Made available in DSpace on 2014-06-11T19:27:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-28Bitstream added on 2014-06-13T20:47:49Z : No. of bitstreams: 1 schuch_v_me_jabo.pdf: 3089029 bytes, checksum: 0835ef08e49e97cfdf7ad571bdfc3671 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Metabólitos secundários são compostos bioativos, com grande importância para a indústria farmacêutica e agropecuária, produzidos por certos grupos de microrganismos e plantas. Os policetídeos, que são sintetizados por complexos enzimáticos denominados policetídeos sintases (PKSs), desatacam-se entre os metabólitos secundários conhecidos e compõe a estrutura química básica de vários antibióticos. Todos os genes envolvidos na biossíntese de um policetídeo se encontram agrupados fisicamente no cromossomo, e contém genes que são altamente conservados, comumente chamados d~ pks mínima. Os métodos tradicionais para pesquisa de novas drogas, que envolvem o cultivo de microrganismos isolados do solo, não são mais tão promissores, devido à alta taxa de redescoberta de antibióticos já conhecidos, que chega a 99,9%, e à pequena parcela de microrganismos do solo que são cultiváveis pelas técnicas padrões de cultivo, cerca de 1 %. A Metagenômica é uma abordagem promissora que permite acessar o genoma desses organismos incultiváveis, pois consiste na extração de DNA diretamente do ambiente e construção de uma biblioteca com este genoma misto. Neste trabalho descrevemos a construção de uma biblioteca feita com DNA de alto peso molecular isolado diretamente de solo coletado sob arboreto de eucaliptos no Estado de São Paulo, Brasil. A biblioteca possui 9.320 clones e foi construída em vetor cosmídeo, com insertos de tamanho variando entre 30 e 45kb... / Secondary metabolites are bioactive compounds with great importance in the pharmaceutical and agriculture industries, procuced by a few groups of microrganisms and plants. The polyketides that are synthetized by enzimatic complexes, denominated polyketides synthases, outstand among the secondary known metabolites, which are part of the main structure of many antibiotics. Ali genes involved in the biosynthesis of antibiotics are found as clusters in the chromossome. The traditional methods for the research of new drugs that are made from microrganisms cultures isolated from the soil are not so promissing, due to the high rate of rediscorevy of already known species, reaching 99.9%. The other small piece of microrganisms are culturable by standards culture methods, reaching 1 % maximum. Metagenomics is a promissing approach that allows the access to genom of these organisms that are not culturable, as it is carried out by DNA extraction directly from the environment and construction of a mixed genomic library. In this work, we describe the construction of a library made from high molecular weight DNA isolated directly form the soi! undemeath a pinus forest in the State of São Paulo, Brazil. The library shows 9.320 dones and it was constructed in a cosmideo vector, with insert size ranging from 30 to 45 kb. Digestion with difterent restriction enzymes of cosmidial DNA randomly chosen allowed to visualize evident difterences in the restriction fragments among the clones, as does the possibility to determine the average insert size. The initial evaluation of the presence of genes involved in the biosynthesis of antibiotics synthesized by the enzymatic system PKS of kind I, was accomplished by the PCR amplification of clones from the library using specific primers. We studied 4.320 clones and the results suggest a great variety of these genes. The PCR products obtained were sequenced for the determination of identity of the amplified gene.

