Upon recognition of the pathogen, plants initiate defense responses that can include the production of antimicrobial compounds such as phytoalexins. Nectria haematococca mating population VI (MPVI) is a filamentous ascomycete that contains a cluster of genes known as the pea pathogenicity (PEP) cluster in which the pisatin demethylase (PDA) gene resides. PDA is responsible for the detoxification of the phytoalexin, pisatin, produced by the pea plant (Pisum sativum). Thus, PDA allows the fungus to colonize and become a pea pathogen. It has been proposed that the evolutionary origin of PDA and the PEP cluster is explained by a horizontal gene transfer (HGT) event. The observations supporting this hypothesis include the location of the PEP cluster on a conditionally dispensable (CD) chromosome, the phylogenetically discontinuous distribution of the cluster among closely related species, and bias in GC content and codon usage. This study used a three-pronged approach to test the hypothesis that PDA and the PEP cluster were inherited via HGT. Percent identities of conserved genes, along with GC content analysis and phylogenetics support vertical inheritance.

Horizontal gene transfer (HGT) and gene loss are important processes in the evolution of prokaryotic lineages. HGT involves the movement of genetic material between distantly related species, and can facilitate adaptation when gained genes confer advantageous phenotypes to recipient lineages. However, high levels of gene gain and loss are predicted to obfuscate patterns of vertical descent and homogenise nucleotide diversity across ecological and phylogenetic boundaries. Thus, a holistic understanding of the role of genome fluctuation in the emergence and maintenance of genetically and ecologically cohesive bacterial groups remains to be fully elucidated. In this thesis, I use the plant-associated bacterium Pseudomonas syringae as a model system to investigate the impact of HGT and gene loss on evolutionary processes such as adaptation, diversification and speciation. The Gram-negative Gammaproteobacterium P. syringae is an opportunistic plant pathogen, and has been used for decades as a model system with which to study the interaction between plants and their microbial pathogens. Recently, the diversification of lineages within this species has involved a number of host jumps onto a range of woody host plant species, resulting in the emergence of diseases such as bacterial canker of kiwi and bleeding canker of the European horse chestnut. Using whole-genome sequence data and a range of comparative genomics and phylogenetics methods, I quantitatively reconstruct the history of gene gain and loss in P. syringae and show HGT to be the predominant evolutionary force in this species. Genomes of this species are under constant permutation, are subject to a highly diverse HGT genepool and show marked differences in patterns of codon usage between imported and core genes. I then generate additional genome data for 26 strains of P. syringae that are pathogenic on a range of different woody plants, and investigate the contribution from HGT to the adaptation of these strains into the woody niche. Using a method that accounts for the underlying phylogenetic relationships among P. syringae strains, I look for the correlated evolution between gained genes and the woody niche, and find that a substantial proportion of the genome is associated with this ecological niche. I then investigate the recent adapitation of P. syringae pv. aesculi onto the European horse chestnut, and show that a number of genomic events that include both homologous and non-homologous recombination are likely to have led to the evolution of this bacterium onto its host, where it has become the causal agent of the bleeding canker disease that is currently epidemic across much of northern and central Europe. Overall, this thesis is an investigation into how HGT contributes to niche adaptation in P. syringae, and aims to further our understanding of the mechanisms that underlie bacterial evolution.

