In aquatic environments the availability of nitrogen (N) generally limits primary production. N2-fixing prokaryotes (diazotrophs) can convert N2 gas into ammonium and provide significant input of N into the oceans. Cyanobacteria are thought to be the main N2-fixers but diazotrophs also include a wide range of heterotrophic bacteria. However, their activity and regulation in the water column is largely unknown. In this thesis the distribution, diversity, abundance, and activity of marine and estuarine heterotrophic diazotrophs was investigated. With molecular methods targeting the nifH gene, encoding the nitrogenase enzyme for N2 fixation, it was shown that diverse nifH genes affiliating with heterotrophic bacteria were ubiquitous in surface waters from ten marine locations world-wide and the estuarine Baltic Sea. Through enrichment cultures of Baltic Sea surface water in anaerobic N-free medium, heterotrophic N2 fixation was induced showing that there was a functional N2-fixing community present and isolates of heterotrophic diazotrophs were obtained. In Sargasso Sea surface waters, transcripts of nifH related to heterotrophic bacteria were detected indicating heterotrophic N2-fixing activity. Nitrogenase expression is thought to be highly regulated by the availability of inorganic N and the presence of oxygen. Low oxygen zones within the water column can be found in association with plankton. The presence of diazotrophs as symbionts of heterotrophic dinoflagellates was investigated and nifH genes related to heterotrophic diazotrophs rather than the cyanobacterial symbionts were found, suggesting that a symbiotic co-existence prevailed. Oxic-anoxic interfaces could also be potential sites for heterotrophic N2 fixation. The Baltic Sea contains large areas of anoxic bottom water. At the chemocline and in anoxic deep water heterotrophic diazotrophs were diverse, abundant and active. These findings extend the currently known regime of N2 fixation to also include ammonium-rich anaerobic waters. The results of this thesis suggest that heterotrophic diazotrophs are diverse and widely distributed in marine and estuarine waters and that they can also be active. However, limits in the knowledge on their physiology and factors which regulate their N2 fixation activity currently prevent an evaluation of their importance in the global marine N budget.

The plant genus Tolpis (Asteraceae) is a predominantly insular plant lineage. It inhabits four of the five archipelagoes that comprise the Atlantic region of Macaronesia and also occurs in Mediterranean Europe and North Africa. Twelve species are currently recognized in Tolpis, of which ten are insular and two continental. The majority of the insular species inhabit the five western Canarian islands, where they constitute endemics to specific ecological habitats. A comprehensive molecular phylogeny of Tolpis is generated via DNA sequences of one nuclear ribosomal and two low-copy nuclear DNA markers. Considerable phylogenetic uncertainty among inferred tree topologies is detected, and incongruence between these topologies is resolved via statistical hypotheses testing. The extant diversity of the genus is identified to be the result of two independent colonization pathways and adaptive radiations on several islands. Moreover, potential hybridization is detected between species that inhabit different islands and archipelagoes, indicating a more widespread historical distribution of the genus. Details of the biogeographic history of Tolpis are inferred via ancestral area reconstructions under parsimony and likelihood optimality criteria. The hypothesis that Tolpis may have undergone a back-dispersal from an island to a continental habitat is also tested. Uncertainty in taxon cladograms owing to the presence of hybrid or allopolyploid taxa is characterized and a potential adjustment strategy evaluated. Averaging reconstruction results over all optimal phylogenetic trees and the manual pruning of cloned DNA sequences are found potential adjustment strategies against the impact of topological uncertainty owning to hybrid or allopolyploid taxa. Adjusted ancestral area reconstructions in Tolpis do not support the scenario that the genus has undergone a reverse colonization of the continent. In addition to the phylogenetic and biogeographic history of the genus, the diversity of symbiotic mycorrhizal fungi associated with Tolpis is characterized. A molecular survey using two nuclear ribosomal DNA markers and 454 pyrosequencing is performed. Particular emphasis is placed on the quality filtering of resulting fungal DNA sequences, the generation of operational taxonomic units, and their taxonomic assignment via similarity searches against DNA sequence databases. Numerous potentially novel fungal genotypes are identified. / text