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No. of bitstreams: 1 pavani_cd_dr_jabo.pdf: 4981240 bytes, checksum: c45fa10351f84e64eb25cd201dcc69c9 (MD5) Previous issue date: 2017-06-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A comunidade mundial busca pela obtenção de biocombustível celulósico, embora ainda seja um grande desafio a desconstrução do arranjo lignocelulósico para obtenção de açúcares livres de forma eficiente e economicamente viável. A fim de superar tais desafios, avanços conquistados pela metagenômica ressaltam sua aplicação como alternativa para compreender e desvendar o grande potencial metabólico presente nos ambientes, buscando encontrar em tais ambientes novas enzimas que atuem de forma eficiente na desconstrução do material lignocelulósico. Neste trabalho utilizou-se, a abordagem metagenômica para caracterização de genes com potencial para degradar biomassa vegetal e também para avaliação da diversidade taxonômica do ambiente ruminal bovino (Cow_1), bem como comparar os dados coletados com outros metagenomas disponíveis. Amostras de conteúdo ruminal de três bovinos machos da raça Nelore foram coletadas para a obtenção da amostra de DNA metagenômico, que posteriormente foi sequenciada pelo sequenciador HiScan SQ (Illumina). Foram obtidas aproximadamente 63 milhões de sequências (Cow _1), 2 x 100 pb. Dentre os 26 filos encontrados o filo Bacteroidetes foi o mais abundante, seguido de Firmicutes e Proteobacteria. Para a anotação gênica foi utilizado os bancos de dados Pfam, GO, KEGG e CAZy. Foram associadas ORFs relacionadas a 86 famílias de Glycoside Hydrolases (GHs), 52 famílias de Carbohydrate-Binding Modules (CBMs), 4 famílias de Auxiliary Activities (AAs), 16 famílias de Carbohydrate Esterases (CEs), 55 famílias de Glycosyl Transferases (GTs), 16 famílias de Polysaccharide Lyases (PLs), 1 família de S-layer homology (SLH) domain e 1 família de cohesina e dockerina, estes genes estão relacionados a formação do complexo enzimático para desconstrução da biomassa vegetal (celulossoma). O metagenoma da cow_1 analisado apresentou maior quantidade de genes relacionados à desconstrução da biomassa vegetal, quando comparados com outros 11 metagenomas. Além disso, os metagenomas dos ambientes lignocelulósicos apresentaram, principalmente, maior abundância de GH3, GH5, GH8, GH10 e GH11, genes que são importantes na degradação da lignocelulose quando comparados com o grupo de metagenomas não lignocelulósico. Os ambientes lignocelulósicos também revelaram maior abundância de sequências de domínios que se ligam na estrutura da celulose (CBM1, CBM10, CBM11, CBM16, CBM3, CBM63, CBM79 e CBM56), domínios que se ligam na estrutura da hemicelulose (CBM35, CBM61, CBM62 e CBM67) e domínios que atuam na estrutura da celulose e xilana (CBM37, CBM44 e CBM59). O metagenoma ruminal mostrou uma grande diversidade de enzimas responsáveis pela degradação da biomassa vegetal, demonstrando seu potencial como fonte de enzimas com potencial biotecnológico. Os metagenomas lignocelulósicos mostraram uma maior abundância de GH3, GH5, GH8, GH10 e GH11, genes que são importantes na degradação de celulose, celobiose, manana, xilana e xilo-oligossacarídeos quando comparados ao grupo não-lignocelulósico. / The world community search to obtain cellulosic biofuel; however the deconstruction of the lignocellulosic arrangement to obtain free sugars in an efficient and economically viable way is still a great challenge. Advances achieved through the metagenomic emphasize its application as an alternative to unfold the great metabolic potential present in the environments in order to overcome such challenges. Thus, a metagenomic approach was used to characterize genes with potential to degrade plant biomass and to evaluate the taxonomic diversity of the Nelore bovine ruminal environment. Samples were collected from three bovine males to obtain the DNA sample, which were sequentially sequenced by the HiScan SQ (Illumina) sequencer. About 63 million sequences, 2 x 100 bp, were obtained. Among the 26 phyla found, Bacteroidetes was the most abundant, followed by Firmicutes and Proteobacteria. For gene annotation was used the Pfam, GO, KEGG and CAZy databases. A total of 86 families of Glycoside Hydrolases (GHs), 52 families of Carbohydrate Binding Modules (CBMs), 4 families of Auxiliary Activities (AAs), 16 Esterase Carbohydrate (CEs), 55 GlycosylTransferases (GTs) Polysaccharide Lyases (PLs), 1 S-homology homology (SLH) domain and 1 family of cohesin and dockerine was associated. these genes are related to the formation of the enzymatic complex for deconstruction of plant biomass (cellulosome). The cow_1 metagenome presented a greater number of genes related to deconstruction of plant biomass when compared to other 11 metagenomes. In addition, the metagenomes of the lignocellulosic environments presented, mainly, greater abundance of GH3, GH5, GH8, GH10 and GH11, genes that are important in the degradation of lignocellulose when compared with the group of non-lignocellulosic metagenomes. Focusing on the CBMs, the metagenomes of the lignocellulosic group showed a greater abundance of CBM1, CBM10, CBM11, CBM16, CBM3, CBM63, CBM79 and CBM56 that bind in the cellulose structure; CBM35, CBM61, CBM62 and CBM67 which bind to the hemicellulose structure; CBM37, CBM44 and CBM59 that act on the structure of cellulose and xylan. The ruminal metagenome showed a great diversity of enzymes responsible for the degradation of the plant biomass, demonstrating its potential as a source of enzymes with biotechnological potential. Lignocellulosic metagenomes showed a greater abundance of GH3, GH5, GH8, GH10 and GH11, genes that are important in the degradation of cellulose, cellobiose, mannan, xylan and xylo-oligosaccharides when compared to the non-lignocellulosic group.

