Em concentrações subletais, agentes antimicrobianos modulam a expressão gênica bacteriana, sendo que o conjunto de genes que é modulado depende tanto da cepa bacteriana, como da natureza do agente antimicrobiano. Neste trabalho, avaliamos o perfil de expressão gênica de Xylella fastidiosa cepa 9a5c em resposta ao tratamento por até 60 minutos com dose subletal do antibiótico estreptomicina. Esta é uma cepa virulenta, originalmente isolada de laranjeiras com sintomas de clorose variegada dos citros. A hibridização de microarranjos de DNA representando 2608 das 2848 sequências codificadoras (CDS) previamente anotadas no genoma desta cepa, revelou que 136 CDS apresentaram expressão gênica diferencial em resposta à exposição à estreptomicina, sendo que destas 109 foram negativamente moduladas e 27 positivamente moduladas. Realizamos, também, ensaios de PCR quantitativo precedido de transcrição reversa (RTqPCR) de 21 CDS para confirmar a modulação observada na análise global da expressão gênica. O perfil de expressão gênica de X. fastidiosa em resposta à estreptomicina foi analisado de forma integrada com outros perfis de expressão gênica desta bactéria. Entre as CDS positivamente moduladas, destacamos aquelas codificadoras das chaperoninas GroEL e GroES, que estão associadas a resposta de choque térmico, e CDS associadas à tradução, tais como proteínas ribossomais e fatores de tradução. Interessantemente, a exposição à estreptomicina induz a expressão da CDS que codifica poligalacturonase, que é um fator de virulência em algumas cepas de X. fastidiosa. Por outro lado, o tratamento com estreptomicina promoveu a modulação negativa de CDS relacionadas à formação e manutenção de biofilme ao contrário do observado quando estas bactérias foram submetidas ao tratamento com gomesina, um peptídeo antimicrobiano. O conjunto destas observações sugere que a exposição à dose subletal de estreptomicina possa promover um fenótipo de maior virulência, contrariamente ao efeito previamente observado com a gomesina. Neste trabalho, também descrevemos o pirosequenciamento do genoma da cepa J1a12 de Xylella fastidiosa, que exibe fenótipo menos virulento em citros e tabaco em relação à cepa 9a5c. A comparação da sequência genômica destas duas cepas confirma diferenças anteriormente observadas utilizando-se microarranjos de DNA e destaca genes potencialmente importantes para virulência de Xylella fastidiosa. / At sublethal concentrations, antimicrobials compounds modulate bacterial gene expression and the gene set that is modulated depends not only on the bacterial strain but also on the nature of antimicrobial agent. In this study, we evaluated changes in gene expression profile of Xylella fastidiosa strain 9a5c exposed up to 60 min to sublethal concentration of streptomycin. This a virulent strain originally isolated from orange trees with symptoms of citrus variegated chlorosis. Hybridization of DNA microarrays representing 2,608 out of 2848 coding sequences (CDS) previously annotated in strain 9a5c genome revealed 136 CDS differentially expressed upon streptomycin treatment. Of which 109 were down-regulated and 27 up-regulated. Differential expression for a subset of 21 CDS was further evaluated by reverse transcriptionquantitative PCR (RT-qPCR). In addition, we performed an integrated analysis of the gene expression profile of X. fastidiosa in response to streptomycin along with other gene expression profiles available for this bacterium. Among the up-regulated CDS, we highlight those encoding chaperonins GroEL and GroES, which are associated with heat shock response, and those CDS related to translation, such as ribosomal proteins and translation factors. Interestingly, exposure to streptomycin induces the expression of a CDS encoding polygalacturonase, which is a virulence factor for some X. fastidiosa strains. Furthermore, treatment with streptomycin down-regulates some CDS related to biofilm formation oppositely to treatment with gomesin, an antimicrobial peptide. Together, these observations suggest that exposure to sublethal dose of streptomycin might promote a higher virulent phenotype, in contrast to the effect previously observed with gomesin. In the present work, we also describe the pyrosequencing of J1a12 genome, a X. fastidiosa strain that exhibits a less virulent phenotype in citrus and tobacco if compared to strain 9a5c. A comparison of genome sequences of these two strains confirms differences previously observed using DNA microarrays and highlights important genes for virulence of X. fastidiosa.

