Thesis advisor: Tim van Opijnen / Each year, the opportunistic pathogen Streptococcus pneumoniae causes millions of illnesses and nearly 300,000 deaths worldwide. Despite widespread vaccination campaigns, S. pneumoniae persists as a public health risk in large part to its high genomic diversity. In previous work, our group has shown that functional pathways, including stress response to antibiotics, are not necessarily conserved between pneumococcal strains. Thus, a holistic pangenome view of S. pneumoniae is a promising avenue to gain understanding of the species and to inform clinical treatment methods. Our group has selected 36 strains, covering 78% of known pneumococcal genetic diversity, for S. pneumoniae pangenome studies. We have previously constructed transposon libraries and performed Tn-seq for 22 of these strains in both in vitro and in vivo conditions. From these studies, our group has constructed pangenome profiles of genes essential for reproduction in culture conditions, infection in a mouse model, and attachment in a human nasopharyngeal epithelial cell line. In this study, we develop and execute a pipeline to construct iSP20, a set of in silico metabolic models for 34 S. pneumoniae strains. We employ these models to predict nutrient and metabolic gene essentiality on both the strain and pangenome level, demonstrating that key patterns in the strains’ essentialomes translate to a metabolic context. Additionally, we perform a functional analysis of the metabolic models, revealing a highly connected metabolic genome and essentialome. We uncover differences in the in vitro and in silico core essentialomes and identify potential sources of discrepancy between the two datasets. Overall, this work demonstrates the utility of strain-specific metabolic models in pangenome essentiality studies and provides enhanced understanding of metabolism in S. pneumoniae. / Thesis (BS) — Boston College, 2020. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

Marseilleviridae est une famille de virus géants isolés initialement à partir de prélèvements environnementaux, dont Marseillevirus est le membre fondateur. La présence des marseillevirus chez l’Homme a été démontrée dans quelques études. Les objectifs sont de mieux documenter la présence des marseillevirus chez l’Homme, de modéliser l’infection par Marseillevirus chez la souris, et enfin, de décrire les génomes des marseillevirus. Nous rapportons un cas d’infection par Marseillevirus chez une patiente atteinte d’un cancer des ganglions, soulevant la question d’un éventuel lien entre Marseillevirus et cancer, à l’instar de l’association existant entre d’autres virus et les cancers. L’infection des souris par Marseillevirus montre que celui-ci persiste un mois au niveau des «amygdales», confirmant le portage pharyngé chronique observé chez un deuxième patient. Enfin, nous identifions deux nouveaux groupes au sein de la famille, soulignant l’importante diversité génétique de la famille. / Marseilleviridae is a new family of giant viruses primarily isolated from environmental samples and whose Marseillevirus is the founding member. The presence of marseilleviruses in humans has been demonstrated in few studies. The aims are to better document the presence of marseilleviruses in humans, to develop a model of infection of mice with Marseillevirus, and to describe the genomes of marseilleviruses. We report a first caes of infection by Marseillevirus in apatient with a lymph nodes cancer, raising the question of a potential link between Marseillevirus and cancer, as the well established association between some viruses and cancers. The infection of miceshows that Marseillevirus persist one month in the “tonsils”, confirming the chronic pharyngeal carriage reported in a second patient. Finally, we identify two new subgroups in the family, highlighting the considerable genetic diversity of the family.

The positional Burrows-Wheeler transform (PBWT) is a foundational data structure for representing haplotype matches of biobank scale. Once the PBWT panel of a set of haplotypes are constructed, efficient algorithms are available for “All vs. All” positional substring matching, finding exact matches of substrings in pre-aligned strings, for haplotypes within the panel, and “One vs. All” positional substring match query for an out-of-panel haplotype against all haplotypes in the panel. While the original PBWT was designed from linear reference genomes, GBWT was proposed to extend PBWT to genome graphs that allow large insertions and deletions. However, there are no GBWT algorithms for haplotype matching. In this work, we develop the efficient algorithms for “All vs. All” and “One vs. All” haplotype set-maximal and long matching algorithms for GBWT. For a GBWT containing a panel of paths P, we show algorithms similar to the matching algorithms of PBWT. Our algorithms achieves theoretically optimal time complexity to output all “All vs. All” matches in time linear to the size of the input panel (O(∑|Pi| + |out put|)), and quasilinear time to the length of the query path for “One vs. All” path match queries (O(|Q| log σ + |out put| log σ ), where σ is the maximum out- degree in the GBWT and out put is the set of discovered path matches). Under the constant σ assumption made by gPBWT and GBWT, these algorithms are in fact linear. Our algorithms open the possibilities for applications of efficient positional substring matching in pangenome references such as identical-by-descent (IBD) segment identification and genotype imputation.

