Thesis (M.S.)--West Virginia University, 2009. / Title from document title page. Document formatted into pages; contains v, 61 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 50-56).

Thesis (MSc (Genetics))--University of Stellenbosch, 2005. / Withering syndrome is a severe disease of abalone, Haliotis species that has been associated with mortality ranging from 99% in black, H. cracherodii Leach and 30% in red abalone, H. rufescens Swainson. The disease was first observed in California, along the west coast of North America and is an economically important disease that has led to the closure of the black abalone fishery throughout the southern California State. The causative agent of withering syndrome is a gram-negative intracellular Rickettsiales-like prokaryote designated Candidatus xenohaliotis californiensis. The geographical range of C. xenohaliotis californiensis is broad, besides red and black abalone it has also been reported in yellow, H. corrugate and blue abalone, H. fulgens all caught in Baja California, Mexico. In 2000 a Rickettsiales-like prokaryote resembling the disease-causing agent was observed in the digestive gland of the South African abalone H. midae. In this study we aimed to determine the relationship of the bacterium observed in the local abalone species, H. midae to the disease-causing agent of withering syndrome. A specific PCR and in situ hybridization test using primers and probes specific for the C. xenohaliotis californiesis 16S rDNA gene were used to screen H. midae digestive gland tissues showing classical features of the Rickettsiales-like prokaryote. Both analyses indicated that C. xenohaliotis californiensis is not present in the local abalone species. We therefore aimed the identification of the organism parasiting the local abalone species by DNA sequence analysis of the 16S rDNA gene. The 16S rDNA gene was amplified by PCR, cloned and sequenced. Phylogenetic trees, constructed by maximum parsimony analysis revealed a diverse community comprised of α - Proteobacteria, Mollicutes and Spirochaetes. In the class α - Proteobacteria a novel group of sequences showing phylogenetic affinities to the order Rickettsiales was identified as likely candidate for forming the Rickettsiales-like inclusions in the digestive gland of H. midae. Oligonucleotide probes that bind to four variable regions of the novel group were used to confirm their presence in infected H. midae digestive gland tissue by in situ hybridization. Although these probes did not recognize the inclusions formed by the Rickettsiales-like organisms, they revealed the presence of a group of free-living bacteria abundant in the host tissue. We therefore conclude that (1) C.xenohaliotis californiensis is not present in the South- African abalone, H. midae; (2) the organisms isolated from the digestive gland of H. midae are part of the normal microflora and (3) the group of sequences showing phylogenetic affinities to the order Rickettsiales is not responsible for the Rickettsiales-like inclusions in infected digestive gland tissues but represent a novel group of organisms that are abundant in the host tissue.

Fitoplasmas são organismos procarióticos desprovidos de parede celular, parasitas intracelulares obrigatórios de floema e patogênicos às plantas. Numerosas doenças associadas a este tipo de agentes patogênicos têm sido frequentemente relatadas em espécies cultivadas e plantas daninhas. No presente estudo, sintomas tipicamente induzidos por fitoplasmas, tais como clorose e avermelhamento foliar, redução no tamanho das folhas, diminuição dos órgãos florais, superbrotamento de ramos e declínio da planta, foram observados em pomares de fruteiras de caroço como pessegueiro e nectarineira. Além disto, em campos de couve-flor, plantas daninhas conhecidas como mostarda-do-campo também manifestaram sintomas que pareciam ter sido provocados por fitoplamas, expressados por superbrotamento de ramos finos e redução no tamanho de folhas e flores. Assim, o presente trabalho teve por objetivos detectar, identificar e classificar a nível