O gênero Xylella é composto de uma única espécie, Xylella fastidiosa, bactéria Gram-negativa, não flagelada, que coloniza o xilema de uma diversidade de plantas cultivadas e silvestres em várias partes do mundo. Em algumas dessas plantas, a bactéria é considerada agente causal de doenças, como a Clorose Variegada do Citros em laranjeiras, a Doença de Pierce das videiras e escaldadura da folha de cafeeiro. Onze diferentes cepas de X. fastidiosa, isoladas de distintos hospedeiros, já tiveram seus genomas sequenciados, entre essas, as cepas 9a5c, isolada de laranjeira, e Temecula 1, isolada de videira. Análises desses genomas indicam uma razoável variabilidade entre suas respectivas sequências e evidenciam vários genes associados a mecanismos de virulência e patogenicidade desta bactéria. No presente trabalho descrevemos o sequenciamento, a montagem e a anotação dos genomas das cepas U24d e Fb7, isoladas de laranjeiras, e da cepa 3124 isolada de cafeeiro, os quais apresentam, respectivamente 2.681.334 pb, 2.733.974 pb e 2.748.594 pb. Destas, apenas a cepa U24d apresenta um plasmídeo, o qual é idêntico ao pXF51 previamente identificado na cepa 9a5c. O genoma da cepa U24d é praticamente colinear ao genoma da cepa 9a5c enquanto que os genomas das cepas Fb7 e 3124 apresentaram maior colinearidade com a cepa Temecula1. Entre as diversas alterações encontradas nas análises comparativas destes genomas, destacamos a inserção no gene pilQ verificada no genoma da cepa U24d. Essa mutação causa ausência do pilus do tipo IV com consequente deficiência na motilidade twitching, sendo que plantas infectadas com a cepa U24d apresentam sintomas localizados restritos ao ponto de inoculação. Na cepa Fb7, detectamos a ausência de formação de biofilme no cultivo in vitro possivelmente devido ausência da expressão dos transcritos de mrkD e pspA, que codificam respectivamente adesina do pilus curto e adesina similar à hemaglutinina. Postulamos que estes genes não são expressos em decorrência de um defeito na via de sinalização de DSF (Fator de Sinalização Difusível) reflexo de uma mutação em rpfC no genoma de Fb7. Assim como as demais cepas de X. fastidiosa, também os genomas de U24d, Fb7 e 3124 apresentaram elevado conteúdo de Elementos Genéticos Móveis (EGM), que aparecem em maior número nas cepas sul-americanas. Os estudos do pangenoma de X. fastidiosa mostraram que essa espécie tem um genoma aberto e grande parte dos genes de EGMs correspondem a genes acessórios. A grande quantidade de EGMs em X. fastidiosa pode estar relacionada a falta do sistema CRISPR/cas completo, um provável resultado de eventos de erosão do genoma desta espécie. A inferência filogenética por MSLA mostrou uma clara distinção dos grupos de cepas da América do Norte em relação às do Sul, sugerindo a ocorrência de mais eventos de recombinações genéticas nas cepas sul-americanas, provavelmente pela falta de isolamento geográfico. Assim, é possível que as cepas norte e sul-americanas sofreram divergência alopátrica e simpátrica, respectivamente. / The genus Xylella consists of a single species, Xylella fastidiosa, a Gram-negative and non-flagellated bacterium that colonizes the xylem of a diversity of cultivated and wild plants in several parts of the world. In some of these plants, this bacterium is considered causal agent of diseases such as the Citrus Variegated Cholorosis in orange trees, Pierce\'s Disease of grapevines and coffee leaf scald. Eleven different strains of X. fastidiosa isolated from different hosts had their genomes sequenced, including 9a5c and Temecula1 strains, respectively isolated from orange tree and grapevine. Analyses of these genomes indicate a reasonable variability in their sequences and showed several genes associated with pathogenicity and virulence mechanisms of this bacterium. In this work we describe the genome sequencing, assembly and annotation of the strains U24d and Fb7, isolated from orange trees, and 3124 isolated from coffee, which have, respectively, 2,681,334 bp, 2,733,974 bp and 2,748,594 bp. Of these, only strain U24d has a plasmid, identical to pXF51 from strain 9a5c. The genome of U24d strain is almost collinear to the genome of strain 9a5c while the genomes of strains Fb7 and 3124 had higher collinearity to Temecula1 strain. Among many changes found in the comparative analysis of these genomes, we highlight an on insertion in pilQ gene that was found in U24d strain genome. This mutation causes lack of type IV pilus with a consequent deficiency in twitching motility. Moreover orange trees infected with U24d strain showed localized symptoms near to the inoculation point. We verified that Fb7 strain does not form biofilm in vitro possibly due to the absence of expression of mrkD and pspA transcripts, which encode, respectively, a short pilus adhesin and a hemagglutinin-like adhesin. We postulate that these genes are not expressed due to a defect in the signaling pathway of DSF (Diffusible Signal Factor) reflecting a mutation on rpfC in the Fb7 genome. Similarly to other X. fastidiosa strains, the genomes of U24d, Fb7 and 3124 also showed high content of mobile genetic elements (MGE), which appear in larger numbers in South American strains. Pan genome studies of X. fastidiosa showed that this species has a open genome and that most of MGE genes correspond to accessory genes. The large number of MGE in X. fastidiosa may be related to the lack of a complete system CRISPR/cas, likely a result of erosion events of the genome of this species. The phylogenetic reconstruction by MLSA showed a clear distinction between groups of strains from North and South America, suggesting the occurrence of more recombination events in South American strains, probably due to lack of geographical isolation. Thus it is possible that North and South American strains underwent allopatric and sympatric divergence, respectively.