Horizontal gene transfer (HGT) is one of the man driving forces of evolution in prokaryotes, and can also promote within-strain variation of bacterial species. The genomes of three previously sequenced Bacteroides fragilis strains, NCTC9343, 638R and YCH46 displayed evidence of extensive HGT, demonstrated by the presence of 28 divergent capsular polysaccharide-associated biosynthesis loci. The genomes of a further four B. fragilis strains, LS66, GNAB92, RD48 and BE1 were sequenced and analysed. Genomic comparisons of BE1 and GNAB92 with NCTC9343, 638R and YCH46 identified ten new divergent polysaccharide biosynthesis loci. There is consequently, the potential to express 38 different polysaccharides amongst these five strains. Such a high level of variation in capsular polysaccharides, in so few strains has not been previously observed. HGT has occurred in B. fragilis despite the presence of diverse Restriction-Modification systems. The genome sequences of NCTC9343 and 638R contained a gene, ubb, the product of which, BfUbb, has 63% identity to human ubiquitin. The closest DNA sequence homology is to a migratory grasshopper entomopox virus, suggesting acquisition of this gene was via inter-kingdom HGT. The ubb gene was also identified in the newly sequenced genomes of B. fragilis strains LS66 and RD48. BfUbb had a predicted signal sequence; both full-length and processed forms were detected in whole-cell extracts by Western blot analysis. The inability to detect BfUbb in periplasmic extracts isolated from a B. fragilis strain containing an ubb signal sequence deletion construct, supported the periplasmic location of the processed form of the protein and the requirement for the signal peptide for transport from the cytoplasm. BfUbb was also detected in concentrated supernatants containing outer membrane vesicles, suggesting a mechanism by which the protein may be delivered to the host. This is the first example of ubiquitin being produced by a prokaryote. Transduction by bacteriophages is one mechanism by which horizontal gene transfer can occur and can also be a useful tool for genetic manipulation. Fifteen potentially new B. fragilis-specific bacteriophages were isolated from filtered sewage and characterised by phage titres and restriction endonuclease cleavage profiles. Of the fifteen, seven phages appeared to be different to the previously identified phage ФED01. None of the bacteriophages were capable of transduction. B. fragilis is a predominant member of the gastrointestinal microbiota. To survive within this specific niche, bacteria must successfully compete with other organisms for nutrients and space, and withstand attacks from bacteriophages. HGT may aid in the survival of B. fragilis as a commensal.

Geotrichum candidum est une levure hémiascomycète ubiquitaire longtemps considérée comme un champignon filamenteux. C’est l’une des levures les plus fréquemment trouvées dans les fromages dans les quelles elle contribue à l’affinage. Dans le cadre du projet ANR ALIA Food Microbiomes en partenariat avec des industriels fromagers et producteur de levain, nous avons caractérisé l’espèce G. candidum par une étude phylogénétique et placé de manière non ambigüe G. candidum parmi les levures hémiascomètes. Une analyse MLST a permis de séparer les souches étudiées en deux groupes. Le premier contient essentiellement des souches environnementales tandis que le second ne contient que des souches isolé du fromage. Cela suggère une certaine sélection ou spécialisation d’un groupe de souche dans la fabrication du fromage. Une méthode de typage inter LTR plus discriminante a permis de typer l’ensemble des souches et peut fournir aux industriels un outil robuste pour le suivi d’une souche en production. Le génome de G. candidum CLIB 918 = ATCC 204307 a été séquencé. Les premières analyses ont mis en évidence des discontinuités évolutives parmi les gènes qui le composent. Parmi les 6802 gènes identifiés, 315 gènes présentent des orthologues chez les champignons filamenteux et non chez les levures. Cela suggère que durant l’évolution, G. candidum a conservé un grand nombre de gènes qui a été perdu chez les autres levures ou en a reçu certain par transfert horizontal de gènes. L’existence de ce même type de gènes chez d’autres levures ayant une position basale dans l’arbre des hémiascomycètes, suggère que G. candidum et ces levures ont une position intermédiaire lors de la transition évolutive champignon vers levure. Il est à noter que certains d’entre eux sont impliqués dans le métabolisme et pourraient jouer un rôle dans l’adaptation de cette levure à la fabrication du fromage. / Geotrichum candidum is a hemiascomycetous yeast frequently found in the environment and foodstuffs. It is one of the main yeasts in cheese and it is widely used as adjunct culture in the maturation of cheese. Within ANR project ALIA Food Microbiomes in partnership with industry, we characterized the species the species G. candidum by a multigene phylogenetic study. MLST analysis allowed us to separate the studied strains into two groups. The first contains mainly environmental strains while the second contains only strains isolated from cheese. This suggests a specialization or a selection of a group of strains within industry. We developed a typing method by inter LTR profiles, which can provide a robust tool for an industrial monitoring of strains. The genome of G. candidum CLIB 918 = ATCC 204307 was sequenced. Preliminary analyses revealed evolutionary discontinuities among genes. 6802 genes where identified in which 315 genes have orthologs in filamentous fungi and not in yeast. This suggests that during evolution, G. candidum has retained a large number of genes which have been lost in other yeasts or has received some by horizontal gene transfer. The existence of this other yeasts also having a basal position in hemiascomycetous tree suggests that G. candidum and these other yeasts have an intermediate position during the evolutionary transition fungus to yeast. It is noteworthy that some of them are involved in the metabolism and may play a role in the adaptation of the yeast to the cheese environment.