Este trabalho teve como objetivo analisar, através de uma abordagem metagenômica/computacional, a variação do conteúdo gênico e do potencial metabólico das comunidades microbianas associadas a dois ambientes da Fundação Parque Zoológico de São Paulo: um reservatório artificial, o Lago São Francisco e o sistema de compostagem de resíduos do parque. Para o estudo do primeiro ambiente, amostras foram coletadas mensalmente no Lago São Francisco de outubro de 2012 a setembro de 2013 e submetidas ao sequenciamento metagenômico. Esse estudo mostrou que agrupamentos de amostras de uma mesma estação são estatisticamente suportados. As coocorrências de espécies, com suporte estatístico alto, foram estabelecidas e representadas em uma rede separada em 60 grupos ou módulos. A maioria dos grandes módulos foram formados quase exclusivamente por espécies do mesmo filo, indicando possíveis mecanismos de resposta a fatores ambientais e à presença de nutrientes ao invés da pura interação entre espécies. Os fatores que levaram à variação dos táxons também influenciaram o potencial metabólico da comunidade: embora os módulos metabólicos de forma geral estivessem distribuídos ao longo dos meses, alguns se destacaram pela variação intensa, principalmente na amostra de novembro de 2012. Para o estudo do segundo ambiente, foram analisados dois conjuntos de dados de sequenciamento metagenômico gerados a partir de amostras seriadas coletadas ao longo do processo de compostagem. Nos dias iniciais do processo de compostagem, os mais discrepantes, houve uma super-representação de módulos metabólicos relacionados principalmente ao metabolismo de carboidratos e à síntese de aminoácidos. Em conjunto, os dados obtidos nesse estudo indicaram uma comunidade microbiana com potencial metabólico variando direcionalmente em função dos compostos inicialmente presentes ou oriundos do substrato, em função da presença de oxigênio e em função da etapa do processo. / This project aimed to analyze, through a computational approach, the change of gene content and metabolic potential in metagenomes from two microbiomes from São Paulo Zoo Park Foundation: an artificial reservoir, Lake São Francisco, and the compost systems in the park. For the study on the first microbiome, samples were monthly collected in Lake São Francisco from October 2012 to September 2013 and submitted to metagenomic sequencing. This study showed that clustering of the samples from a same season is statistically supported. The species co-occorrences, with high statistical support, were established and represented in a network composed by 60 groups or modules. The most biggest modules were formed almost exclusively by species of the same phylum, pointing possible response mechanisms to environmental factors and to presence of nutrients instead of the simple species interaction. The factors which leaded to taxa variation also influenced the metabolic potential of the community: although the metabolic modules generally are spreaded through the months, some highlighted because the intense variation, mainly in the sample from November 2012. For the latter environment, two metagenomic datasets were analyzed from serial samples collected throughout the composting process. In the initial days of composting process, the most discrepant, there was an uprepresentation of metabolic modules related to carbohydrates metabolism and amino acids synthesis. All data in this study indicated a microbial community with metabolic potential varying according to nutrientes, oxygen presence and process stage.