Xylella fastidiosa é uma bactéria Gram-negativa, do subgrupo das Gama-Proteobactérias, não-flagelada, que coloniza o xilema de diversas plantas cultivadas e silvestres, podendo ser causadora de doenças. Sua disseminação é feita por insetos conhecidos como cigarrinhas. Genomas de cepas de X. fastidiosa isoladas de distintos hospedeiros já foram sequenciados completa ou parcialmente: 9a5c de citros; Temecula-1 e GB514 de videira; Dixon, M12 e M23 de amendoeira; Ann-1 de espirradeira e EB92-1, isolada de sabugueiro e utilizada como bio-controle para Doença de Pierce de videiras. Estudos de genômica comparativa associados a abordagens de genômica funcional e de genética molecular têm possibilitado o estudo detalhado de mecanismos potencialmente relevantes tanto para a colonização de plantas e insetos por este fitopatógeno como para o desenvolvimento de sintomas associados a doenças específicas em seus respectivos hospedeiros vegetais. Exceto o genoma de 9a5c, todos os demais genomas conhecidos são de cepas isoladas na América do Norte. Neste trabalho descrevemos o pirossequenciamento dos genomas da cepa J1a12, que exibe fenótipo não-virulento em citros, e das cepas Pr8x e Hib4, isoladas, respectivamente, de ameixeira e hibisco. A cepa J1a12 possui além de seu cromossomo principal de 2.788.789 pb dois plasmídeos, pXF51 e pXF27, respectivamente de 51.180 pb e 27.268 pb. pXF51 já foi descrito também na cepa de citros 9a5c e pXF27 tem similaridade com outros plasmídeos de cepas de X. fastidiosa norte-americanas isoladas de amoreira e videira. A cepa Pr8x possui além de seu cromossomo principal de 2.666.242 pb um plasmídeo, pXF39, de 39.580 pb, o qual contém a maioria das CDS presentes no pXF51. A cepa Hib4, isolada de hibisco, tem o maior cromossomo (2.813.297 pb) e também o maior plasmídeo (pXF64 com 64.251 pb) já descritos para X. fastidiosa. pXF64 apresenta extensa similaridade com o plasmídeo pBVIE04 de Burkholderia vietnamensis cepa G4, sendo descrito pela primeira vez em cepas de X. fastidiosa. Análises comparativas destes genomas possibilitaram a identificação de alterações que podem ser correlacionadas com os fenótipos exibidos por estas cepas, além da variedade e diversidade de regiões relacionadas a bacteriófagos e de plasmídeos que co-existem nas diferentes cepas deste fitopatógeno. / Xylella fastidiosa is a Gram-negative bacteria, of the Gamma-proteobacterium subgroup, non-flagellated that colonizes the xylem of several cultivated and wild plants, where may cause disease. The bacterium is spread by insects known as sharpshooters. Genomes of X. fastidiosa strains isolated from different hosts have been completely or partially sequenced: 9a5c from citrus; Temecula-1 and GB514 from grapevine; Dixon, M12 and M23 from almond tree; Ann-1 from oleander and EB92-1, isolated from elderberry and used as bio-control for Pierce\'s disease of grapevines. Comparative genomics studies associated with approaches from functional genomics and molecular genetics have allowed a detailed study of mechanisms potentially relevant to the colonization of plants and insects by this pathogen as well as to the development of symptoms associated with specific diseases in their respective host plants. Except for 9a5c, all other known genomes are from strains isolated in North America. Here we describe the pyrosequencing of the genomes of strain J1a12, which displays non-virulent phenotype in citrus and of Pr8x and Hib4 strains isolated, respectively, from plum and hibiscus. J1a12 has a main chromosome of 2,788,789 bp and two plasmids, pXF51 and pXF27, respectively of 51,180 bp and 27,268 bp. pXF51 has been described also in the citrus strain 9a5c and pXF27 has similarity with other plasmids found in North American strains isolated from mulberry tree and grapevine. The strain Pr8x has a main chromosome of 2,666,242 bp and one plasmid, pXF39, of 39,580 bp which present similarities with pXF51. Hib4, the strain isolated from hibiscus, has the largest chromosome (2,813,297 bp) and the largest plasmid (pXF64 with 64,251 bp) described for X. fastidiosa. pXF64 shows extensive similarity with the plasmid pBVIE04 of Burkholderia vietnamensis G4 strain and is described for the first time in X. fastidiosa. Comparative analyzes of these genomes have identified several differences that may be correlated with the phenotypes displayed by these strains, in addition to the variety and diversity of regions related to bacteriophages and plasmids that co-exist in different strains of this pathogen.