Human biotherapy (HBT) or fecal transplants have been shown to be an effective treatment for patients with recurrent Clostridium difficile infection (CDI). This study examines the microbial populations present in CDI patients pre- and post-HBT by extracting bacterial DNA from stool samples and performing pyrosequencing of the 16S rRNA gene. We then compared these microbial populations to those of the donors. We examined 19 pairs of patient samples, of which 14 were clinically cured of CDI, and 5 patients were failures. The successful treatment of CDI was associated with an increase in diversity and richness of the patient's fecal microbiome. The majority of those cured showed an increase in the proportion of Firmicutes and decrease in the proportion of Proteobacteria, although varying antibiotic exposure and innate variability between patients was observed. / MSc thesis / NSERC, CIHR, St. Joseph's Healthcare Hamilton

Oligotrophic ecosystems can be loosely defined as environments that exhibit low ambient nutrient levels. During my thesis, I used 454 DNA pyrosequencing of partial 16S rDNA to explore the bacterial diversity in three different oligotrophic environments, including A. surface desert soil, B. Asian sandstorm dust and C. a section of the city of Paris's drinking water distribution system.A. Arid regions represent nearly 30% of the Earth's terrestrial surface. The living conditions at the surface of deserts are a challenge for microorganisms, as there is little available water and/or carbon, a very large range of temperatures and high exposure to UV irradiation from the Sun. In surface sand samples from two large Asian deserts, unexpectedly large bacterial diversity residing was revealed. Sequences belonging to the Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria phyla were the most abundant. An increase in phylotype numbers with increasing C/N ratio was noted, suggesting a possible role in the bacterial richness of these desert sand environments.B. Desert sandstorms are a meteorological phenomenon which have been postulated affect the Earth's climate and public health. We examined the particle-associated (dust and sand-associated) bacterial populations of atmospheric sand in the absence (as control) and presence of sandstorms in five Asian cities. Greater than 90% of the sequences can be classified as representing bacteria belonging to four phyla: Proteobacteria, Bacteriodetes, Actinobacteria and Firmicutes. Principal component analyses showed that the sandstorm-associated bacterial populations were clustered by sampling year, rather than location. Members belonging to nine bacterial genera (Massilia, Planococcus, Carnobacterium, Planomicrobium, Pontibacter, Pedobacter, Lysobacter, Sanguibacter, Ohtaekwangia) were observed to increase in sand-associated samples from sandstorms, versus the controls. C. We characterized the bacterial communities in three water and three biofilm samples from one part of the Parisian drinking water distribution system. A dramatic change in bacterial population in the water during flow through the distribution system from the water treatment plant to the exit from the reservoir was found. The richness of the bacterial population was reduced from the water treatment plant to the reservoir (from 336 to 165 OTUs for water samples leaving the reservoir and from 947 to 275 for biofilm samples in the network). Several OTUs belonging to pathogenic genera were detected in our samples, mostly in the biofilm samples, thus suggesting that the biofilms may be an important source of bacteria during water distribution to the consumers.