Orientador: Laura Maria Mariscal Ottoboni / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T16:24:46Z (GMT). No. of bitstreams: 1 Carlos_Camila_D.pdf: 3834562 bytes, checksum: ae067ac8bf3731e5d1ab6af674e63c67 (MD5) Previous issue date: 2014 / Resumo: Os recifes de corais são ecossistemas sensíveis que estão ameaçados pelas mudanças climáticas. Estudos têm demonstrado a importância da microbiota associada aos corais na resistência às doenças e aos estresses. Neste trabalho, a caracterização taxonômica e funcional da microbiota associada a corais encontrados no litoral de São Paulo permitiu a identificação de associações espécie-específicas entre corais e bactérias e a identificação de funções bacterianas responsáveis pelo estabelecimento de associações coral-bactéria. A composição taxonômica associada a ambientes coralíneos (muco, água e sedimento do entorno) de quatro espécies de corais encontradas no litoral de São Paulo foi avaliada por pirosequenciamento do gene de rRNA 16S. Os resultados indicam que a comunidade microbiana do muco não é apenas distinta do ambiente do entorno, todavia é, também, mais estável ao longo das estações do ano. A composição taxonômica da microbiota do coral mole Palythoa caribaeorum foi bastante distinta das demais espécies, pertencentes estas à ordem Scleractinia, indicando a influência das relações filogenéticas na moldagem das comunidades microbianas associadas aos corais. O metagenoma de Madracis decactis e da espécie brasileira endêmica Mussismilia hispida foi sequenciado por pirosequenciamento. A maior parte das sequências obtidas não pôde ser anotada, o que pode indicar a abundância de micro-organismos, especialmente vírus, ainda não estudados. Entre as sequências anotadas, foi observada uma grande abundância de sequências de origem viral em ambas as bibliotecas. Os metagenomas de M. hispida e M. decactis foram comparados a outros metagenomas disponíveis no banco de dados do MG-RAST e foi possível identificar genes ou funções mais abundantes nessas bibliotecas, como genes de resistência a antibióticos da classe dos aminoglicosídeos, que podem estar sujeitos à transferência horizontal nesse ambiente. Por último, foi sequenciado o genoma de uma bactéria isolada de muco de M. hispida, Paracoccus sp. SM22M-07. A análise comparativa desse genoma com outros genomas do gênero Paracoccus revelou funções únicas do isolado de muco de coral, por exemplo, genes do sistema de secreção do tipo IV podem exercer a função de contribuir com o estabelecimento de interações bactéria-coral. Todos os resultados aqui apresentados e analisados apontam para uma grande diversidade tanto taxonômica quanto funcional da comunidade bacteriana associada aos corais brasileiros. A estabilidade sazonal dessas comunidades é uma propriedade importante que contribui para a prevenção da colonização de bactérias patogênicas. Os resultados aqui apresentados representam um extenso inventário da diversidade microbiana em um ecossistema ameaçado pelas mudanças climáticas globais e permitirão futuros estudos comparativos e a criação de modelos e estimativas das taxas de redução da diversidade microbiana em ambientes marinhos / Abstract: Coral reefs are one of the ecosystems most threatened by global climate changes. Studies have shown the importance of the coral microbiota in stress and disease resistance. In this work, the taxonomic and functional characterization of the bacterial communities associated with corals from the coast of São Paulo State, Brazil, allowed the identification of species-specific interactions between corals and bacteria and the identification of the bacterial functions responsible for the establishment of these interactions. The taxonomic composition of mucus, water and surrounding sediment of four coral species found in the coast of Sao Paulo State was assessed by 16S rDNA pyrosequencing of metagenomic DNA. The microbial communities found in samples of mucus, water, and sediment differed according to the taxonomic composition, and the coral mucus community seemed to be more stable to seasonal changes. The taxonomic composition of the microbiota of the soft coral Palythoa caribaeorum was distinct from the other species belonging to the order Scleractinia, indicating the influence of phylogenetic relationships in shaping the microbial communities associated with the coral. The metagenome of Madracis decactis and the Brazilian endemic species Mussismilia hispida was sequenced by pyrosequencing. Most of the sequences obtained could not be annotated, which might indicate an abundance of unknown microorganisms, especially viruses. Among the annotated