Débutée en 2005, l'ère du pangénome a connu un important essor ces dernières années, notamment grâce aux progrès des techniques de séquençage haut débit. Le pangénome, qui est divisé en deux grandes parties, le core génome et le génome accessoire, offre un grand éventail d'utilisation. Au cours de ces trois dernières années, nous avons étudié cette gamme de possibilités en nous basant sur des pathogènes humains tel que Coxiella burnetii, Kingella kingae et Bacillus anthracis. Ainsi, outre la découverte d'une nouvelle espèce de Kingella et l'étude de quelques génomes spécifiques, nous nous sommes attardés sur le lien entre pangénome et pathogénicité, sur l'importance des SNPs (Single Nucleotide Polymorphism), ainsi que sur la corrélation entre pangénome et taxonomie et donc, par extension, nous avons étudié la notion d'espèce bactérienne. / The pangenome area began in 2005 and had known a huge increase thanks to the improvement of the Next Generation Sequencing methods. The pangenome, which is divided into two parts, the core and the accessory genome, offer a large panel of uses. During the last three years, we have studied all these possibilities. We based our work on human pathogens as Coxiella burnetii, Kingella kingae and Bacillus anthracis. Thus, in addition to the discovery of a new Kingella species and the study of some specific genomes, we studied in details the link between pangenome and pathogenicity, the importance of SNPs (Single Nucleotide Polymorphism) and the correlation between pangenome and taxonomy. Finally, we worked on the bacterial species definition.

Thesis advisor: Kenneth Williams / Due to overuse and misuse of antibiotics, the global threat of antibiotic resistance is a growing crisis. Three critical issues surrounding antibiotic resistance are the lack of rapid testing, treatment failure, and evolution of resistance. However, with new technology facilitating data collection and powerful statistical learning advances, our understanding of the bacterial stress response to antibiotics is rapidly expanding. With a recent influx of omics data, it has become possible to develop powerful computational methods that make the best use of growing systems-level datasets. In this work, I present several such approaches that address the three challenges around resistance. While this body of work was motivated by the antibiotic resistance crisis, the approaches presented here favor generalization, that is, applicability beyond just one context. First, I present ShinyOmics, a web-based application that allow visualization, sharing, exploration and comparison of systems-level data. An overview of transcriptomics data in the bacterial pathogen Streptococcus pneumoniae led to the hypothesis that stress-susceptible strains have more chaotic gene expression patterns than stress-resistant ones. This hypothesis was supported by data from multiple strains, species, antibiotics and non-antibiotic stress factors, leading to the development of a transcriptomic entropy based, general predictor for bacterial fitness. I show the potential utility of this predictor in predicting antibiotic susceptibility phenotype, and drug minimum inhibitory concentrations, which can be applied to bacterial isolates from patients in the near future. Predictors for antibiotic susceptibility are of great value when there is large phenotypic variability across isolates from the same species. Phenotypic variability is accompanied by genomic diversity harbored within a species. I address the genomic diversity by developing BFClust, a software package that for the first time enables pan-genome analysis with confidence scores. Using pan-genome level information, I then develop predictors of essential genes unique to certain strains and predictors for genes that acquire adaptive mutations under prolonged stress exposure. Genes that are essential offer attractive drug targets, and those that are essential only in certain strains would make great targets for very narrow-spectrum antibiotics, potentially leading the way to personalized therapies in infectious disease. Finally, the prediction of adaptive outcome can lead to predictions of future cross-resistance or collateral sensitivities. Overall, this body of work exemplifies how computational methods can complement the increasingly rapid data generation in the lab, and pave the way to the development of more effective antibiotic stewardship practices. / Thesis (PhD) — Boston College, 2020. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