Reverse genetics systems that are based on either viral transcripts or cDNA genome segments cloned in plasmids have recently been reported for some of the dsRNA viruses of the Reoviridae family, namely African horsesickness virus, bluetongue virus and orthoreovirus. For rotaviruses, three reverse genetics systems which only allow the manipulation of a single genome segment have been described. These rotavirus single genome segment reverse genetics systems are not true stand-alone systems because they require a helper virus and a recombinant virus selection step. A true selection-free, plasmid- only or transcript-based reverse genetics system for rotaviruses is lacking. This study sought to identify and characterise the factors that need to be understood and overcome for the development of a rotavirus reverse genetics system using mRNA derived from the in vitro transcription of a consensus nucleotide sequence as well as from double-layered particles. The consensus whole genome sequence of the prototype rotavirus DS-1 and SA11 strains was determined using sequenceindependent whole genome amplification and 454® pyrosequencing. For the rotavirus DS-1 strain, a novel isoleucine in a minor population variant was found at position 397 in a hydrophobic region of VP4. NSP1 contained seven additional amino acids MKSLVEA at the N-terminal end due to an insertion in the consensus nucleotide sequence of genome segment 5. The first 34 nucleotides at the 5'- terminus and last 30 nucleotides at the 3'-terminal end of genome segment 10 (NSP4) of the DS-1 strain were determined in this study. The consensus genome segment 11 (NSP5/6) sequence was 821 bp in length, 148 bp longer than previously reported. The 454® pyrosequence data for a rotavirus SA11 sample with no known passage history revealed a mixed infection with two SA11 strains. One of the strains was a reassortant which contained genome segment 8 (NSP2) from the bovine rotavirus O agent. The other ten consensus genome segments of the two strains could not be differentiated. Novel minor population variants of genome segments 4 (VP4), 9 (VP7) and 10 (NSP4) were identified. Molecular clock phylogenetic analyses of the rotavirus SA11 genomes showed that the two SA11 strains were closely related to the original SA11-H96 strain isolated in 1958. Plasmids containing inserts of the consensus cDNA of the rotavirus DS-1 strain were purchased and used to generate exact capped transcripts by in vitro transcription with a T7 polymerase. Wild-type transcripts of rotavirus SA11 were obtained from in vitro transcription using purified rotavirus SA11 double-layered particles. The purified rotavirus DS-1 and SA11 transcripts were transfected into BSR, COS-7 and MA104 cells. Work on MA104 cells was discontinued due their very low transfection efficacy. In BSR and COS-7 cells, rotavirus DS-1 and SA11 transcripts induced cell death. However, no viable rotavirus was recovered following attempts to infect MA104 cells with the BSR and COS-7 transfected cell lysates. The cell death was determined to be due to apoptotic cell death mechanisms. Immunostaining showed that the DS-1 genome segment 6 (VP6) and SA11 transcripts were translated in transfected BSR and COS-7 cells. Based on visual inspection, the translation seemed to be higher in the retinoic acid-inducible gene-I (RIG-I) deficient BSR cells than in COS-7 cells. This suggested that the transfection of rotavirus transcripts induced an innate immune response which could lead to the development of an antiviral state. Therefore, the innate immune response to rotavirus transcripts was investigated in HEK 293H cells using qRT-PCR and western blot analyses. Results of this investigation showed that RIG-I, but not MDA5 sensed rotavirus transcripts in transfected HEK 293H cells. Furthermore, rotavirus transcripts induced high levels of cellular mRNA encoding the cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α. Other cytokines namely, IFN-α, IL-10, IL-12 p40 and the kinase RIP1 were not significantly induced. Inhibiting the RNA-dependent protein kinase R (PKR) reduced the induction of cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α, but the expression levels were not abrogated. The importance of a consensus sequence and the insights gained in the current study regarding the role of the innate immune response after transfection of rotavirus transcripts into cells in culture, should aid the development of a true rotavirus reverse genetics system. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2013

Reverse genetics systems that are based on either viral transcripts or cDNA genome segments cloned in plasmids have recently been reported for some of the dsRNA viruses of the Reoviridae family, namely African horsesickness virus, bluetongue virus and orthoreovirus. For rotaviruses, three reverse genetics systems which only allow the manipulation of a single genome segment have been described. These rotavirus single genome segment reverse genetics systems are not true stand-alone systems because they require a helper virus and a recombinant virus selection step. A true selection-free, plasmid- only or transcript-based reverse genetics system for rotaviruses is lacking. This study sought to identify and characterise the factors that need to be understood and overcome for the development of a rotavirus reverse genetics system using mRNA derived from the in vitro transcription of a consensus nucleotide sequence as well as from double-layered particles. The consensus whole genome sequence of the prototype rotavirus DS-1 and SA11 strains was determined using sequenceindependent whole genome amplification and 454® pyrosequencing. For the rotavirus DS-1 strain, a novel isoleucine in a minor population variant was found at position 397 in a hydrophobic region of VP4. NSP1 contained seven additional amino acids MKSLVEA at the N-terminal end due to an insertion in the consensus nucleotide sequence of genome segment 5. The first 34 nucleotides at the 5'- terminus and last 30 nucleotides at the 3'-terminal end of genome segment 10 (NSP4) of the DS-1 strain were determined in this study. The consensus genome segment 11 (NSP5/6) sequence was 821 bp in length, 148 bp longer than previously reported. The 454® pyrosequence data for a rotavirus SA11 sample with no known passage history revealed a mixed infection with two SA11 strains. One of the strains was a reassortant which contained genome segment 8 (NSP2) from the bovine rotavirus O agent. The other ten consensus genome segments of the two strains could not be differentiated. Novel minor population variants of genome segments 4 (VP4), 9 (VP7) and 10 (NSP4) were identified. Molecular clock phylogenetic analyses of the rotavirus SA11 genomes showed that the two SA11 strains were closely related to the original SA11-H96 strain isolated in 1958. Plasmids containing inserts of the consensus cDNA of the rotavirus DS-1 strain were purchased and used to generate exact capped transcripts by in vitro transcription with a T7 polymerase. Wild-type transcripts of rotavirus SA11 were obtained from in vitro transcription using purified rotavirus SA11 double-layered particles. The purified rotavirus DS-1 and SA11 transcripts were transfected into BSR, COS-7 and MA104 cells. Work on MA104 cells was discontinued due their very low transfection efficacy. In BSR and COS-7 cells, rotavirus DS-1 and SA11 transcripts induced cell death. However, no viable rotavirus was recovered following attempts to infect MA104 cells with the BSR and COS-7 transfected cell lysates. The cell death was determined to be due to apoptotic cell death mechanisms. Immunostaining showed that the DS-1 genome segment 6 (VP6) and SA11 transcripts were translated in transfected BSR and COS-7 cells. Based on visual inspection, the translation seemed to be higher in the retinoic acid-inducible gene-I (RIG-I) deficient BSR cells than in COS-7 cells. This suggested that the transfection of rotavirus transcripts induced an innate immune response which could lead to the development of an antiviral state. Therefore, the innate immune response to rotavirus transcripts was investigated in HEK 293H cells using qRT-PCR and western blot analyses. Results of this investigation showed that RIG-I, but not MDA5 sensed rotavirus transcripts in transfected HEK 293H cells. Furthermore, rotavirus transcripts induced high levels of cellular mRNA encoding the cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α. Other cytokines namely, IFN-α, IL-10, IL-12 p40 and the kinase RIP1 were not significantly induced. Inhibiting the RNA-dependent protein kinase R (PKR) reduced the induction of cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α, but the expression levels were not abrogated. The importance of a consensus sequence and the insights gained in the current study regarding the role of the innate immune response after transfection of rotavirus transcripts into cells in culture, should aid the development of a true rotavirus reverse genetics system. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2013