Xanthomonas albilineans est la bactérie responsable de l'échaudure des feuilles, une maladie vasculaire et létale de la canne à sucre. Cette plante, domestiquée depuis plus de 5000 ans, est aujourd'hui cultivée sur près de 26 millions d'hectares. Depuis le début du 20ème siècle, les variétés naturelles de canne à sucre ont été remplacées par des variétés hybrides issues de croisements artificiels et présentant des caractères agronomiques plus intéressants. La transmission de X. albilineans est essentiellement liée au mode de multiplication par bouturage de la canne à sucre, les boutures produites à partir d'un plant infecté ayant de fortes chances d'être contaminées. L'utilisation d'outils de coupe favorise également la transmission de la bactérie à partir d'un plant infecté. Une transmission aérienne a toutefois été décrite pour au moins un groupe génétique de souches de X. albilineans appelé PFGE-B et caractérisé à l'aide de la technique d'électrophorèse en champ pulsé. Mon sujet de thèse visait, par une étude de génomique comparative et évolutive sur plusieurs souches de X. albilineans, à, d'une part, comprendre l'histoire de l'association de cette bactérie avec la canne à sucre, et, d'autre part, identifier des gènes candidats qui pourraient expliquer la pathogénie de X. albilineans et la capacité des souches du groupe PFGE-B à être transmises par voie aérienne.En comparant 12 souches de X. albilineans appartenant à neuf groupes PFGE différents, j’ai construit une phylogénie solide proposant l'existence de huit lignées regroupées en deux clades. Cette phylogénie a été confirmée en utilisant trois méthodes de phylogénomique différentes. De façon intéressante, cette phylogénie permet de proposer des hypothèses sur l'évolution de X. albilineans et sur l'introduction de cette bactérie chez la canne à sucre. L'analyse des gènes présents dans le génome de ces 12 souches appartenant à neuf groupes PFGE différents m'a également permis d'identifier un système RM (restriction/modification) qui n'est présent que chez les souches PFGE-B et qui pourrait expliquer pourquoi ces souches sont plus résistantes aux phages que celles appartenant aux autres groupes PFGE. En facilitant la survie des bactéries dans l'environnement, cette résistance aux phages pourrait jouer un rôle important dans la transmission aérienne.La comparaison de 11 souches de X. albilineans appartenant au groupe PFGE-B m'a permis de construire, à partir de 67 SNP (Single Nucleotide Polymorphism), une phylogénie qui indique que les Caraïbes, où la canne à sucre n'a été introduite qu'à la fin du 15ème siècle, pourraient être un centre de dispersion des souches PFGE-B. Cette étude a également mis en évidence un polymorphisme au niveau des spacers d'un locus CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats system). L'étude de ce polymorphisme sur 18 souches de X. albilineans non séquencées a permis de démontrer que les souches de Floride sont originaires des Caraïbes et de proposer des hypothèses concernant l'origine des souches PFGE-B d’autres régions du monde, même si le séquençage d'un plus grand nombre de souches sera nécessaire pour confirmer ces hypothèses.En analysant la séquence du génome de deux souches qui avaient été initialement classées dans l'espèce X. albilineans mais qui n'avaient pas été isolées à partir de tiges de canne à sucre, j’ai mis en évidence une nouvelle espèce que nous avons appelée 'Xanthomonas pseudalbilineans'. Enfin, la comparaison du génome fini et annoté de la souche PFGE-B GPE PC73 avec le génome d'autres espèces bactériennes phytopathogènes m'a permis de proposer des hypothèses pour expliquer la pathogénie de X. albilineans. / Xanthomonas albilineans is the bacterium causing leaf scald, a vascular disease being lethal for sugarcane. This plant, domesticated for over 5,000 years, is nowadays grown across almost 26 million hectares. Since the beginning of 20th century, natural varieties of sugarcane have been replaced by hybrid varieties resulting from artificial crossings. The transmission of X. albilineans is mainly linked to the fact that sugarcane is propagated by taking cuttings, with the cuttings from an infected plant likely to be contaminated. The use of cutting instruments also facilitates transmission of the bacteria from an infected plant. An aerial transmission has nevertheless been described, at least for one genetic group of strains from X. albilineans, namely PFGE-B, characterized using the pulsed-field gel electrophoresis technique. My thesis topic consisted in performing a