sequences, an abundance of viral sequences in both libraries was observed. The metagenomas M. decactis and M. hispida were compared with metagenomes datasets available in the MG-RAST database and through these comparisons, the most abundant genes or functions of the corals¿ metagenomes were identified, for example, genes for resistance to antibiotics of the aminoglycoside class. Finally, the genome of an isolate of mucus of M. hispida, Paracoccus sp . SM22M - 07 was sequenced. The comparative analysis of this genome with other genomes of the genus Paracoccus revealed unique functions of the bacteria isolated from coral mucus, such as genes of the type IV secretion system, which may contribute to the establishment of coral-bacteria interactions. All results presented and analyzed in this work show a great diversity, both taxonomic and functional, of the bacterial community associated with Brazilian corals. The seasonal stability of these communities is an important property that contributes to preventing the colonization by pathogenic bacteria. The results presented here represent an extensive inventory of microbial diversity in an ecosystem threatened by global climate change and will allow future comparisons, modeling and estimates of the rates of reduction of microbial diversity in marine environments / Doutorado / Genetica de Microorganismos / Doutora em Genética e Biologia Molecular

Orientador: Valeria Maia Merzel / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T20:30:53Z (GMT). No. of bitstreams: 1 Tomazetto_Geizecler_D.pdf: 2728814 bytes, checksum: 5ca8845e4db01805f12ccff354b0a0b0 (MD5) Previous issue date: 2013 / Resumo: O hidrogênio é apontado como o candidato mais promissor para substituição do combustível fóssil devido a sua maior eficiência na conversão de energia útil e ausência de emissão de substâncias tóxicas. A produção de hidrogênio a partir de resíduos orgânicos é realizada por meio de digestão anaeróbica, tornando-se uma alternativa ecologicamente correta para atender à futura demanda por hidrogênio. No entanto, os micro-organismos e os processos metabólicos envolvidos estão longe de serem exaustivamente caracterizados. Nesse trabalho, amostras de uma planta de tratamento de esgoto doméstico foram analisadas em dois estudos complementares visando à caracterização de sua diversidade filogenética e a descrição de novas hidrogenases. O primeiro trabalho combinou a análise dos genes de RNAr 16S e FeFehidrogenase (hydA) com ferramentas estatísticas para estimar a riqueza e diversidade da comunidade procariótica em nível filogenético e funcional. As análises filogenéticas e de diversidade das bibliotecas gênicas demonstraram que todas as sequências de arquéias foram afiliadas a Euryarchaeota não cultivadas e, com relação ao Dominio Bactéria, Proteobacteria foi grupo filogenético predominante apresentando os maiores índices de diversidade e riqueza. As sequências putativas de hydA foram identificadas como sequências de genes de FeFehidrogenases ainda não descritas. Na segunda abordagem, a biblioteca metagenômica de fosmideo construída nesse estudo foi analisada empregando a tecnologia de pirosequenciamento 454 e resultou em aproximadamente 218 Mb de dados. Os três diferentes classificadores aplicados permitiram uma visão geral dos grupos taxonômicos mais abundantes devido ao enorme número de sequências metagenômicas não classificadas. Contudo, análises taxonômicas revelaram Gammaproteobacteria e Deltaproteobacteria, respectivamente, como as classes taxonômicas predominantes, enquanto que as espécies do gênero Methanospirillum foram dominantes entre as arquéias metanogênicas. A análise do metabolismo da comunidade microbiana através das bases de dados COG e Carma revelou que a degradação da biomassa depende de diferentes grupos filogenéticos, como por exemplo, Bacteroidia e Gammaproteobacteria, os quais foram indicados como envolvidos na degradação de carboidratos e proteínas, respectivamente. Além disso, as análises sugerem Clostridia e Methanomicrobiales e Methanosarcinales como principais micro-organismos produtores de hidrogênio e metano, respectivamente. As análises das seis sequências codificantes de FeFehidrogenase identificadas no conjunto de dados metagenômicos revelaram que essas representam novas sequências do gene alvo. Contudo, quatro dessas sequências foram identificadas na biblioteca de fosmídeo pela triagem gênica baseada no uso de PCR. O conjunto de resultados obtido nesse estudo permitiu elucidar a composição e o potencial metabólico dos micro-organismos residentes na planta de tratamento de esgoto analisada e sugere esse