molecular os possíveis fitoplasmas associados às plantas sintomáticas citadas anteriormente, utilizando as técnicas de PCR, análise de RFLP virtual e o método de classificação on-line denominado de iPhyclassifier. Para tanto, o DNA das amostras das fruteiras de caroço e da erva daninha foi extraído com o auxílio de um kit comercial ou através do protocolo 2X CTAB. A detecção dos fitoplasmas foi conduzida por duplo PCR com os primers universais R16mF2/mR1 e SN910601/SN011119 na primeira reação e com o par R16F2n/R2 na segunda reação. Para a mostarda, a identificação dos fitoplasmas também foi realizada através de duplo PCR com os primers e R16(III)F2/R16(III)R1 específicos para fitoplasmas do grupo 16SrIII. A aplicação de PCR com primers universais permitiu a detecção consistente desses agentes nos tecidos das plantas sintomáticas de pessegueiro, nectarineira e mostarda-do-campo pela amplificação de um fragmento genômico de 1250 pb, correspondente ao gene 16S rRNA. Para todos os hospedeiros analisados, os produtos amplificados por duplo PCR com os primers universais foram purificados, clonados em Escherichia coli e submetidos ao sequenciamento. A análise de RFLP virtual e iPhyclassifier permitiram classificar os fitoplasmas encontrados em mostarda nos grupos 16SrIII e 16SrVII. Os fitoplasmas detectados na mostarda-do-campo, foram definitivamente classificados como representantes dos subgrupos 16SrIII-B, 16SrIII-J e 16SrIII-U e 16SrVII-B. Em relação aos fitoplasmas encontrados no pessegueiro e nectarineira, foi possível classificar os fitoplasmas nos grupos 16SrI e 16SrIII, sendo definitivamente classificados como representantes dos subgrupos 16SrI-B em pessegueiro, e em nectarineira, classificados em 16SrI-B e 16SrIII-J. / Phytoplasmas are prokaryotic organisms devoid of cell wall, obligate intracellular phloem parasites and pathogenic to plants. Numerous diseases associated with this type of pathogen have been frequently reported in cultivated species of plants and weeds. In this present study, symptoms typically induced by phytoplasmas, such a yellowing an reddeling leaves, small leaves and flowers, \"witches\' broom\" appearance on terminal new bud growth, and death in plants, were observed in orchards of stone fruit trees such as peach and nectarines. In addition, in fields of cauliflower, weeds known as mustard-field also exhibited symptoms that appeared to have been caused by phytoplasmas, expressed by thin branches and reduced leaf and flower size. The objective of this present study was to detect, identify and classify at molecular level the phytoplasmas possibly associated with the symptomatic plants. PCR techniques, virtual RFLP analysis and the online method known as iPhyclassifier were used. Total DNA was extracted using a commercial kit or through the CTAB protocol. The detection of phytoplasmas was conducted by nested PCR with the universal primers R16mF2/mR1 and SN910601/SN011119 in the first reaction and R16F2n/R2 pair in the second reaction. For mustard-field, the identification of the phytoplasmas was also performed through nested PCR with the primers R16(III) F2/R16(III) R1, which are specific to detection of 16SrIII group phytoplasmas. The application of PCR with universal primers allowed the consistent detection of these agents in the tissues in symptomatic plants of peach, nectarine and mustard-field by the amplification of a 1250 bp genomic fragment, corresponding to the 16S rRNA gene. For all hosts analyzed, the products amplified by nested PCR with the universal primers were purified, cloned in Escherichia coli and sequenced. The analysis of virtual RFLP and iPhyclassifier allowed to classify the phytoplasmas found in mustard in the 16SrIII and 16SrVII groups. The phytoplasmas detected in mustard-field were definitively classified as representatives of the subgroups 16SrIII-B, 16SrIII-J and 16SrIII-U and 16SrVII-B. The phytoplasmas found in the peach and nectarine were classified within the 16SrI and 16SrIII groups, as representatives of the subgroups 16SrI-B for peach and 16SrI-B and 16SrIII-J for nectarine.