Petroleum hydrocarbons are the most widespread contaminants in the environment. Interest in the biodegradation of polycyclic aromatic hydrocarbons and compounds (PAHs/PACs) is motivated by their ubiquitous distribution, their low bioavailability, high persistence in soils and their potentially deleterious effects to human health. Identifying the diversity of microorganisms that degrade PAHs/PACs can be utilised in the development of bioremediation techniques. Understanding the mechanisms of bacterial populations to adapt to the presence of pollutants and the extent that lateral transfer of key functional genes occurs, will allow the exploitation of microbial PAC/PAH-degradative capabilities and therefore enhance the successful application of bioremediation strategies. A key aim of this study was to isolate and identify PAC-degrading bacteria for potential use in future bioremediation programmes. A series of PAC enrichments were established under the same experimental conditions from a single sediment sample taken from a highly polluted estuarine site. Distinct microbial community shifts were directly attributable to enrichment with different PAC substrates. The findings of this study demonstrate that five divisions of the Proteobacteria and Actinobacteria can degrade PACs. By determining the precise identity of the PAC-degrading bacteria isolated, and by comparing these with previously published research, this study showed how bacteria with similar PAC degrading capabilities and 16S rRNA signatures are found in similarly polluted environments in geographically very distant locations e.g. China, Italy, Japan and Hawaii. Such a finding suggests that geographical barriers do not limit the distribution of key PAC-degrading bacteria. This is significant when considering the diversity and global distribution of microbes with PAC-degradative capabilities and the potential for utilising these microbial populations in future bioremediation strategies. In the laboratory, enrichment of bacteria able to utilise PAHs has commonly been performed in liquid media, with the PAH dissolved in a carrier solvent. This study found the presence of a carrier solvent significantly affects the resultant microbial population. Although the same sediment sample was used as the bacterial source in all enrichments, different bacterial strains were obtained depending upon the presence of the carrier solvent and the PAH. This is important when considering appropriate methodology for the isolation of PAH-degrading bacteria for future bioremediation programmes. Additionally, the species comprising the resultant population of the enrichment when a carrier solvent was present were similar to previously reported PAH-degrading species. Such a finding necessitates review of previously reported PAH-degrading bacterial species that have been isolated and identified from enrichments using a carrier solvent. Understanding how bacteria acclimatise to environmental pollutants is vital for exploiting these mechanisms within clear up strategies of contaminated sites. Two major lineages of the α subunit of PAH dioxygenases were identified: Actinobacteria and Proteobacteria. Comparison of the α subunit phylogeny with the 16S rRNA phylogeny implies that the PAH-dioxygenases evolved prior to the separation of these phyla or that lateral transfer occurred in the very distant past. No evidence for lateral transfer of the α subunit between the Actinobacteria and Proteobacteria was found in the phylogenetic analyses of this research. Multiple lateral transfer events were inferred between the species of the Actinobacteria and between the classes of the Proteobacteria. The clustering of the taxa within the α subunit phylogeny indicates that lateral transfer of the α subunit gene occurred after the separation of the classes of Proteobacteria and also after the speciation of the γ-Proteobacteria. These findings reveal how bacteria have acclimatised to PAH pollutants through multiple lateral transfer events of a key PAH-degradative gene. This knowledge of the transfer of genetic material will broaden our prospects of exploiting microbial populations.