Agrobacterium are Alphaproteobacteria common in most soils that closely interact with plants in two respects. Firstly, they are rhizospheric bacteria saprophytically living in the rhizosphere of numerous plants and they are likely beneficial to plants. Secondly, when they harbor a dispensable Ti plasmid (i.e. tumor inducing plasmid), agrobacteria are plant pathogens able to cause the crown gall disease to most dicots and gymnosperms and some monocots. An epidemiological survey of crown gall thus also requires expert determination of the Agrobacterium taxonomy. In this thesis we evaluated the usefulness of MALDI-TOF MS technique as a high throughput tool to determine and classify agrobacteria. Then we set up a recA-based PCR method to accurately and exhaustively assess agrobacterial diversity either of isolated agrobacteria or directly in various biotopes. We applied standard biochemical, recA-based and Ti plasmid-based identification methods to study the prevalence of pathogenic and non-pathogenic agrobacteria at the country and local scales. Finally, we tested whether analyzing the internal composition of recA amplicons could be a way to directly assess the micro-diversity of agrobacterial populations using cloning sequencing or pyrosequencing approaches. The later methodology was applied to establish the actual field diversity of Agrobacterium and to evaluate whether plant genotypes differentially select agrobacteria in their root systems, providing first data upon biotic factors shaping the population structure of agrobacteria

There are a variety of archaeological questions, which are difficult to assess by traditional archaeological methods. Similarly, there are genetic and population genetic questions about human evolution and migration that are difficult to assess by studying modern day genetic variation. Archaeological genetics can directly study the archaeological remains, allowing human history to be explored by means of genetics, and genetics to be expanded into historical and pre-historical times. Examples of archaeological questions that can be resolved by genetics are determining biological sex on archaeological remains and exploring the kinship or groups buried in close proximity. Another example is one of the most important events in human prehistory – the transition from a hunter-gatherer lifestyle to farming - was driven through the diffusion of ideas or with migrating farmers. Molecular genetics has the potential to contribute in answering all these questions as well as others of similar nature. However, it is essential that the pitfalls of ancient DNA, namely fragmentation, damage and contamination are handled during data collection and data analysis. Analyses of ancient DNA presented in this thesis are based on both mitochondrial DNA and nuclear DNA through the study of single nuclear polymorphisms (SNPs). I used pyrosequencing assays in order to identify the biological sex of archaeological remains as well as verifying if fragmented remains were human or from animal sources. I used a clonal assay approach in order to retrieve sequences for the HVRI of a small family-like burial constellation from the Viking age. By the use of low coverage shotgun sequencing I retrieved sequence data from 13 crew members from the 17th century Swedish man-of-war Kronan. This data was used to determine the ancestry of the crew, which in some cases was speculated to be of non-Scandinavian or non-European origin. However, I demonstrate that all individuals were of European ancestry. Finally, I retrieved sequence data from a Neolithic farmer from the Iberian Peninsula, which added one more facet of information in exploring the Neolithization process of Europe. The Neolithic Iberian individual was genetically similar to Scandinavian Neolithic farmers, indicating that the genetic variation of prehistoric Europe correlated with subsistence mode rather than with geography.