comparative and evolutive genomic study of several strains of X. albilineans in order, on one hand, to better understand the history of association of this bacterium with the sugarcane, and, on the other hand, to identify candidate genes which may explain the pathogenicity of X. albilineans as well as the ability of PFGE-B strains to be aerially transmitted. Comparing 12 strains of X. albilineans belonging to nine different PFGE groups, I built a robust phylogeny which proposes the existence of eight lineages clustered in two clades. This phylogeny has been confirmed by three different phylogenomic methods. Interestingly, this phylogeny allows to propose hypotheses about the evolution of X. albilineans and the introduction of this bacterium in sugarcane. The analysis of genes from the genomes of these 12 strains belonging to nine different PFGE groups also allowed me to identify a RM (Restriction/Modification) system which is present only in PFGE-B strains and which may explain why these strains are more resistant to phages that those belonging to other PFGE groups. By facilitating the survival of bacteria in the environment, this resistance to phages may play an important role in aerial transmission. The comparison of 11 strains of X. albilineans belonging to the PFGE-B group allowed me to build, based on 67 SNPs (Single Nucleotide Polymorphism), a phylogeny which indicates that the Caribbean area, where sugarcane was introduced not before the end of the 15th century, might be a center for dispersion of PFGE-B strains. This study also revealed a polymorphism of spacers of a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats system) locus. The study of this polymorphism in 18 non-sequenced strains of X. albilineans allowed to demonstrate that strains from Florida originated in the Caribbean region, and to propose hypotheses regarding the origin of PFGE-B strains from other regions of the world, even if the sequencing of a higher number of strains will be required to confirm these hypotheses.Analyzing the sequence of the genome of two strains, initially thought to belong to the X. albilineans species but which weren’t isolated from sugarcane stalks, I revealed a new species that we called ‘Xanthomonas pseudalbilineans’. Finally, the comparison of the finished and annotated genome of the PFGE-B strain GPE PC73 with the genome of other phytopathogenic bacteria allowed me to propose hypotheses to explain X. albilineans’ pathogenicity.

>Magister Scientiae - MSc / Tsetse flies (Glossina spp) are vectors of African trypanosome (Trypanosoma spp) parasites, causative agents of Human African trypanosomiasis (sleeping sickness) and Nagana in livestock. Research suggests that tsetse fly immunity factors are key determinants in the success and failure of infection and the maturation process of parasites. An analysis of tsetse fly immunity factors is limited by the paucity of genomic data for Glossina spp. Nevertheless, completely sequenced and assembled genomes of Drosophila melanogaster, Anopheles gambiae and Aedes aegypti provide an opportunity to characterize protein families in species such as Glossina by using a comparative genomics approach. In this study we characterize thioester-containing proteins (TEPs), a sub-family of immunity-related proteins, in Glossina by leveraging the EST data for G.morsitans and the genomic resources of D. melanogaster, A. gambiae as well as A.aegypti.A total of 17 TEPs corresponding to Drosophila (four TEPs), Anopheles (eleven TEPs) and Aedes aegypti (two TEPs) were collected from published data supplemented with Genbank searches. In the absence of genome data for G. morsitans, 124 000 G.morsitans ESTs were clustered and assembled into 18 413 transcripts (contigs and singletons). Five Glossina contigs (Gmcn1115, Gmcn1116, Gmcn2398, Gmcn2281 and Gmcn4297) were identified as putative TEPs by BLAST searches. Phylogenetic analyses were conducted to determine the relationship of collected TEP proteins.Gmcn1115 clustered with DmtepI and DmtepII while Gmcn2398 is placed in a separate branch, suggesting that it is specific to G. morsitans.The TEPs are highly conserved within D. melanogaster as reflected in the conservation of the thioester domain, while only two and one TEPs in A. gambiae and A. aegypti thioester domain show conservation of the thioester domain suggesting that these proteins are subjected to high levels of selection. Despite the absence of a sequenced genome for G. morsitans, at least two putative TEPs where identified from EST data.

No description available.