<p dir="ltr">Open-access bioinformatics data enables accessible public health research for a variety of stakeholders, including teachers and low-resourced researchers. This project outlines two case studies utilizing open-access bioinformatics data sets and analysis software as proofs of concept for the types of research projects that can be adapted for workforce development purposes. The first case study is a spatial temporal analysis of Lyme disease rates in the United States from 2008 to 2020 using freely available data from the United States Department of Agriculture and Centers for Disease Control and Prevention to determine how urbanization and other changes in land use have impacted Lyme disease rates over time. The second case study conducts a pangenome analysis using bacteriophage data from the Actinobacteriophage Database to determine conserved gene regions related to host specificity.</p>

Le modèle étudié est l’agent phytopathogène vasculaire Ralstonia solanacearum, en portant une attention particulière aux souches de phylotype II. Cette bactérie d’origine tellurique est très diversifiée, tant au plan génétique que phénotypique. Sa classification en constante évolution témoigne d’une volonté de clarifier cette biodiversité inhabituellement forte, tout en cherchant à reconnaître les écotypes structurant ce complexe d’espèces, i.e., des groupes de souches partageant à la fois des traits génotypiques et biologiques spécifiques. Dans le cadre de ce pathosystème modèle, nous nous sommes attachés dans un premier temps à revisiter de façon précise les pathotypes au sein d’écotypes bien décrits dans la littérature, ou à en faire la description (phylotype III africain). Nous avons observé une forte convergence phénotypique entre les souches de phylotype III des hauts plateaux africains et les souches Brown rot de phylotype IIB-1, capables de flétrir la pomme de terre et d’autres Solanacées à température froide. L’adaptation de souches aussi diverses pour la tolérance au froid nous a conduits à dresser un bilan de la situation R. solanacearum en Europe et in extenso dans le bassin méditerranéen. Cette approche a permis d’apprécier les degrés de divergence significative dans le pouvoir pathogène (virulence et agressivité) sur Solanaceae au sein de souches quasi clonales unifiant l’écotype Brown rot, qui s’établissent aussi sous forme d’infections latentes dans les tissus vasculaires de bananiers (Musacées). Dans le même temps, le phénotype de souches pathogènes du bananier, unifiant l’écotype Moko, a aussi été revisité sur Solanaceae qu’elles parviennent à flétrir, y compris des ressources génétiques résistantes au flétrissement bactérien. L’ensemble de ces données expérimentales a permis de dégager les critères de sélection pour le choix de trois nouvelles souches du complexe d’espèces R. solanacearum, dont nous avons obtenu les séquences génomiques. Notre approche en génomique comparative a permis de décrire le premier pangénome chez cet agent pathogène : l’ensemble les gènes repérés de l’espèce. Ces données ont été exploitées par différentes approches bio-informatiques et permettent de concevoir une refonte pertinente du complexe d’espèces R. solanacearum en trois nouvelles espèces génomiques, regroupant les souches de phylotypes I (Asie) et III (Afrique) d’une part, puis les souches de phylotype II (Amérique), et enfin les souches de phylotype IV (Indonésie) d’autre part. Ce pangénome a ensuite été exploité en concevant et développant une puce à ADN, un outil permettant l’exploration à haut débit d’une grande quantité de souches. La densité des données expérimentales accumulées permet une démarche vers l’écologie moléculaire et de reconstituer certains pans du passé évolutif des souches de phylotype II chez R. solanacearum. Par ailleurs, l’analyse approfondie de ces données de génomique, associant phylogéographie et structuration des populations de l’écotype Brown rot, montre une double situation épidémiologique en Europe, recoupant des influences andines et africaines. De la même façon, l’écotype Moko présente trois structures génétiques distinctes. Ces données ont été analysées de manière à retracer les principaux flux de gènes dans les états ancestraux des phylotypes et de dégager la forte contribution de la partie mobile du génome, des gènes relatifs à l’adaptation environnementale et à la pathogénie, comme moteurs dans l’évolution de cet important organisme phytopathogène. / The studied model is the vascular plant pathogen Ralstonia solanacearum, with a particular focus on phylotype II strains. This telluric bacterium has a wide diversity, both on genotypic and phenotypic levels. Its evolving classification reflects the need to clarify its unusual biodiversity and seek to identify ecotype structure in this species complex, i.e., groups of strains with both genotypic and specific biological traits. Within the framework of this model pathosystem, we initially focused on deeply revisiting pathotypes among ecotypes, although well described in the literature, or describing new ecotypes (African phylotype III). We observed high phenotypic convergence between strains from phylotype III from the African highlands and Brown rot strains from phylotype IIB-1, both able to trigger wilt symptoms on potato and other Solanaceae at cold temperatures. Adaptation of diverse strains for cold tolerance led us to investigate the R. solanacearum situation in Europe and more specifically in the Mediterranean regions. This strategy allowed us to appreciate the significant divergence towards pathogenicity (virulence and aggressiveness) on Solanaceae within clonal-like structure of strains in the Brown rot ecotype, which also established latent interactions in the banana vascular system. In the mean time, phenotypes of banana pathogenic strains unifying the Moko ecotype, was also revisited on Solanaceae, and was able to trigger symptoms on both susceptible and resistant genetic resources to bacterial wilt. All these experimental data yielded selection criteria for choosing three new candidate strains in the R. solanacearum species complex for complete genome sequencing. Our genomic comparative approach allowed us to describe the first pangenome of this pathogen: all targeted identified genes of this species complex. These data were analyzed by various bioinformatic approaches and allowed us to design a complete reshaping of R. solanacearum species complex into three distinct genomic species, firstly clustering strains from phylotype I (Asia) with strains from phylotype III (Africa); strains from phylotype II (America); and lastly, strains from phylotype IV (Indonesia). This pangenome was then used for designing a DNA microarray, a high resolution tool that allowed us to explore a wide set of genomes. The density of accumulated data allowed for a molecular ecological approach to retrieve a certain amount of the evolutionary past of R. solanacearum phylotype II strains. Furthermore, a deeper analysis of these genomic data, combining phylogeography with population structure analysis of the Brown rot ecotype, revealed a dual epidemic situation in Europe, both across Andean and African influences. Similarly, the Moko ecotype presents three distinct genetic structures. These data were analyzed within the purpose of tracking the main gene flows in the ancestral states of phylotypes and to unravel the strong contribution of the mobile elements, genes related to environmental adaptation, and pathogenicity as a major driving force into the evolution of this successful plant pathogen.