Au cours des dernières anneés, la taxonomie bactérienne a subi de profonds changements mais peu de consensus existent quant à la description précise des procaryotes. La diversité des procaryotes a été estimeé à 107 espèces, et la classification actuelle contient plus de 12 900 espèces officiellement reconnues. Ceci souligne l'absence d'un répertoire des procaryotes isolés chez l'homme. Nous avons constitué ce répertoire qui contient 2 156 espèces différentes réparties en 12 phyla,Notre second travail est la caractérisation du microbiote digestif par 5 approches varieés (cytométrie de flux, coloration de gram, TEM, qPCR, pyroséquençage). Nous avons analysé 16 selles de patients en comparant le taux de procaryotes de type gram positif/négatif. La moitié des procaryotes de type gram négatif n'est pas détecté par le pyroséquençage,alors qu'ils sont décrits comme les constituants majeurs de ce microbiote d'après les 1ères études réaliseés utilisant la culture.Dans notre 3ème travail, nous avons voulu montrer que l'utilisation de la culture bactérienne n'est pas inférieure aux techniques de séquençage pour étudier la diversité du microbiote digestif. Au total, depuis 4 ans, 685 échantillons ont été analysés et plus de 500 000 colonies ont été identifieés par MALDI-TOF. Ce travail a permis d'augmenter de 77,5 % le nombre d'espèces identifieés dans le tube digestif. Les nouvelles espèces sont décrites suivant le concept « taxonogenomics » incluant des donneés phénotypiques et le séquençage du génome. / Bacterial taxonomy has undergone tremendous changes over time, with little historical consensus regarding specific descriptions of prokaryotes. The prokaryotes have been estimated about 107 species, and the current classification contained more than 12,900 species. This highlights the absence of an exhaustive and specific database listing all prokaryotes associated with humans. We found than the human prokaryotic repertoire contained 2,156 species, divided among 12 different phyla.The second aim of our work was to characterize the human gut microbiota using 5 different techniques, including morphologic and molecular approaches (flow cytometry, gram staining, electron microscopy, qPCR and pyrosequencing). We analyzed 16 stools samples of patient and we copared the rate of gram-positive and gram-negative prokaryotes obtained with each technique. We found than by pyrosequencing only a half of gram-negative prokaryotes was detected.Our third goal was to demonstrate that bacterial culture was not inferior to pyrosequencing to describe the gut diversity. Culturomics concept created during the pioneer study has revolutionized the approach of the microbiota exploration. Since 4 years, we have performed the analyze of 685 different samples and identified more than 500,000 colonies using the MALDI-TOF mass spectrometry. We have increased by 77.5% the number of species isolated in the gut. Each new species will be described following our new concept named "taxonogenomics", including phenotypic data and genome sequencing.

Microbial-root associations are important to help plants cope with abiotic and biotic stressors. Managing these interactions offers an opportunity for improving the efficiency and sustainability of agricultural production. By characterizing the bacterial and archaeal community (via 16S rRNA sequencing) associated with the bulk and rhizosphere soil of sixteen strawberry cultivars in two controlled field studies, we explored the relationships between the soil microbiome and plant resistance to two soilborne fungal pathogens of strawberry (Verticillium dahliae and Macrophomina phaseolina). Overall, the plants had a distinctive rhizosphere microbiome relative to the bulk soil, with higher abundances of known beneficial bacteria such as Pseudomonads and Rhizobium. Plant genotype, biomass, leaf nutrient content and mortality were influenced differently by the rhizosphere microbiome in each of the two trials. In the V. dahliae trial, the rhizosphere microbiome was associated with plant biomass and leaf nutrient content and only indirectly to the disease resistance. In the M. phaseolina trial, the rhizosphere microbiome was associated to plant biomass, but not nutrient content; furthermore, resistant cultivars had larger abundances of Pseudomonas and Arthrobacter in their rhizosphere relative to susceptible cultivars. The mechanisms involved in these beneficial plant-microbial interactions and their plasticity in different environments should be studied further for the design of low-input disease management strategies.