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Non-coding RNAs (ncRNAs) are transcripts that function directly as RNA molecule without ever being translated to protein. The transcriptional output of eukaryotic cells is diverse, pervasive, and multi-layered. It consists of spliced as well as unspliced transcripts of both protein-coding messenger RNAs and functional ncRNAs. However, it also contains degradable non-functional by-products and artefacts - certainly a reason why ncRNAs have long been wrongly disposed as transcriptional noise. Today, RNA-controlled regulatory processes are broadly recognized for a variety of ncRNA classes. The thermoresponsive ROSE ncRNA (repression of heat shock gene expression) is only one example of a regulatory ncRNA acting at the post-transcriptional level via conformational changes of its secondary structure. Bioinformatics helps to identify novel ncRNAs in the bulk of genomic and transcriptomic sequence data which are produced at ever increasing rates. However, ncRNA annotation is unfortunately not part of generic genome annotation pipelines. Dedicated computational searches for particular ncRNAs are veritable research projects in their own right. Despite best efforts, ncRNAs across the animal phylogeny remain to a large extent uncharted territory. This thesis describes a comprehensive collection of exploratory bioinformatic field studies designed to de novo predict ncRNA genes in a series of computational screens and in a multitude of newly sequenced genomes. Non-coding RNAs can be divided into subclasses (families) according to peculiar functional, structural, or compositional similarities. A simple but eligible and frequently applied criterion to classify RNA species is length. In line, the thesis is structured into two parts: We present a series of pilot-studies investigating (1) the short and (2) the long ncRNA repertoire of several model species by means of state-of-the-art bioinformatic techniques. In the first part of the thesis, we focus on the detection of short ncRNAs exhibiting thermodynamically stable and evolutionary conserved secondary structures. We provide evidence for the presence of short structured ncRNAs in a variety of different species, ranging from bacteria to insects and higher eukaryotes. In particular, we highlight drawbacks and opportunities of RNAz-based ncRNA prediction at several hitherto scarcely investigated scenarios, as for example ncRNA prediction in the light of whole genome duplications. A recent microarray study provides experimental evidence for our approach. Differential expression of at least one-sixth of our drosophilid RNAz predictions has been reported. Beyond the means of RNAz, we moreover manually compile sophisticated annotation of short ncRNAs in schistosomes. Obviously, accumulating knowledge about the genetic material of malaria causing parasites which infect millions of humans world-wide is of utmost scientific interest. Since the performance of any comparative genomics approach is limited by the quality of its input alignments, we introduce a novel light-weight and performant genome-wide alignment approach: NcDNAlign. Although the tool is optimized for speed rather than sensitivity and requires only a minor fraction of CPU time compared to existing programs, we demonstrate that it is basically as sensitive and specific as competing approaches when applied to genome-wide ncRNA gene finding and analysis of ultra-conserved regions. By design, however, prediction approaches that search for regions with an excess of mutations that maintain secondary structure motifs will miss ncRNAs that are unstructured or whose structure is not well conserved in evolution. In the second part of the thesis, we therefore overcome secondary structure prediction and, based on splice site detection, develop novel strategies specifically designed to identify long ncRNAs in genomic sequences - probably the open problem in current RNA research. We perform splice site anchored gene-finding in drosophilids, nematodes, and vertebrate genomes and, at least for a subset of obtained candidate genes, provide experimental evidence for expression and the existence of novel spliced transcripts undoubtedly confirming our approach. In summary, we found evidence for a large number of previously undescribed RNAs which consolidates the idea of non-coding RNAs as an abundant class of regulatory active transcripts. Certainly, ncRNA prediction is a complex task. This thesis, however, rationally advises how to unveil the RNA complement of newly sequenced genomes. Since our results have already established both subsequent computational as well as experimental studies, we believe to have enduringly stimulated the field of RNA research and to have contributed to an enriched view on the subject.