Coxiella burnetii est l’agent pathogène responsable de la fièvre Q. Dans le cadre de cette thèse nous nous sommes intéressés à l'étude de souches de C.burnetii responsables d'événements épidémiques. Nous avons séquencé une souche de génotype MST33 (Z3055), proche de la souche responsable de l'épidémie de fièvre Q aux Pays-Bas, et une souche de génotype MST17 (Cb175) clone provoquant l'une des formes les plus virulentes de fièvre Q aiguë jamais décrite auparavant et retrouvée à ce jour uniquement en Guyane Française. Les résultats de ces analyses montrent que le génome de la souche Z3055 était très similaire à celui de la souche de référence Nine Mile I. Les différences observées sont liées à la présence de mutations non synonymes dans le génome de Z3055. Le pourcentage élevé de protéines membranaires mutées pourrait expliquer l’ampleur de cette épidémie en Hollande. En effet, le changement de profil antigénique pourrait être à l’origine de la formation d’un nouveau sérotype capable d'échapper à la réponse immunitaire de l'hôte et de diffuser facilement dans une population au système immunitaire naïf. Nous avons d’ailleurs montré que la souche responsable de la fièvre Q en Guyane (Cb175) présente des différences chromosomiques importantes par rapport à NMI. Ces différences se manifestent principalement par la présence d’une délétion d’une région de 6105pb contenant l’opéron hlyCABD du système de sécrétion de type 1 (T1SS). Ce résultat est cohérent avec ce qui a été observé chez les bactéries épidémiques les plus dangereuses comparées à leurs espèces non-épidémiques plus proches qui ont un génome réduit et contiennent moins de protéines du système de sécrétion. / Coxiella burnetii is a human pathogen that causes the zoonotic disease Q fever. In this work, we focused on the study of strains responsible for epidemic events. Particularly, we sequenced the clone of the strain responsible for Netherlands outbreak having genotype MST33 (Z3055), and strain having MST17 (Cb175) responsible for one of the most severe form of acute Q fever never reported in literature and uniquely described in French Guiana. Our findings showed that the Netherlands outbreak responsible strain (clone Z3055) was highly similar to the reference strain Nine Mile I. Only slight differences were observed, which were related to non-synonymous mutations in Z3055 genome. The high proportion of mutated membrane proteins could explain this large-scale outbreak. Change of antigenic profile may have led to a new serotype, conferring to the novel clone the capacity to escape the host immune response and to disseminate easily in a immunologically naïve population. On the contrary, the type strain responsible for Q fever in Guiana (Cb175) showed an important difference in its chromosome sequence compared to the reference NMI because of the deletion of a sequence of 6105bp containing the Type 1 secretion systems (T1SS) hlyCABD operon. This result appear consistent with previous findings that showed the most dangerous epidemic bacteria compared with their closest non-epidemic species are characterized by reduced genomes accompanied by significant decrease in ORF content and contain less secretion system proteins.