Os manguezais são ecossistemas costeiros de transição formados entre o ambiente marinho e terrestre de zonas tropicais e intertropicais, e estão sujeitos ao regime de marés. Devido a tais condições, os microrganismos que vivem nos sedimentos sofrem constante pressão adaptativa. Nesse contexto, os Elementos Genéticos Móveis (EGMs) (fagos, plasmídeos e transposons) são fatores genéticos diretamente relacionados com a transferência horizontal de genes e podem facilitar processos de adaptação. Neste trabalho foram estudados os EGMs dos sedimentos de três áreas de manguezais do estado de São Paulo, duas localizadas no município de Bertioga e uma na reserva ambiental da Ilha do Cardoso, em Cananeia. Foram avaliadas a frequência e expressão dos diferentes tipos de EGMs no microbioma dos manguezais a partir de metagenomas e metatranscriptomas dos sedimentos, além da utilização de uma biblioteca de fosmídeos, montada a partir de DNA do sedimento de uma das áreas de Bertioga. As sequências de DNA e mRNA foram obtidas de três pontos amostrados por área, sequenciadas em equipamento Illumina HiSeq2000 e anotadas automaticamente na plataforma do MG-RAST. Para a biblioteca de fosmídeo, foi sequenciado um pool de todos os clones em equipamento Illumina HiSeq 2000. Contigs dessas sequências foram montados e fagos e profagos foram preditos e anotados automaticamente pelo pipeline VirSorter. As sequências preditas como fagos ou profagos foram anotadas manualmente. A partir da análise das sequências de metagenômica foi possível verificar que a participação dos EGMs no metabolismo total do microbioma dos manguezais corresponde de 6 a 15 % dos genes preditos, sendo que fagos e profagos correspondem a 90% dos genes associados aos EGMs. A partir dos contigs montados para as sequências da biblioteca, foram preditos e anotados automaticamente 27 fagos, dos quais nove fagos foram anotados manualmente em etapa posterior. Foram encontrados genes que codificam proteínas típicas de fagos, como capsídeo, transposase e integrase, além de genes acessórios potencialmente importantes, como relacionados ao sistema de transporte ABC e sistema de transporte tipo II. A partir da anotação manual, genes conservados de fagos foram utilizados como base para predição da linhagem viral. Foram identificados 6 tipos de vírus pertencentes à ordem Caudovirales, que infectam Proteobacteria e Firmicutes. Esse trabalho sustenta a hipótese de que os fagos infectam os grupos bacterianos mais abundantes nos manguezais e podem carregar genes importantes consigo, desempenhando papel chave na evolução das bactérias nesse ambiente. / Mangroves are coastal transitional ecossystems between marine and terrestral environments of tropical and intertropical zones and are subject to tidal regime. Due those condictions, microorganisms that lives in the sediments suffer constant adaptative pressure. In this context, Mobile Genetic Elements (MGEs) (phages, plasmids and transposons) are genetic fators directly related with horizontal gene transfer and can facilitate adaptation processes. In this work MGEs were studied in the sediments of three mangrove areas in São Paulo state, two located in Bertioga county and one in environmental reserve of Ilha do Cardoso, in Cananeia. Frequency and expression were avaliated for diferente EGMs types in mangrove microbiome from the sediments metagenomes and metatranscriptomes, besides utilization os a fosmid library assembled from a Bertioga area sediment DNA. The sequences of DNA and mRNA were obtained from three sampled points, sequenced in Illumina HiSeq2000 equipment and automatically anotaded in MG-RAST plataform. A pool of all clone were sequenced for fosmid library in Illumina HiSeq2000 equipment. Contigs were assembled and phages and prophages were automatically predicted in VirSorter pipeline. The phages or prophages predicted sequences were manually anotaded. From the analisys os metagenomic sequences was possible to verify that the MGEs participation in total microbiome metabolism stands for 6 to 15% of predicted genes, wich 90% corresponds to phage associated genes. From contigs assembled of library sequences, 27 phages were predicted and annotaded, wich nine code for phage typical proteins, as capsid, transposase and integrase, besides accessory genes potencially important, as ABC transporter and type II transport related. Phage conserved genes were used as base for viral lineage prediction. 6 viral types belonging do Caudovirales order were identified, which infects Proteobacteria and Firmicutes. This work supports the hypothesis that phages infect the most abundant bacterial groups in mangroves and can carry important genes playing a key role in bacterial evolution in this environment.