The canonical model of transcriptional regulation in prokaryotes restricted binding site locations to promoter regions and suggested that the binding sequences serve as the main determinants of binding. In this dissertation, I challenge these assumptions. As a member of the TB Systems Biology Consortium, I analyzed and validated ChIP-Seq and microarray experiments for over 100 transcription factors (TFs). In order to study the transcriptional functions of predicted binding sites, I integrated binding and expression data and assigned potential regulatory roles to 20% of the binding sites. Stronger binding sites were more often associated with regulation than weaker sites, suggesting a correlation between binding strength and regulatory impact. Seventy-six percent of the sites fell into annotated coding regions and a significant proportion was assigned to regulatory functions. To study the importance of binding sequences, I compared experimental sites with computational motif predictions. Although a conservative binding motif was found for most TFs, only a fraction of the observed motifs appeared bound in the experiment. Some low-affinity binding sites appeared occupied by the corresponding TF while many high-affinity binding sites were not. Interestingly, I found exactly the same nucleotide sequences (up to 15 residues long) bound in one area of the genome but not bound in another area, pointing to DNA accessibility as an important factor for in vivo binding. To investigate the evolutionary conservation of binding-site occupancy, sequence, and transcriptional impact, I analyzed ChIP-Seq and expression experiments for five conserved TFs for two-to-four Mycobacterial relatives. The regulon composition showed significantly less conservation than expected from the overall gene conservation level across Mycobacteria. Despite expectations, sequence conservation did not serve as a good indicator of whether or not a computationally predicted motif was bound experimentally; and in some cases, a fully conserved motif was bound in one relative but not in the other. Conservation of genic binding sites was higher than expected from the random model, adding to the evidence that at least some genic sites are functional. Understanding the evolutionary story of binding sites allowed me to explain unusual site configurations, some of which indicated a role for DNA looping.

Bacteria live under constant assault by bacteriophages and have evolved a diverse array of defense strategies. CRISPR-Cas systems are prokaryotic adaptive immune systems that rely on RNA-guided binding for the recognition and degradation of invading nucleic acids. Intriguingly, some bacteria also encode divergent CRISPR-Cas systems that can bind to — but cannot degrade — target nucleic acids. In this dissertation, I describe the study of nuclease-deficient CRISPR-Cas systems alongside the evolutionary pressures that led to their persistence in bacterial genomes. I present experimental data for the existence of CRISPR-associated transposons (CASTs) that utilize the RNA-guided DNA binding ability of Type I-F CRISPR-Cas systems to direct transposition to new target sites in a heterologous Escherichia coli host. This RNA-guided DNA integration pathway can tolerate large cargos of up to 10 kilo-base pairs in size, and is highly specific for the programmed target site, as determined by deep sequencing experiments. We further reveal the physical link between the CRISPR-Cas and transposition machineries through biochemical experiments and by determining cryo-EM structures of the transposition protein TniQ in complex with the CRISPR-Cas effector. After bioinformatic analyses and experimental validation we established an array of twenty diverse CAST systems for which a subset works completely orthogonally. This dataset revealed the modular nature of CASTs by showcasing the horizontal acquisition of targeting modules and by characterizing a system that encodes both a programmable, RNA-dependent pathway, and a fixed, RNA-independent pathway. Further analysis of the transposon-encoded cargo genes uncovered the striking presence of anti-phage defense systems, suggesting a role in transmitting innate immunity between bacteria. Finally, we exploit high-throughput screening assays to determine the specific sequence and spacing requirements of the transposon ends, and use this knowledge to develop a CAST-mediated endogenous gene-tagging approach. Intriguingly, our experiments uncover the involvement of a previously unknown cellular protein, integration host factor (IHF), which is critical for transposition of VchCAST, but not other homologous systems. Collectively, the work presented in this dissertation describes the discovery of RNA-guided DNA integration employed by CASTs, substantially advances our biological understanding of these systems, and expands the suite of RNA-guided transposases for programmable, large-scale genome engineering.