ambiente como um reservatório potencial de novos genes de hidrogenases para a exploração biotecnológica / Abstract: Hydrogen appears to be the most promising candidate for the replacement of fossil fuel due to its potentially higher efficiency of conversion to usable power and no toxic emission production. The production of hydrogen from organic wastes is performed through the anaerobic digestion, making it an environmentally friendly alternative for satisfying future hydrogen demands. Nonetheless, the microorganisms and metabolic processes involved are far from being exhaustively characterized. In this work, samples of a domestic sewage treatment plant were analyzed in two complementary studies aiming at the characterization of its phylogenetic diversity and the description of new hydrogenases. The first one, combined the analysis of 16S rRNA and [FeFe]-hydrogenase (hydA) genes with statistical tools to estimate richness and diversity of the prokaryotic community at the phylogenetic and functional levels. Phylogenetic analysis showed that all archaeal sequences were affiliated with yet uncultured Euryarchaeota and that Proteobacteria were the most predominant and diversified phylogenetic group within the bacterial library. The putative hydA sequences were identified as hitherto undetected [Fe-Fe]- hydrogenase gene sequences. Diversity statistical analysis confirmed a great richness and diversity of bacterial and hydA sequences retrieved from the sewage sludge sample. In the second approach, a fosmid metagenomic library was constructed and analyzed employing 454- pyrosequencing technology, resulting in approximately 218 Mb of data. Three different classifiers applied allowed a broad overview of the most abundant taxonomic groups due to a huge number of metagenome reads remained unidentified. However, taxonomic analysis revealed Gammaproteobacteria and Deltaproteobacteria, respectively, as the most abundant classes, whereas species of the genus Methanospirillum were dominant among methanogenic Archaea. The analyzes of the microbial community metabolism by means of COG and Carma databases revealed that the degradation of biomass depends on different phylogenetic groups, for instance, Bacteroidia and Gammaproteobacteria were indicated as involved into the degradation of carbohydrate and proteins. Furthermore, the analysis suggested Clostridia and Methanomicrobiales and Methanosarcinales as the main microorganisms producing hydrogen and methane, respectively. Analysis of the six coding sequences of FeFe-hydrogenases identified into the dataset revealed that they represented novel target gene sequences. However, only four of these coding sequences could be detected into the fosmid library by PCR screening. The combined results obtained in this study allowed us to have an insight of the composition and potential metabolism of the microbes residing in the analyzed domestic sewage treatment plant and suggested such environment as a potential reservoir for new hydrogenase genes to biotechnological exploration / Doutorado / Genetica de Microorganismos / Doutora em Genética e Biologia Molecular

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22477号 / 農博第2381号 / 新制||農||1074(附属図書館) / 学位論文||R2||N5257(農学部図書室) / 京都大学大学院農学研究科森林科学専攻 / (主査)准教授 舘野 隆之輔, 教授 北島 薫, 教授 德地 直子 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The discovery of large DNA viruses has challenged the traditional perception of viral complexity due to their enormous genome size and physical dimensions. Previously, viruses were considered small, filterable agents until the discovery of large DNA viruses. Among large DNA viruses, the phylum Nucleocytoviricota and its members, which are often called "giant viruses" have large genome sizes (up to 2.5 Mbp) and virion sizes (up to 1.5 um). Due to having large virion and genome sizes, these viruses were often excluded from viral surveys and remained understudied for years. Luckily, the advancement of metagenomic analysis has facilitated the study of large DNA viruses by analyzing them directly from their environment without cultivating them in the lab, which could be challenging for viruses. In the first chapter of the thesis, I investigated 11 metagenome-assembled genomes (MAGs) of giant viruses previously surveyed from Station ALOHA in the Pacific Ocean. St. ALOHA is located near Hawaii and represents oligotrophic gyres which the majority of the ocean is made of them. I focused on 11 MAGs of giant viruses to get insight into their phylogenetic characteristics, genomic repertoire, and global distribution patterns. Despite the fact that metagenomic analysis has