmodel simulating the seasonal growth of F. vesiculosus in the Kiel Outdoor Benthocosms (KOBs) was developed. Nitrogen and carbon cycling in the KOBs were considered and relevant physiological and ecological processes were implemented. To run simulations under present and global change scenarios (e.g. warming, ocean acidification) the model was forced with atmospheric and hydrographic data of the Kiel fjord. DIN and DIC concentration in the water and Fucus growth as carbon and nitrogen increase were explicitly modelled. For instance, the following processes were implemented: (1) Storage of carbon and nitrogen assimilates by Fucus, leading to a temporal decoupling of assimilation and growth. (2) Shading effects of epiphytes. (3) Grazing by Idotea, Gammarus and Littorina on both Fucus and epiphytes, but with species-specific rates and preferences. At present, the model is a suitable scientific tool capable of integrating our knowledge about macroalgal processes, their growth and productivity in coastal areas. It further facilitates the communication of complex knowledge to lay persons. Ultimately, the development of a predictive model, which can be coupled to a 3D-high resolution western Baltic Sea model, is anticipated. This will allow observations on the consequences of global change for the wellbeing and distribution of F. vesiculosus in the western Baltic Sea. Understanding responses of macroalgae and of the associated community is important because changing global temperatures and elevated CO2 may affect the ecological role of Fucus as primary producer, carbon sink, water purifier, and ecosystem engineer in the coastal ecosystem of the Baltic Sea

Polyphasic taxonomy of thermophilic actinomycetes

PhD ThesisMolecular systematic methods were applied in a series of studies designed to resolve the taxonomic relationships of thermophilic actinomycetes known to be difficult to classify using standard taxonomic procedures. The test strains included representatives of clusters defined in an extensiven umerical phenetic survey of thermophilic streptomycetesa nd twelve marker strains. The resultant genotypic data together with the results of corresponding phenotypic studies were used to highlight novel taxa and to improve the circumscription of validly described species. The most comprehensive study was undertaken to clarify relationships within and between representative alkalitolerant, thermophilic and neutrophilic, thermophilic streptomycetes isolated from soil and appropriate marker strains. The resultant data, notably those from DNA: DNA relatedness studies, supported the taxonomic integrity of the validly described species Streptomyces thermodiastaticus, Streptomyces thermoviolaceus and Streptomyces thermovulgaris. However, the genotypic and phenotypic data clearly show that Streptomyces thermonitrificans Desai and Dhala 1967 and Streptomyces thermovulgaris (Henssen 1957) Goodfellow et al. 1987 represent a single species. On the basis of the priority, Streptomyces thermonitrificans is a later subjective synonym of Streptomyces thermovulgaris. Similarly, eight out of eleven representative alkalitolerant, thermophilic isolates and three out of sixteen representative neutrophilic, thermophilic isolates had a combination of properties consistent with their classification as Streptomyces thermovulgaris. One of the remaining alkalitolerant, thermophilic isolate, Streptomyces strain TA56, merited species status. The name Streptomyces thermoalcalitolerans sp. nov. is proposed for this strain. A neutrophilic, thermophilic isolate, Streptomyces strain NAR85, was identified as Streptomyces thermodiastaticus. Four other neutrophilic thermophilic isolates assigned to a numerical phenetic cluster and a thermophilic isolates from poultry faeces were also considered to warrant species status; the names Streptomyces eurythermophilus sp. nov. and Streptomyces thermocoprophilus sp. nov. are proposed to accommodate these strains. It was also concluded that additional comparative taxonomic studies are required to clarify the relationships between additional thermophilic streptomycete strains included in the present investigation. A corresponding polyphasic approach was used to clarify the taxonomy of six thermophilic isolates provisionally assigned to either the genera Amycolatopsis or Excellospora. Two of the isolates, strain NT202 and NT303, had properties consistent with their classification in the genus Amycolatopsis. However, the genotypic and phenotypic data also showed that these strains formed a new centre of taxonomic variation for which the name Amycolatopsis eurythermus sp. nov. is proposed. Similarly, the four remaining strains formed two new centre of taxonomic variation within the genus Excellospora. It is proposed that isolates TA113 and TA114 be designated Excellospora alcalithermophilus sp. nov. Similarly, the name Excellospora thermoalcalitolerans sp. nov. is proposed for strains TA86 and TA111. An emended description is also given for the genus Excellospora.Overseas Research Student Award

27th Fungal Genetics Conference

Program and abstracts from the 27th Fungal Genetics Conference Asilomar, March 12-17, 2013