Prokaryotic microorganisms, which are at the base of deep-sea hydrothermal vent food webs, adapt rapidly to environmental fluctuations. This study aimed at comparing bacterial communities in contrasting hydrothermal habitats to better understand compositional adaptations to local conditions. I first used small subunit (SSU) ribosomal RNA (rRNA) gene sequences to compare mat-forming bacterial communities associated with iron oxides at two hydrothermal vent sites on the Tonga Arc, southwest Pacific. Operational taxonomic units (OTUs), defined at 97% sequence similarity, were affiliated to a great diversity of autotrophic and heterotrophic groups. Metabolically diverse Gammaproteobacteria dominated the sample from Volcano 19, collected at 992 m depth. The sample from Volcano 1, collected at 197 m depth, was dominated by iron-oxidizing bacteria from the class Zetaproteobacteria. The depth of the sampling sites was proposed to explain clone library dissimilarities. In the following studies, I compared bacterial communities associated with the vestimentiferan tubeworm Ridgeia piscesae, a foundation species at the Juan de Fuca Ridge. Samples of the polychaete were collected from tubeworm habitats in contrasting flow regimes that influenced temperature and hydrogen sulphide concentrations. Free-living bacteria were analyzed using both sequencing and 454 pyrosequencing of the SSU rRNA gene. Statistical analyses suggested a predictable pattern of bacterial community composition for the two habitats, with higher proportions of sulphur and hydrogen oxidizers in High Flow and more heterotrophic groups in Low Flow environments. Temperature, available energy for metabolism, and stability of the habitat were suggested to explain these distinctive bacterial communities. Symbiotic assemblages were investigated using the same sequencing methods together with catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH). Gammaproteobacteria dominated all sequence libraries, followed by Epsilonproteobacteria. CARD-FISH confirmed the co-occurrence of these groups within R. piscesae trophosomes. Statistical analyses indicated distinctive membership and structure of trophosome assemblages between sampling sites. Analysis of R. piscesae juvenile showed distinctive structural properties when compared to adult individuals, but similar membership, within sampling sites. These results suggested that the composition of trophosome assemblages might be affected by specific physical and chemical conditions at each vent site and that a selection process might occur during R. piscesae’s development. / Graduate / 0410 / 0416 / 0329 / nathalieforget@gmail.com

There are generally two contrasting alternatives to what limits recruitment in plants, namely the availability of seeds (seed limitation) or the quality or quantity of suitable sites (microsite limitation). Dust seeds, the smallest existing seeds, lack or have minimal nutrient reserves. During germination and initial development they consequently parasitize on mycorrhizal fungi. This is called mycoheterotrophy, and can vary in degree of fungal dependency in adult plants from full, partial or initial mycoheterotrophy. The aim of this thesis was to investigate the recruitment ecology of mycoheterotrophic Ericaceae (tribe Pyroleae) species with dust seeds, and to determine what limits their recruitment. The investigated species were: Chimaphila umbellata, Moneses uniflora, Orthilia secunda, Pyrola chlorantha, P. minor and P. rotundifolia. This aim was achieved by combining field experiments (seed sowing) with isotope analysis and fungal host pyrosequencing. Results provide evidence that the species in Pyroleae are heterogeneous, not only with regard to their degree of mycoheterotrophy, but also concerning germination and early seedling development. A combination of microsite and seed limitation is thus likely to be of importance for all studied species, but the relative importance of these limitations varies among species. Despite having adaptations for wind dispersal the majority of the seeds were deposited in close vicinity of the seed source. But with high seed production at least some seeds should be able to disperse long-distance. Seedlings of all studied species were found to associate with a wide range of ectomycorrhizal fungi, at least during their initial developmental stages. There seems to be a tendency for host narrowing in some Pyroleae species, but not as strict as the host specialization seen in fully mycoheterotrophic Monotropa hypopitys, supporting the hypothesis of geographical and developmental host shifts. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: In press. Paper 4: Manuscript.</p>