Os manguezais são ecossistemas costeiros de transição formados entre o ambiente marinho e terrestre de zonas tropicais e intertropicais, e estão sujeitos ao regime de marés. Devido a tais condições, os microrganismos que vivem nos sedimentos sofrem constante pressão adaptativa. Nesse contexto, os Elementos Genéticos Móveis (EGMs) (fagos, plasmídeos e transposons) são fatores genéticos diretamente relacionados com a transferência horizontal de genes e podem facilitar processos de adaptação. Neste trabalho foram estudados os EGMs dos sedimentos de três áreas de manguezais do estado de São Paulo, duas localizadas no município de Bertioga e uma na reserva ambiental da Ilha do Cardoso, em Cananeia. Foram avaliadas a frequência e expressão dos diferentes tipos de EGMs no microbioma dos manguezais a partir de metagenomas e metatranscriptomas dos sedimentos, além da utilização de uma biblioteca de fosmídeos, montada a partir de DNA do sedimento de uma das áreas de Bertioga. As sequências de DNA e mRNA foram obtidas de três pontos amostrados por área, sequenciadas em equipamento Illumina HiSeq2000 e anotadas automaticamente na plataforma do MG-RAST. Para a biblioteca de fosmídeo, foi sequenciado um pool de todos os clones em equipamento Illumina HiSeq 2000. Contigs dessas sequências foram montados e fagos e profagos foram preditos e anotados automaticamente pelo pipeline VirSorter. As sequências preditas como fagos ou profagos foram anotadas manualmente. A partir da análise das sequências de metagenômica foi possível verificar que a participação dos EGMs no metabolismo total do microbioma dos manguezais corresponde de 6 a 15 % dos genes preditos, sendo que fagos e profagos correspondem a 90% dos genes associados aos EGMs. A partir dos contigs montados para as sequências da biblioteca, foram preditos e anotados automaticamente 27 fagos, dos quais nove fagos foram anotados manualmente em etapa posterior. Foram encontrados genes que codificam proteínas típicas de fagos, como capsídeo, transposase e integrase, além de genes acessórios potencialmente importantes, como relacionados ao sistema de transporte ABC e sistema de transporte tipo II. A partir da anotação manual, genes conservados de fagos foram utilizados como base para predição da linhagem viral. Foram identificados 6 tipos de vírus pertencentes à ordem Caudovirales, que infectam Proteobacteria e Firmicutes. Esse trabalho sustenta a hipótese de que os fagos infectam os grupos bacterianos mais abundantes nos manguezais e podem carregar genes importantes consigo, desempenhando papel chave na evolução das bactérias nesse ambiente. / Mangroves are coastal transitional ecossystems between marine and terrestral environments of tropical and intertropical zones and are subject to tidal regime. Due those condictions, microorganisms that lives in the sediments suffer constant adaptative pressure. In this context, Mobile Genetic Elements (MGEs) (phages, plasmids and transposons) are genetic fators directly related with horizontal gene transfer and can facilitate adaptation processes. In this work MGEs were studied in the sediments of three mangrove areas in São Paulo state, two located in Bertioga county and one in environmental reserve of Ilha do Cardoso, in Cananeia. Frequency and expression were avaliated for diferente EGMs types in mangrove microbiome from the sediments metagenomes and metatranscriptomes, besides utilization os a fosmid library assembled from a Bertioga area sediment DNA. The sequences of DNA and mRNA were obtained from three sampled points, sequenced in Illumina HiSeq2000 equipment and automatically anotaded in MG-RAST plataform. A pool of all clone were sequenced for fosmid library in Illumina HiSeq2000 equipment. Contigs were assembled and phages and prophages were automatically predicted in VirSorter pipeline. The phages or prophages predicted sequences were manually anotaded. From the analisys os metagenomic sequences was possible to verify that the MGEs participation in total microbiome metabolism stands for 6 to 15% of predicted genes, wich 90% corresponds to phage associated genes. From contigs assembled of library sequences, 27 phages were predicted and annotaded, wich nine code for phage typical proteins, as capsid, transposase and integrase, besides accessory genes potencially important, as ABC transporter and type II transport related. Phage conserved genes were used as base for viral lineage prediction. 6 viral types belonging do Caudovirales order were identified, which infects Proteobacteria and Firmicutes. This work supports the hypothesis that phages infect the most abundant bacterial groups in mangroves and can carry important genes playing a key role in bacterial evolution in this environment.