La première partie de ma thèse est une revue donnant un aperçu du répertoire génomique des bactéries intracellulaires et de leurs symbiotes. L'objectif de cette étude est d'explorer le processus permettant aux bactéries intracellulaires d'acquérir leur mode de vie spécifique. Nous avons commencé par examiner les données à propos de l'existence ancienne de bactéries intracellulaires, leur adaptation à leur hôte et les différences entre sympatrie et allopatrie. Une comparaison du contenu génomique de plusieurs bactéries avec différents modes de vie a révélé la capacité des bactéries à échanger des gènes à des degrés différents, en fonction de l'écosystème. La deuxième partie de ma thèse porte sur la séquence du génome de la souche Diplorickettsia massiliensis 20B qui est une bactérie intracellulaire obligatoire à Gram négatif isolée à partir des tiques de Slovaquie Ixodes ricinus. Dans ma troisième et dernière partie, nous exploré le répertoire du génome de Diplorickettsia massiliensis en le comparant aux génomes de bactéries phylogénétiquement très proches de Diplorickettsia massiliensis, issues de différentes niches. Ceci a permis de révélé son mode de vie allopatrique. Dans cette étude, nous avons comparé les caractéristiques du génome de Diplorickettsia massiliensis avec vingt-neuf espèces séquencées de Gammaproteobacteria (Legionella, Coxiella burnetii, Francisella tularensis et Rickettsiella grylli) en utilisant l'approche pangénomique multi-genre. Ce travail de thèse fournit des données originales et permet d’apporter plus de lumière sur la diversité des bactéries intracellulaires. / The initial purpose of my thesis is to understand with the help of comparative genomics, genomic variations based on coexistence, by examining data on the ancient existence of intracellular bacteria, their host adaptation and the differences between sympatry and allopatry. The first part of my thesis is a review giving insight into intracellular bacterial genome repertoire and symbionts. The goal of this review is to explore how intracellular microbes acquire their specific lifestyle. Due to their different evolutionary trajectories, these bacteria have different genomic compositions. We reviewed data on the ancient existence of intracellular bacteria, their host adaptation and the differences between sympatry and allopatry. A comparison of the genomic contents of bacteria with certain lifestyles revealed the bacterial capacity to exchange genes to different extents, depending on the ecosystem. The second part of my thesis present about the genome sequence of Diplorickettsia massiliensis strain 20B which is an obligate intracellular, gram negative bacterium isolated from Ixodes ricinus ticks collected from Slovak. In the third part, we investigated the genome repertoire of Diplorickettsia massiliensis compared to closely related bacteria according to its niche, revealing its allopatric lifestyle. In this study, we compared the genomic features of Diplorickettsia massiliensis with twenty-nine sequenced Gammaproteobacteria species (Legionella strains, Coxiella burnetii strains, Francisella tularensis strains and Rickettsiella grylli) using multi-genus pangenomic approach. This thesis work provides original data and sheds light on intracellular bacterial diversity.

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.