Background: MicrO is an ontology of microbiological terms, including prokaryotic qualities and processes, material entities (such as cell components), chemical entities (such as microbiological culture media and medium ingredients), and assays. The ontology was built to support the ongoing development of a natural language processing algorithm, MicroPIE (or, Microbial Phenomics Information Extractor). During the MicroPIE design process, we realized there was a need for a prokaryotic ontology which would capture the evolutionary diversity of phenotypes and metabolic processes across the tree of life, capture the diversity of synonyms and information contained in the taxonomic literature, and relate microbiological entities and processes to terms in a large number of other ontologies, most particularly the Gene Ontology (GO), the Phenotypic Quality Ontology (PATO), and the Chemical Entities of Biological Interest (ChEBI). We thus constructed MicrO to be rich in logical axioms and synonyms gathered from the taxonomic literature. Results: MicrO currently has similar to 14550 classes (similar to 2550 of which are new, the remainder being microbiologically-relevant classes imported from other ontologies), connected by similar to 24,130 logical axioms (5,446 of which are new), and is available at (http://purl.obolibrary.org/obo/MicrO.owl) and on the project website at https://github.com/carrineblank/MicrO. MicrO has been integrated into the OBO Foundry Library (http://www.obofoundry.org/ontology/micro.html), so that other ontologies can borrow and re-use classes. Term requests and user feedback can be made using MicrO's Issue Tracker in GitHub. We designed MicrO such that it can support the ongoing and future development of algorithms that can leverage the controlled vocabulary and logical inference power provided by the ontology. Conclusions: By connecting microbial classes with large numbers of chemical entities, material entities, biological processes, molecular functions, and qualities using a dense array of logical axioms, we intend MicrO to be a powerful new tool to increase the computing power of bioinformatics tools such as the automated text mining of prokaryotic taxonomic descriptions using natural language processing. We also intend MicrO to support the development of new bioinformatics tools that aim to develop new connections between microbial phenotypes and genotypes (i.e., the gene content in genomes). Future ontology development will include incorporation of pathogenic phenotypes and prokaryotic habitats.

Le cycle du carbone est régi principalement par les phénomènes de production et de reminéralisation de la matière organique le long de la colonne d'eau. Les acteurs principaux de cette reminéralisation sont les procaryotes hétérotrophes, dont les actions peuvent être mesurées sur l'ensemble de la colonne d'eau via la respiration procaryotique. Au cours de ce travail de thèse, un suivi à long terme et en temps réel des conditions hydrologiques et biogéochimiques (température potentielle, salinité et oxygène dissous, O2) a été mené entre 2008 et 2010 en Méditerranée Nord Occidentale, sur le site ANTARES. Ces observations ont permis de mettre en évidence les influences d'évènements ponctuels (convection hivernale d'eau profonde) par advection sur ces paramètres hydrologiques et biogéochimiques. Ces influences, directes ou indirectes, vont également avoir des incidences sur la concentration en matière organique et donc sur le potentiel reminéralisateur du milieu profond. Le suivi temporel de la concentration d'O2 a également permis de mettre en évidence une diminution de la concentration globale de 2.6 µmol O2 dm-3 a-1, sur une période de trois ans. Le développement au sein du laboratoire et en collaboration avec le Centre de Physique des Particules de Marseille (CPPM) d'un nouvel outil en équipression, le IODA6000 (In situ Oxygen Dynamics Autosampler), mesurant directement et à haute fréquence la dynamique de l'O2 a permis d'obtenir des vitesses de respiration procaryotique à 2000 m de profondeur depuis décembre 2009 sur le site ANTARES. / The carbon cycle is mostly driven by production and remineralisation processes which are constraining organic matter concentration along the water column. The main actors of the remineralisation are the heterotrophic prokaryotes, which actions can be measured from surface to deep by the prokaryotic respiration. During this PhD thesis, a long term real time monitoring of hydrological and biogeochemical conditions (potential temperature, salinity and dissolved oxygen O2) has been carried out between 2008 and 2010 in the North Occidental Mediterranean Sea, at the ANTARES site. Influence of punctual events has been observed which seem to be related to winter deep sea convection and subsequent advection, changing hydrological and biogeochemical properties observed at the ANTARES site. These direct or indirect modifications will have consequences on the organic matter concentration and therefore on the deep-sea remineralisation potential. The temporal monitoring of O2 concentration has also allow us to estimate the deep water oxygen consumption of 2.6 µmol O2 dm-3 a-1, during a three year period. The development in our laboratory in collaboration with Centre de Physique des Particules de Marseille (CPPM) of a new equipressured tool, the IODA6000 (In situ Oxygen Dynamics Autosampler), measuring directly and at high frequency the O2 concentration, allowed us to measure PR rates at 2000 m depth since December 2009 at the ANTARES site. This unique ongoing time series shows a mean prokaryotic respiration rates higher (0.2 µmol O2 dm-3 d-1) than expected by literature (5.5 10-3 µmol O2 dm-3 d-1), with a high temporal variability (from 8 10-3 to 0.5 µmol O2 dm-3 d-1).