Geotrichum candidum est une levure hémiascomycète ubiquitaire longtemps considérée comme un champignon filamenteux. C'est l'une des levures les plus fréquemment trouvées dans les fromages dans les quelles elle contribue à l'affinage. Dans le cadre du projet ANR ALIA Food Microbiomes en partenariat avec des industriels fromagers et producteur de levain, nous avons caractérisé l'espèce G. candidum par une étude phylogénétique et placé de manière non ambigüe G. candidum parmi les levures hémiascomètes. Une analyse MLST a permis de séparer les souches étudiées en deux groupes. Le premier contient essentiellement des souches environnementales tandis que le second ne contient que des souches isolé du fromage. Cela suggère une certaine sélection ou spécialisation d'un groupe de souche dans la fabrication du fromage. Une méthode de typage inter LTR plus discriminante a permis de typer l'ensemble des souches et peut fournir aux industriels un outil robuste pour le suivi d'une souche en production. Le génome de G. candidum CLIB 918 = ATCC 204307 a été séquencé. Les premières analyses ont mis en évidence des discontinuités évolutives parmi les gènes qui le composent. Parmi les 6802 gènes identifiés, 315 gènes présentent des orthologues chez les champignons filamenteux et non chez les levures. Cela suggère que durant l'évolution, G. candidum a conservé un grand nombre de gènes qui a été perdu chez les autres levures ou en a reçu certain par transfert horizontal de gènes. L'existence de ce même type de gènes chez d'autre levure ayant une position basale dans l'arbre des hémiascomycètes, suggère que G. candidum et ces levures ont une position intermédiaire lors de la transition évolutive champignon vers levure. Il est à noter que certains d'entre eux sont impliqués dans le métabolisme et pourraient jouer un rôle dans l'adaptation de cette levure à la fabrication du fromage.

The bacterial pathogen Vibrio cholerae is responsible for ongoing cholera outbreaks in Haiti and elsewhere. Association of V. cholerae with the human host is responsible for fatal disease, but the bacteria also reside as natural inhabitants of aquatic environments, commonly attaching as biofilms to chitinous surfaces of copepods and crabs. Prior studies in V. cholerae demonstrated that competence for genetic transformation, a mechanism of horizontal gene transfer (HGT), requires the TfoX regulator protein that is triggered by chitin, and the HapR transcription factor that is made in response to quorum sensing (QS) signals produced by V. cholerae and Vibrios. To define regulatory components connecting extracellular signals to natural competence, I first demonstrated that QS molecules produced by Vibrios within multi-species chitinous biofilms are required for DNA uptake by V. cholerae, confirming the critical role of QS signals in HGT. Second, I identified by transposon-mutagenesis a new positive regulator of competence, CytR (cytidine repressor), only studied prior in E. coli as a regulator of nucleoside scavenging. Specific mutations in V. cholerae CytR impaired expression of competence genes and halted DNA uptake; and the addition of exogenous cytidine had similar affects as predicted in E. coli. V. cholerae and other competent Vibrios encode TfoX, HapR, and CytR, although none of these regulators directly controls genes coding for the DNA uptake apparatus. Thus, these results have uncovered a regulatory network, likely used by many Vibrios, that contains additional factors linking several extracellular chemical molecules (cytidine, chitin, and QS signals) to DNA uptake. My study has begun to define a molecular mechanism by which both environment and genetics contribute to genome evolution for this important marine pathogen.

Microsporidia are obligate intracellular pathogens of medical and ecological importance whose genomes have been studied extensively over the last decade. Their parasitic lifestyle has lead them to lose a great number of genes and, thus, biochemical pathways capacities, but these reductive processes have been often offset by the acquisition of several genes by means of horizontal gene transfer (HGT). First, in this thesis, we will describe the complete genomes of Encephalitozoon hellem and Encephalitozoon romaleae. Both species also were found to harbor a number of protein-coding genes absent in other microsporidia, which products assembled complete metabolic pathways. All these genes are functionally related to DNA and folate metabolism, and all appear to have been acquired from HGT events from different eukaryotic and prokaryotic donors. Interestingly in E. romaleae genes involved in de novo synthesis of folate are all pseudogenes, highlighting the transient nature of transferred genes. Secondly, we took a closer look at the ploidy and sexual status of Encephalitozoon cuniculi, a vertebrate pathogen, by mapping Illumina sequence reads against the genomes of four strains of this species. We identified the presence of low level of heterozygosity in all strains investigated; a feature that revealed the diploid nuclear state of the species. This reductive intra-individual genetic diversity could result from the long-term propagation of these strains under laboratory conditions, but we propose that it could also reflect an intrinsic capacity of these vertebrate pathogens to self-reproduce. Overall, the work presented in this thesis resulted in a much greater understanding of the genome evolution of a medically and economically important group of parasites.