facilitated the study of genetic materials of microbes and viruses on a huge scale, it is essential to benchmark the performance of metagenomic tools and understand the associated biases, particularly in viral metagenomics. In the second chapter, I evaluated the performance of metagenomic tools (contigs assembler and binning tool) in recovering viral genomes using annotated dataset. We used a metagenome simulator (CAMISIM) to generate simulated short reads with known composition to assess these processes. Moreover, I emphasized the importance of binning contigs for viral genomes to fully recover the genomes of viruses along with discussing how diversity metrics were differed for contigs, bins populations. / Master of Science / Viruses are generally thought to be small biological agents with small genome (genetic material) sizes and tiny physical structures; for instance, the genome length of a Human Immunodeficiency Virus (HIV) is around 10 kilobase pair (a unit for measuring genetic material in an organism), and the virion size (physical dimension of a virus) can go up to 120 nm. The discovery of large DNA viruses has challenged the idea of considering viruses as small biological entities, as their genome sizes and physical dimensions can be up to 2.5 megabase pairs and 1500 nm, respectively. Famous members of large DNA viruses from the phylum Nucleocytoviricota are often known as "Giant Viruses'' because they have enormous genome sizes and physical dimensions. Due to having large viral particles, these viruses may usually be excluded from viral surveys. For instance, in field studies, samples must be filtered through a fraction (e.g., 0.2 um) to eliminate bacterial and archaeal genomes and cellular debris, which also results in excluding larger viruses. Since these viruses remain understudied for several years because of biases associated with having large viral particles, there is a solid need to discover and investigate more about them. Growing and cultivating viruses in the laboratory may be challenging, as they need specific hosts to be dependent on to produce more viral progeny and some specific laboratory environments. Luckily, with the advancement of biotechnology, scientists could find ways to evade the need for cultivating viruses in the lab and study them with computational tools such as metagenomic analysis and bioinformatic tools. Metagenomics analysis helps to study the genetic materials of microbial or viral populations directly from their habitat without growing them in a laboratory. In short, metagenomic analysis has multiple steps, including collecting and filtering samples, fragmenting DNA within the samples, generating short DNA sequences (short-read sequences) with NGS (Next Generation Sequencing) technology, assembling short-read sequences into large DNA fragments which can be contigs (contiguous DNA fragments) and metagenome-assembled genome (MAGs). With metagenomic analysis, we can recover the genome of multiple organisms, and we name the recovered genome as metagenome-assembled genome (MAGs) as it is generated through metagenomic processes. The metagenomic analysis will allow us to study microbes and viruses in their environment and gain insight into their taxonomic details, genomic content, and how widespread they are. In the first chapter, I studied 11 MAGs of giant viruses previously surveyed from St. ALOHA, Hawaii. St. ALOHA is a good field site for examining microbial processes and diversity and a good representative of oligotrophic waters (low in nutrients). I examined 11 MAGs of giant viruses to investigate their taxonomic characteristics to clarify which order they belong to within their phylum, their genomic content, and their global distribution pattern. Although studies have successfully recovered the genome of large DNA viruses from their habitats and then analyzed them, all these metagenomic processes need to be evaluated so the results will be valid to consider as the genome of our interested organisms. In the second chapter, I developed a workflow for viral metagenomic analysis to assess metagenomic tools' performance in recovering reliable viral genomes, particularly for large DNA viruses. Most of these benchmarking workflows are done for bacterial and archaeal genomes, and in this thesis, I used these metagenomic tools and applied them to recover large DNA viruses genomes. Also, I emphasized the importance of using binning tools to fully recover large DNA viruses genomes, as due to their large genome size, their genomes might remain fragmented into different contigs, which are longer sequences than reads but shorter than MAGs.

