L’insecte phytophage Riptortus pedestris, appartenant au sous-ordre des Hétéroptères, est un ravageur notoire de cultures agricoles en Asie du sud-est qui se nourrit préférentiellement de plants de soja. Cette punaise est associée à une bactérie symbiotique du genre Burkholderia nommée Burkholderia insecticola, localisée dans une région spécifique de l’intestin de l’insecte appelée la région M4. Cette région M4, organisée en cryptes, constitue l’organe symbiotique dans lequel le symbiote prolifère de manière extracellulaire. Cette interaction favorise la croissance et le développement de la punaise. Récemment, il a été montré que Riptortus produit des peptides antimicrobiens au sein des cryptes, appelés “crypt-specific cysteine-rich peptides” ou peptides CCR pour lesquels le symbiote est particulièrement résistant. Il a été proposé que les peptides antimicrobiens de l’hôte,incluant les peptides CCR, participent à la colonisation spécifique de l’organe symbiotique par B. insecticola. Dans ce travail, une approche Tn-seq a été utilisée pour identifier les gènes bactériens impliqués dans la résistance aux peptides antimicrobiens et dans la symbiose. Dans un premier temps, la robustesse de la méthode Tn-seq a été évaluée en identifiant le génome essentiel de B. insecticola. Puis dans un second temps, les facteurs bactériens impliqués dans la résistance aux peptides antimicrobiens ont été caractérisés via une approche gènes-candidats et l’approche Tn-seq. Dans une dernière partie, une expérience de Tn-seq in vivo a permis d’évaluer l’ampleur du goulot d’étranglement sur la population symbiotique lors de l’infection de l’organe symbiotique et d’identifier les facteurs symbiotiques impliqués dans la colonisation de R. pedestris. / The phytophagous insect Riptortus pedestris, belonging to the Heteroptera suborder, is a notorious crop pest in South-Eastern Asia which feeds preferentially on soybean plants. This bean bug is associated with a bacterial symbiont, a specific Burkholderia species named Burkholderia insecticola, located in the M4 region of the insect’s midgut. This M4 region is organized in crypts and constitutes the symbiotic organ where the symbiont proliferates extracellularly. This interaction promotes the growth and the development of the bean bug. Recently, it was demonstrated that Riptortus produces antimicrobial peptides in the midgut crypts called crypt-specific cysteine-rich peptides (CCR) for which the bacterial symbiont demonstrates a high resistance profile. It was proposed that host antimicrobial peptides, including the CCR peptides, contribute to the specific colonization of the symbiotic organ by B. insecticola. In this work, a Tn-seq approach was used to find bacterial fitness genes involved in antimicrobial peptide resistance and symbiosis. First, the robustness of the Tn-seq method was assessed by identifying the essential genome of B. insecticola. Second, the bacterial factors for antimicrobial peptide resistance were characterized, based on both a candidate-gene and the Tn-seq approach. Finally, a Tn-seq in vivo experiment was performed to reveal the infection bottleneck effect on the symbiotic population and to identify the bacterial symbiosis factors for the colonization of R. pedestris.