<p>  </p> <p><em>Fusobacterium necrophorum</em> is a Gram-negative, anaerobic, opportunistic pathogen that causes necrotic infections in cattle leading to liver abscess, foot rot, and calf diphtheria. Particularly, liver abscess in cattle is reported at 20.7% annually, and leads to liver condemnation and an annual economic burden of about 62 million dollars to the feedlot industry. Antibiotic administration is the mainstay of treating these infections, but antibiotic resistance is unavoidable and demand for antibiotic-free, natural, and organic beef has demanded alternative therapies and preventatives. Vaccination is one of the best alternatives to prophylactic antibiotic administration. In this study, we have explored outer membrane proteins (OMPs) and outer membrane vesicles (OMVs) for potential vaccine candidates. OMPs and OMVs are vaccine targets because of their antigenic properties and host specificity. Additionally, we performed comparative genomic analysis of <em>F. necrophorum</em> species to identify additional virulence genes with vaccine potential, unique to the <em>F. necrophorum</em> and its virulent subspecies <em>necrophorum</em>. </p> <p>Protein- protein interaction investigation through binding assay and pulldown assay identified novel OMPs, namely 17kDa, 22kDa, and 66.3 kDa proteins, which were further characterized as OmpH, OmpA and Cell Surface Protein (CSP), respectively. In this study, these novel OMPs including previously characterized 43kDa OMPs were cloned, and recombinant proteins were expressed and purified. These recombinant proteins were used to generate polyclonal antibodies in rabbits, and their efficacy was studied using <em>in vitro</em> adhesion inhibition assays. The combination of two or more antibodies raised against the recombinant OMPs was significantly effective in reducing/neutralizing bacterial binding to bovine endothelial cells compared to individual antibody treatment. This suggests that a multiple subunit vaccine could be effective and provide sufficient evidence to perform <em>in vivo</em> studies. </p> <p>Similarly, we purified OMVs of <em>F. necrophorum</em> subspecies <em>necrophorum</em> 8L1 and analyzed its content using proteomics and lipidomics. Out of 342 proteins identified by tandem liquid chromatography mass spectrometry (LC-MS), OMPs and toxins were the most abundant. These included OMPs and toxins namely, 43 kDa OMP, OmpH, OmpA, CSP, FadA, leukotoxin family filamentous adhesin, N-terminal domain of hemagglutinin and other OMP transport and assembly factor protein. The presence of a subset of these proteins was further confirmed by western blot analysis. Lipidomics analysis showed that OMVs contained phospholipid, sphingolipid, and acetyl carnitine as the main lipid contents. Cytotoxicity assay on BL-3 cell line showed that these OMVs have a toxic effect on host immune cells and could impart immunomodulatory effect. All these findings suggest the vaccine potential of OMVs and demand dose-based <em>in vivo</em> study.</p> <p>In addition, we identified and characterized 5 clinical isolates of <em>F. necrophorum</em> using comparative genomics, UBCG (Up-to-date Bacterial Core Gene) based analysis enabled phylogenetic characterization of 46 <em>F. necrophorum</em> genomes into subspecies specific clades. The pangenome and recombination analysis showed the extensive disparity in accessory genes resulting in species divergence. Strikingly, we detected antimicrobial resistance gene for macrolides and tetracycline in one strain of <em>F. necrophorum</em>, a harbinger of the start of resistance and necessitating search for an alternative prophylactic method. We also noted common virulence genes, including toxins, outer membrane adhesion proteins, cell envelope, type IV secretion system, ABC (ATP-binding cassette) transporters and transporter proteins in <em>F. necrophorum</em> strains. A focused study on these genes could help identify the main genes of virulence and inform effective vaccination strategies against fusobacterial infections. </p> <p>Overall, the studies suggest adhesins and toxin and/or OMV-based subunit vaccine could be potential targets for vaccine development against fusobacterial infections.  </p>

Streptococcus agalactiae is a pathogen of multiple hosts. The bacterium, an aetiological agent of septicaemia and meningo-encephalitis in freshwater and saltwater fish species, is considered a major threat to the aquaculture industry, particularly for tilapia. Cattle and humans are however the main known reservoirs for S. agalactiae. In humans, the bacterium (commonly referred to as Group B Streptococcus or GBS) is a member of the commensal microflora of the intestinal and genito-urinary tracts, but it is also a major cause of neonatal invasive disease and an emerging pathogen in adults. In cattle, S. agalactiae is a well-recognized causative agent of mastitis. Numerous studies focusing on S. agalactiae from human and bovine origins have provided insight into the population structure of the bacterium, as well as the genome content and pathogenic mechanisms through identification of virulence determinants. Concerning S. agalactiae from aquatic origins, scientific information mainly focused on case reporting and/or experimental challenges, with a limited or absence of information in terms of pathogenesis, virulence determinants and genotypes of the strains involved. The objective of this study was to enhance our understanding of the molecular epidemiology, host-adaptation and pathogenicity of S. agalactiae in aquatic species, with particular emphasis on tilapia. Firstly, a collection of 33 piscine, amphibian and sea mammal isolates originating from several countries and continents was assembled, with the aim of exploring the population structure and potential host specificity of aquatic S. agalactiae. Isolates were characterised using pulsed-field gel electrophoresis (PFGE), multi-locus sequence typing (MLST), and a