CRISPR-Cas is the state-of-the-art biological tool that allows precise and fast manipulation of the genetic information of cellular genomes. The translation of the CRISPR-Cas technology from in vitro studies into clinical applications highlighted a variety of limitations: the currently available systems are limited by their off-target activity, the availability of a Cas-specific PAM sequence next to the target and the size of the Cas protein. In particular, despite high levels of activity, the size of the CRISPR-SpCas9 editing machinery is not compatible with an all-in-one AAV delivery system and the genomic sequences that can be targeted are limited by the 3-NGG PAM-dependency of the SpCas9 protein. To further expand the CRISPR tools repertoire we turned to metagenomic data of the human microbiome to search for uncharacterized CRISPR-Cas9 systems and we identified a set of novel small Cas9 orthologs derived from the analysis of reconstructed bacterial metagenomes. In this thesis study, ten candidates were chosen according to their size (less than 1100aa). The PAM preference of all the ten orthologs was established exploiting a bacterial-based and an in vitro platform. We demonstrated that three of them are active nucleases in human cells and two out of the three showed robust editing levels at endogenous loci, outperforming SpCas9 at particular targets. We expect these new variants to be very useful in expanding the available genome editing tools both in vitro and in vivo. Knock-out-based Cas9 applications are very efficient but many times a precise control of the repair outcome through HDR-mediated gene targeting is required. To address this issue, we also developed an MS2-based reporter platform to measure the frequency of HDR events and evaluate novel HDR-modulating factors. The platform was validated and could allow the screening of libraries of proteins to assess their influence on the HDR pathway.

No description available.

Soil microbial communities mediate important ecological processes and play essential roles in biogeochemical cycling. Ecosystem disturbances such as surface mining significantly alter soil microbial communities, which could lead to changes or impairment of ecosystem functions. Reforestation procedures were designed to accelerate the reestablishment of plant community and the recovery of the forest ecosystem after reclamation. However, the microbial recovery during reforestation has not been well studied even though this information is essential for evaluating ecosystem restoration success. In addition, the similar starting conditions of mining sites of different ages facilitate a chronosequence approach for studying decades-long microbial community change, which could help generalize theories about ecosystem succession. In this study, the recovery of microbial communities in a chronosequence of reclaimed mine sites spanning 30 years post reforestation along with unmined reference sites was analyzed using next-generation sequencing to characterize soil-microbial abundance, richness, taxonomic composition, interaction patterns and functional genes. Generally, microbial succession followed a trajectory along the chronosequence age, with communities becoming more similar to reference sites with increasing age. However, two major branches of soil microbiota, bacteria and fungi, showed some contrasting dynamics during ecosystem recovery, which are likely related to the difference in their growth rates, tolerance to environmental change and relationships with plants. For example, bacterial communities displayed more intra-annual variability and more complex co-occurrence networks than did fungi. A transition from copiotrophs to oligotrophs during succession, suggested by taxonomic composition shifts, indicated that the nutrient availability is one important factor driving microbial succession. This theory was also supported by metagenomic analysis of the functional genes. For example, the increased abundance of genes involved in virulence, defense and stress response along ages indicated increased competition between microorganisms, which is likely related to a decrease of available nutrients. Metagenomic analysis also revealed that lower relative abundances of methanotrophs and methane monooxygenase at previously-mined sites compared with unmined sites, which supports previous observations that ecological function of methane sink provided by many forest soils has not recovered after 30 years. Because of the difficulty identifying in situ functional mechanisms that link soil microorganisms with environmental change, modeling can be a valuable tool to infer those relationships of microbial communities. However, the extremely high richness of soil microbial communities can result in extremely complicated models that are difficult to interpret. Furthermore, uncertainty about the coherence of ecological function at high microbial taxonomic levels, grouping operational taxonomic units (OTUs) based on phylogenetic linkages can mask trends and relationships of some important OTUs. To investigate other ways to simplify soil microbiome data for modeling, I used co-occurrence patterns of bacterial OTUs to construct functional groups. The resulting groups performed better at characterizing age-related microbial community dynamics and predicted community structures and environmental factors with lower error. / PHD