Le phytopathogène Agrobacterium tumefaciens est l'agent causal de la maladie appelée galle du collet, et est capable d'infecter plus de 90 familles de plantes dicotylédones. Cette ∝-protéobactérie appartient à la famille Rhizobiaceae. A. tumefaciens est un complexe de différentes espèces regroupées en 10 génomovars (G1 à G8 et G13). A. tumefaciens C58 appartient au groupe du G8. Son génome est constitué de 4 réplicons : 1 chromosome circulaire, 1 chromosome linéaire et des 2 plasmides dispensables : pAt (pour A.tumefaciens) et pTi (pour Tumor inducing, qui est requis pour la virulence). Pour explorer de nouveaux aspects du mode de vie d’A. tumefaciens, et en particulier l'interaction entre la bactérie et sa plante hôte, deux approches différentes ont été utilisées pour identifier, caractériser et analyser les gènes qui pourraient jouer un rôle dans l'adaptation des bactéries à la tumeur. Une expérience de l'évolution par des passages en série de trois souches différentes de l'agent pathogène sur la plante hôte Solanum lycopersicum a été effectuée afin de clarifier la dynamique évolutive du génome au cours de l'infection. Parallèlement, une étude de différents transcriptomes (in planta et in vitro) a été réalisée et étudiée pour élucider des gènes bactériens candidats impliqués dans l'interaction de la bactérie avec la plante et divers composés produits dans la tumeur. Ce travail tente de donner une vue plus générale du processus d'adaptation de la bactérie à la niche écologique qui est la tumeur. / Agrobacterium tumefaciens is the causal agent of the plant disease called crowngall, and it’s able to infect more than 90 families of dicotyledonous plants. It is an α-Proteobacterium and belongs to the Rhizobiaceae family. A. tumefaciens is a complex of different species grouped in 10 genomovars (G1 to G8, and G13). A. tumefaciens C58 belongs to the G8 group. Its genome consists in 4 replicons: 1 chromosome circular, 1 chromosome linear and 2 dispensable plasmids: pAt (for A. tumefaciens) and pTi (for Tumor inducing), which is required for virulence. To explore new aspects of the A. tumefaciens lifestyle, and in particular the interaction between the bacteria and its plant host, two different approaches have been used to identify, characterize and analyze genes that could play a role in the adaptation of the bacteria to tumor lifestyle. An evolution experiment by serial passages of three different strains of thepathogen on the host plant Solanum lycopersicum has been carried out to clarify the evolutionary dynamics of the genome during the course of infection. In parallel, a study of different transcriptomes (in planta and in vitro) was performed and studied to elucidate bacterial candidate genes involved in the interaction of the bacteria with the plant and various compounds produced in the tumor. This work attempts to give a more general view of the process of adaptation of the bacteria to the ecological niche that is the tumor.

Thesis advisor: Michelle Meyer / The primary bacterial targets for most antibiotics are well known. To survive the stress of an antibiotic a bacterium must decrease the antibiotic to target binding ratio to escape from harmful effects. This can occur through a number of different functions including down-regulation of the target, mutation of the binding site on the target, and decreasing the intake or increasing the efflux of the antibiotic. However, it is becoming more evident that an antibiotic stress response influences more than just the primary target, and that a wave of secondary responses can be triggered throughout the bacterium. As a result resistance mutations may arise in genes that are indirectly affected by the initial interaction between the antibiotic and target. These indirect responses have been found to be associated with metabolism, regulation, cell division, oxidative stress, and other critical pathways. One technique recently developed in our lab, called transposon insertion sequencing (Tn-seq), can be used to further understand the complexity of these indirect responses by profiling growth rates (fitness) of mutants at a genome-wide level. However, Tn-seq is normally performed with large libraries of pooled mutants and thus it remains unclear how this may influence fitness of some independent mutants that may be compensated by others in the population. Additionally, since the original method has only utilized planktonic culture, it is also not clear how higher order bacterial structures, such as biofilms or microcolonies, influence bacterial fitness. To better understand the dynamics of pooled versus individual mutant culture, as well as the effect of community structure in microcolony development on the influence of fitness, we adapted a droplet microfluidics-based technique to encapsulate and culture single mutants. We were able to successfully encapsulate at least 7 different species of bacterial pathogens, including Streptococcus pneumoniae, and culture them planktonically, or as microcolonies, in either monodisperse liquid or agarose droplets. These experiments, however, raised an important challenge: the DNA yield from one encapsulation experiment is insufficient to generate samples for sequencing by means of the traditional Tn-seq method. This led us to develop a novel Tn-seq DNA library preparation method, which is able to generate functional Tn-seq library molecules from picogram amounts of DNA. This method is not ideal yet because fitness data generated through the new method currently does not correlate well with data from traditional Tn-seq library preparation. However, we have identified one major culprit that should be easily solvable. We expect by modifying the binding site of the primer used for linear amplification of transposon ends that the new preparation method will be able recapitulate results from the traditional Illumina preparation method for Tn-seq. This will enable us to prepare robust Tn-seq samples from very small amounts of DNA in order to probe stress responses in single mutants as well as in microcolonies in a high-throughput manner. / Thesis (MS) — Boston College, 2016. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Entomopathogenic nematodes (EPNs) (genera Heterorhabditis and Steinernema) kill their invertebrate hosts with the aid of a mutualistic bacterium. The bacteria (Xenorhabdus spp. for steinernematids and Photorhabdus spp. for heterorhabditids) are primarily responsible for killing the host and providing the nematodes with nutrition and defense against secondary invaders. Photorhabdus is a Gram-negative bacterium in the Enterobacteriaceae family with high virulence towards their insect hosts. To achieve high mortality rates Photorhabdus produces a variety of virulence factors such as toxins, lipases, proteases, secretion systems, and fimbriae. EPNs are amenable to laboratory rearing and mass production for biocontrol applications against insects using in vivo or in vitro methods; however, in vitro liquid culture is considered to be the most efficient. In this method the symbiotic bacteria are cultured prior to the addition of their partner EPN. This can leave the bacteria susceptible to a number of problems such as genetic drift and inadvertent selection. Regardless of the culture method the symbiotic bacteria exhibit trait deterioration or changes due to laboratory rearing. This project had three primary aims: 1) investigate the role of nutrition in trait deterioration, 2) examine virulence evolution using a phylogenetic context, and 3) identify genes that are necessary for survival and virulence inside the insect host. Prior to studying these objectives we first determined the optimal conditions for growing and counting viable cells of Photorhabdus. We discovered that growth is enhanced by the addition of pyruvate to growth media. To determine the role of nutrition in trait deterioration we repeatedly sub-cultured Photorhabdus in three different media types. Throughout this study we found that, in contrast to previous studies, trait deterioration does not always happen and the environment influences trait deterioration. Furthermore, based on our phylogenetic studies we found that Photorhabdus spp. are evolving to an increase in insect virulence. Lastly, using Tn-seq we determined a list of 84 genes that are needed for efficient virulence inside the insect host and provide suggestions for ongoing research efforts.

The rhizobia-legume symbiosis is the most agriculturally significant source of naturally fixed nitrogen, accounting for almost 25% of all biologically available nitrogen. Rhizobia-legume compatibility restrictions impose limits on symbiotic nitrogen fixation. In many cases, the molecular basis for symbiotic compatibility is not fully understood. The signals required for establishing a symbiotic partnership between nitrogen-fixing bacteria (e.g. Sinorhizobium meliloti) and leguminous plants (e.g. Medicago truncatula) have been partially characterized at the molecular level. The first stage of successful root colonization is competitive occupation of the rhizosphere (which is poorly understood). Here, the bacteria introduce themselves as potential symbiotic partners through the secretion of glycolipid "Nod" factors. In response, the host facilitates a more exclusive mode of colonization by the formation of a root nodule – a new organ capable of hosting dense intracellular populations of symbiotic rhizobia for nitrogen fixation. This dissertation reports the exhaustive identification of S. meliloti genes that permit competitive colonization of the M. truncatula rhizosphere, and includes a mechanistic study of one particular bacterial signaling pathway that is crucial for both rhizosphere colonization and nodulation. I have made use of Tn-seq technology, which relies on deep sequencing of large transposon mutant libraries to monitor S. meliloti genotypes that increase or decrease in relative abundance after competition in the rhizosphere. This work included the collaborative development of a new computational pipeline for performing Tn-seq analysis. Our analysis implicates a large ensemble of bacterial genes and pathways promoting rhizosphere colonization, provides hints about how the host plant shapes this environment, and opens the door for mechanistic studies about how changes in the rhizosphere are sensed and interpreted by the microbial community. Notable among these sensory pathways is a three-protein signaling system, consisting of FeuQ, FeuP, and FeuN, which are important for both rhizosphere colonization and nodule invasion by S. meliloti. The membrane-bound sensor kinase FeuQ can either positively or negatively influence downstream transcription of target genes by modulating the phosphorylation state of the transcriptional activator FeuP. FeuN, a small periplasmic protein, inhibits the positive mode of FeuPQ signaling by its direct interaction with the extracellular region of FeuQ. FeuN is essential for S. meliloti viability, underscoring the vital importance of controlling the activity of downstream genes. In summary, I have employed several powerful genetic, genomic, computational, and biochemical approaches to uncover a network of genes and pathways that coordinate root colonization and nodulation functions.