standardised 3-set genotyping system comprising molecular serotypes, surface protein gene profiles and mobile genetic element profiles. Two major subpopulations were identified in fish. The first subpopulation consisted of non-haemolytic isolates that belonged to sequence type (ST) 260 or 261, which are STs that have been reported only from teleosts. These isolates exhibited a low level of genetic diversity by PFGE and clustered with other STs that have been reported only in fish. Another common feature was the absence of all surface protein genes or mobile genetic elements targeted as part of the 3-set genotyping and that are usually found in human or bovine isolates. The second subpopulation consisted of β-haemolytic isolates recovered from fish, frogs and sea mammals, and that exhibited medium to high genetic diversity by PFGE. STs identified among these isolates have previously been identified from strains associated with asymptomatic carriage and invasive disease in humans. The human pathogenic strain ST7 serotype Ia was detected in fish from Asia. Moreover, ST283 serotype III-4 and its novel single locus variant ST491 detected in fish from Southeast Asia shared a 3-set genotype identical to that of an emerging ST283 clone associated with invasive disease of adult humans in Asia. These observations suggested that some strains of aquatic S. agalactiae may present a zoonotic or anthroponotic hazard. STs found among the seal isolates (ST23) have also been reported from humans and numerous other host species, but never from teleosts. This work provided an excellent basis for exploration of the virulence of selected strains in experimental challenges. The virulence of two strains of S. agalactiae was experimentally investigated by intra-peritoneal infection of Nile tilapia (Oreochromis niloticus), using an isolate originally recovered from fish and belonging to ST260, and an isolate originating from a grey seal and belonging to ST23. The clinical signs, the in vivo distribution of viable bacteria and bacterial antigens, and the gross and histopathological lesions that developed during the time course of the infection were investigated. The ST260 strain was highly virulent, whereas no major clinical sign or mortalities occurred in the fish challenged with the ST23 strain. After injection, both strains however gained access to the bloodstream and viable bacteria were recovered from all organs under investigation. During the early stages of infection, bacteria were mostly found within the reticulo-endothelial system of the spleen and kidney. Thereafter, the ST260 demonstrated a particular tropism for the brain and the heart, but granulomatous inflammation and associated necrotic lesions were observed in all organs. ST23 was responsible for a mixed inflammatory response associated with the presence of bacteria in the choroid rete and in the pancreatic tissue only. After 7 days post-challenge and for both strain, the formation or containment of bacteria within granulomata or other encapsulated structures appeared to be a major component of the fish response. However, the load of viable bacteria remained high within organs of fish infected with ST260, suggesting that, unlike ST23, this strain is able to survive within macrophages and/or to evade the immune system of the fish. This work demonstrates that the lack of report of ST23 strains in fish is possibly not due to a lack of exposure but to a lack of virulence in this host. The two strains, which differ in prevalence and virulence in fish, provide an excellent basis to investigate genomic differences underlying the host-association of distinct S. agalactiae subpopulations. The genome of the ST260 strain used in challenge studies was sequenced. We therefore provided the first description for the genome sequence of a non-haemolytic S. agalactiae isolated from tilapia (strain STIR-CD-17) and that belongs by multi-locus sequence typing (MLST) to clonal complex (CC) 552, which corresponds to a presumptive fish-adapted subgroup of S. agalactiae. The genome was compared to 13 S. agalactiae genomes of human (n=7), bovine (n=2), fish (n=3) and unknown (n=1) origins. Phylogenetic analysis based on the core genome identified isolates of CC552 as the most diverged of all S. agalactiae studied. Conversely, genomes from β-haemolytic isolates of CC7 recovered from fish were found to cluster with human isolates of CC7, further supporting the possibility that some strains may represent a zoonotic or anthroponotic hazard. Comparative analysis of the accessory genome enabled the identification of a cluster of genes uniquely shared between CC7 and CC552, which encode proteins that may provide enhanced fitness in specific niches. Other genes identified were specific to STIR-CD-17 or to CC552 based on genomic comparisons; however the extension of this analysis through the PCR screening of a larger population of S. agalactiae suggested that some of these genes may occasionally be present in isolates belonging to CC7. Some of these genes, occurring in clusters, exhibited typical signatures of mobile genetic elements, suggesting their acquisition through horizontal gene transfer. It is not possible to date to determine whether these genes were acquired through intraspecies transfer or through interspecies transfer from the aquatic environment. Finally, general features of STIR-CD-17 highlighted a distinctive genome characterised by an absence of well conserved insertion sequences, an abundance of pseudogenes, a smaller genomic size than normally observed among human or bovine S. agalactiae, and an apparent loss of metabolic functions considered conserved within the bacterial species, indicating that the fish-adapted subgroup of isolates (CC552) has undergone niche restriction. Finally, genes encoding recognised virulence factors in human S. agalactiae were selected and their presence and structural conservation was evaluated within the genome of STIR-CD-17.