Os fitoplasmas são organismos procariotos sem parede celular, que habitam as células do floema das plantas e são transmitidos, principalmente, por cigarrinhas sugadoras de floema. Este patógeno é responsável por causar doenças em diversas espécies vegetais, provocando a expressão de diversos sintomas, entre os quais podem ser destacados a redução do tamanho foliar, amarelecimento das folhas, superbrotamento de ramos e enfezamento da planta hospedeira. Entre os anos de 2013 e 2014, plantas de três espécies vegetais com sintomas característicos de infecção causada por fitoplasmas foram observadas no estado de São Paulo. O DNA total foi extraído a partir de plantas de couve-flor com sintomas de nanismo, malformação da inflorescência e necrose dos vasos do floema; de plantas da ornamental conhecida por árvore-da-felicidade com sintomas amarelecimento e redução do limbo foliar; e de plantas de Alho-poró com sintomas de enfezamento. Por meio do teste de PCR conduzido em sua maioria com os primers P1A/16S-SR foi possível detectar fitoplasmas nas três espécies vegetais testadas. Os fragmentos genômicos correspondentes ao 16S rRNA dos fitoplasmas foram sequenciados e identificados. Nas plantas de couve-flor e da árvore-da-felicidade foram identificados fitoplasmas afilados ao grupo 16SrVII-B, através da análise virtual de RFLP, cálculo do coeficientes de similaridade (F) e análise filogenética. Para ambas as espécies, este é o primeiro relato da ocorrência de representantes deste grupo, nas condições brasileiras. Nas plantas de Alho-poró foram identificados fitoplasmas afiliados ao subgrupo 16SrIII-J e ao grupo 16SrXIII, com base na análise dos genes 16S rRNA, SecY e proteína ribossomal, conduzidas através de análise virtual de RFLP, valores de coeficientes de similaridade e análise filogenética. Para o fitoplasma membro do grupo 16SrXIII foram identificadas quatro estirpes, uma delas afiliada ao subgrupo 16SrXIII-E e as demais como prováveis representantes de novos subgrupos. O presente estudo se constitui em uma contribuição ao conhecimento de novos patossistemas envolvendo fitoplasmas, bem como à caracterização molecular de fitoplasmas baseadas em diferentes genes marcadores, atualmente usados para a classificação de representantes deste grupo emergente de agentes causais de doenças de plantas. / The phytoplasmas are prokaryotic organisms without cell walls, which inhabit the phloem cells of plants and are transmitted mainly by leafhoppers sucking the phloem. This pathogen is responsible for causing diseases in various plant species, leading to expression of many symptoms, among which can be highlighted the reduction of leaf size, leaf yellowing, shoots proliferation and stunting of the plant host. Between the years 2013 and 2014, three plant species with characteristic symptoms of infection caused by phytoplasma were observed in São Paulo. Total DNA was extracted from cauliflower plants with symptoms of stunting, malformation of the inflorescence and necrosis of the phloem; ornamental plants known for Ming Aralia with symptoms yellowing and little leaves; and leek plants with symptoms of stunting. Through the PCR test conducted mostly with the primers P1A/16S-SR was possible to detect phytoplasmas in the three species of plants. The genomic fragments corresponding of 16S rRNA gene of the phytoplasma were sequenced and identified. In plants of cauliflower and Ming Aralia were identified phytoplasmas belonging to 16SrVII-B group through virtual RFLP analysis, calculation of similarity coefficients (F) and phylogenetic analysis. For both species, this is the first report of the occurrence of representatives of this group, in Brazilian conditions. In leek plants were identified phytoplasmas affiliated with 16SrIII-J subgroup, and the 16SrXIII group, based on the analysis of the 16S rRNA, SecY and ribosomal protein genes, conducted through virtual analysis of RFLP, similarity coefficient values and phylogenetic analysis. For the phytoplasma member of group 16SrXIII were identified four strains, one affiliated with 16SrXIII-E subgroup and the others as probable representatives of new subgroups. This study constitutes a contribution to knowledge of new pathosystems involving phytoplasmas and the molecular characterization of phytoplasma based on different marker genes, currently used for the classification of representatives of this emerging group of plant pathogens.