Thesis advisor: Tim van Opijnen / Streptococcus pneumoniae is a prominent gram-positive commensal and opportunistic pathogen which possesses a large pan-genome. Significant strain-to-strain variability in genomic content drives the use of varied pathways to perform similar processes between strains. Considering this variation, we employ a set of 36 strains, representative of 78% of total pan-genome diversity, with which to perform functional studies. We previously determined the set of genes required by 22 of the 36 strains to maintain successful infection in a host, or the virulome. In this work, we sought to parse from the virulome the genes required specifically for nasopharyngeal adhesion, a crucial step in S. pneumoniae colonization and transmission, and often a precursor to invasive disease, as well as gene requirements for subversion of the macrophage. We performed in vitro attachment Tn-seq in the 22 strains to D562 human nasopharyngeal epithelial cells, identifying thirteen factors that exhibit requirements for adhesion, and preliminarily validated a proposed universal requirement for survival of the macrophage by a killing assay using J774A.1 murine migratory macrophages. / Thesis (BS) — Boston College, 2020. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: A&S Honors. / Discipline: Biology.

Les bactéries environnementales font face à de très nombreux stress dans leur milieu devie. Ces dernières doivent s’acclimater rapidement pour faire face à des variations detempérature, des changements d’osmolarité, des changements de pH ou encore descarences nutritives afin de survivre et de prospérer dans leur environnement naturel. Cetravail de thèse s’inscrit dans l’étude de la réponse aux stress chez les bactériesenvironnementales en prenant l’exemple du stress engendré par les métaux chez labactérie modèle Pseudomonas putida et du stress rencontré en cours d’infection deplante chez le phytopathogène Dickeya dadantii. La présence d’un excès d’ionsmétalliques ainsi que les défenses d’une plante sur un phytopathogène entrainent toutdeux un stress oxydatif et peut provoquer une altération membranaire ainsi queprotéique. Ces stress, à première vue éloignés, pourraient ainsi induire des mécanismesde réponses similaires chez les bactéries. La plupart des études de résistance aux stresschez les bactéries portent sur l’étude de gènes sur ou sous exprimés et peuvent alorsmanquer des gènes importants pour la réponse à un stress donné dont l’expressionreste inchangée. Afin d’identifier de nouveaux facteurs de résistance aux stressmétalliques chez P. putida ainsi que de nouveaux facteurs de résistance/virulence chezle phytopathogène D. dadantii, un crible Tn-seq (Transposon-Sequencing) a été réalisé àl’aide de banques de mutants très denses. Les deux cribles ont permis de mettre enévidence le rôle majeur de régulateurs de transcription ainsi que l’importance desmécanismes d’efflux dans la réponse à un stress. Les deux cribles ont aussi mis enévidence l’importance des voies de biosynthèses en acides aminées et acides nucléiques.Enfin, de nombreux gènes à fonction inconnue participent à la survie des deux bactériesen condition de stress. Les résultats obtenus pourraient permettre de développer denouveaux antimicrobiens chez D. dadantii. Les mécanismes de résistance aux métaux etaux antibiotiques peuvent être similaires. Nos travaux améliorent la compréhension desmécanismes de résistance aux métaux et pourraient ainsi permettre de trouver denouvelles molécules à activité antimicrobienne / Environmental bacteria have to deal with a number of stresses in their livingenvironment. The bacteria have to adapt quickly to changes in temperature, osmolarity,pH changes or nutrient deficiencies in order to survive and thrive in their naturalenvironment. This thesis work is related to the study of stress response inenvironmental bacteria by taking the example of stress caused by metals in thebacterium Pseudomonas putida model and stress encountered during plant infection inthe phytopathogen Dickeya dadantii. The presence of an excess of metal ions as well asthe defences of a plant on a phytopathogen both cause oxidative stress and can causemembrane and protein alteration. These stresses, at first sight distant, could thus inducesimilar response mechanisms in bacteria. Most stress resistance studies in bacteriafocus on the study of over- or under-expressed genes and may then lack genesimportant for the response to a given stress whose expression remains unchanged. Inorder to identify new metal stress resistance genes in P. putida and newresistance/virulence factors in D. dadantii phytopathogen, a Tn-seq (Transposon-Sequencing) screen was performed using very dense mutant banks. Both screenshighlighted the major role of transcription regulators as well as the importance of effluxmechanisms in the response to stress. Both screens also highlighted the importance ofamino acid and nucleic acid biosynthesis pathways. Finally, many genes with unknownfunction participate in the survival of both bacteria in stress conditions. The resultsobtained could lead to the development of new antimicrobials in D. dadantii. Themechanisms of resistance to metals and antibiotics may be similar. Our work isimproving our understanding of metal resistance mechanisms and could lead to newmolecules with antimicrobial activity

The peptidoglycan of many Gram-positive bacteria is densely functionalized with anionic glycopolymers called wall teichoic acids (WTAs). Recent studies have shown that these polymers play crucial roles in cell shape determination, regulation of cell division, and other fundamental aspects of Gram-positive bacterial physiology. Furthermore, in pathogens they are important in host infection and play key roles in antibiotic resistance. In many cases, precise mechanisms for WTA involvement in these processes have not been established. In order to better understand the roles of WTAs in the biology of the human pathogen Staphylococcus aureus, we sought to identify their interactions with other cellular pathways. By employing a transposon screen, we found that lipoteichoic acid (LTA) synthesis, D-alanylation of teichoic acids, cell wall stress sensors, CAAX-like proteases, and peptidoglycan biosynthesis were all synthetically lethal with depletion of WTAs in Staphylococcus aureus . Further investigations revealed that several genes required when WTAs were depleted were not essential when LTAs were removed. Unexpectedly, TA D-alanylation, became essential in the absence of WTAs, but not LTAs. Examination of terminal phenotypes following WTA depletion revealed that strains lacking LTA D-alanine esters died from envelope rupture during ongoing cell division whereas strains lacking LTAs were unable to form Z rings, stopped dividing, and had altered PG biosynthesis. Finally, we designed and implemented parallel, pathway-specific chemical screens to identify inhibitors that specifically kill mutants deficient in WTAs or D-alanylation of TAs. In addition to elucidating new interactions between cell envelope pathways, and establishing distinct roles LTAs and WTAs in the cell envelope of S. aureus, these experiments provide a list of potential targets and a strategy for identifying inhibitors for these targets, in compound combinations as therapeutics against antibiotic-resistant S. aureus infections.

Escherichia coli is an incredibly diverse group of bacteria that consist of both commensal and pathogenic strains that cause disease in a wide variety of tissues in many different animals. The current dogma, based on years of extensive molecular and genetic studies, is that individual strains have adapted to specific environments through acquisition of specific genes or come from lineages that are particularly suited to a unique tissue or host. However, mastitis-associated E. coli (MAEC) have thus far resisted such descriptions. The fitness and virulence factors of MAEC are poorly understood and molecular tools are rarely applied. This dissertation reports new approaches to assess virulence of MAEC strains, enabling comparative genomic studies across multiple strains as well as genome-wide analysis of specific successful MAEC isolates. I outline the identification of the first virulence factor of MAEC, a ferric dicitrate receptor that is essential for colonization of a lactating mammary gland in a murine model. Genes previously studied in the contexts of other extraintestinal E. coli infections were also implicated in mastitis. These include a type III capsule found in the MAEC strain M12, which is crucial for dissemination from the mammary gland to the spleen. A mutant unable to produce capsule had diminished lethality in Galleria mellonella and decreased kidney colonization in a mouse urinary tract infection. I also report a link between zinc uptake, bile salts, and capsule production. I have utilized a transposon mutant library paired with deep sequencing of transposon junctions to elucidate the fitness factors needed to grow in milk and colonization of both murine and insect models. This analysis implicates a broad set of genes and metabolic pathways pertinent to these conditions. In addition to Tn-seq, I sequenced 94 MAEC genomes and identified genes associated with disease severity, growth in milk, and colonization of mammary glands in cow and mouse models. Employing bioinformatic tools to interrogate the pan-genome, I identified genes that are involved in biofilm formation and adhesion that were specifically associated with either mild or severe disease. In summary, I have employed several powerful genetic, genomic, computational, and molecular approaches to the characterization of mastitis associated E. coli. This work provides the groundwork for future experiments to better understand the host-pathogen interface and a model for mastitis-associated E. coli.