To diversify their genetic material, allowing adaptation to environmental disturbances and colonization of new ecological niches, bacteria use various evolutionary processes, including the acquisition of new genetic material by horizontal transfer mechanisms such as conjugation, transduction and transformation. Electrotransformation mediated by lightningrelated electrical phenomena may constitute an additional gene transfer mechanism occurring in nature. The presence in clouds of bacteria capable of forming ice nuclei that lead to precipitations and are involved in the triggering of lightning, such as the global phytopathogen Pseudomonas syringae, led us to postulate that natural electrotransformation in clouds may affect bacteria, by contributing to increase their adaptive potential. We first determined if the ice nucleator bacterium P. syringae could survive when in clouds and acquire exogenous genetic material through lightning shock-simulating in vitro electroporation. In comparison to two other bacteria, P. syringae appears to be best adapted for survival and for genetic electrotransformation under these conditions, which suggests that this bacterium would be able to survive and evolve whilst being transported in clouds. Secondly, we evaluated the impact of lightning shock-simulating in vitro electroporation on the survival, the electrotransformation potential and the diversity of bacteria collected from rain samples. These isolates better resisted lightning than the laboratory strains and some were able to electrotransform exogenous DNA. The rain bacteria we isolated were of different origins and were representative of life modes of the various sources of bacterial emissions on Earth. Our study suggests that bacteria aerosolized from diverse terrestrial ecosystems can spread to new habitats through clouds whilst also being able to acquire new genetic material via lightning-based electrotransformation, thereby potentially enhancing their genetic diversity. The final part of our work consisted of evaluating whether electrotransformation could be applied to the engineering of indigenous soil bacteria in order to develop a tool for the bioremediation of lindane, a once widely used pesticide. Optimized experiments revealed that both natural and electrotransformation contributed to the incorporation of a plasmid harboring a gene encoding the first lindane dechlorination steps by indigenous soil bacteria. In conclusion, we showed that natural electrotransformation mediated by electrical discharges such as those occurring in clouds or reaching soils can be involved in the horizontal gene transfer process among bacteria and, considering the importance of lightning worldwide, may play a role in the adaptation and evolution of these organisms.

Reducing the production cost of industrial biofuels will greatly facilitate their proliferation and co-integration with fossil fuels. The cost of feedstock is the largest cost in most fermentation bioprocesses and therefore represents an important target for cost reduction. Meanwhile, the biorefinery concept advocates revenue growth through complete utilization of by-products generated during biofuel production. Taken together, the production of biofuels from low-cost crude glycerol, available in oversupply as a by-product of bioethanol production, in the form of thin stillage, and biodiesel production, embodies a remarkable opportunity to advance affordable biofuel development. However, few bacterial species possess the natural capacity to convert glycerol as a sole source of carbon and energy into value-added bioproducts. Of particular interest is the anaerobe Clostridium pasteurianum, the only microorganism known to convert glycerol alone directly into butanol, which currently holds immense promise as a high-energy biofuel and bulk chemical. Unfortunately, genetic and metabolic engineering of C. pasteurianum has been fundamentally impeded due to a complete lack of genetic tools and techniques available for the manipulation of this promising bacterium. This thesis encompasses the development of fundamental genetic tools and techniques that will permit extensive genetic and metabolic engineering of C. pasteurianum. We initiated our genetic work with the development of an electrotransformation protocol permitting high-level DNA transfer to C. pasteurianum together with accompanying selection markers and vector components. The CpaAI restriction-modification system was found to be a major barrier to DNA delivery into C. pasteurianum which we overcame by in vivo methylation of the recognition site (5’-CGCG-3’) using the M.FnuDII methyltransferase. Systematic investigation of various parameters involved in the cell growth, washing and pulse delivery, and outgrowth phases of the electrotransformation procedure significantly elevated the electrotransformation efficiency up to 7.5 × 104 transformants µg-1 DNA, an increase of approximately three orders of magnitude. Key factors affecting the electrotransformation efficiency include cell-wall-weakening using glycine, ethanol-mediated membrane solubilization, field strength of the electric pulse, and sucrose osmoprotection. Following development of a gene transfer methodology, we next aimed to sequence the entire genome of C. pasteurianum. Using a hybrid approach involving 454 pyrosequencing, Illumina dye sequencing, and single molecule real-time sequencing platforms, we obtained a near-complete genome sequence comprised of 12 contigs, 4,420,100 bp, and 4,056 candidate protein-coding genes with a GC content of 30.0%. No extrachromosomal elements were detected. We provide an overview of the genes and pathways involved in the organism’s central fermentative metabolism. We used our developed electrotransformation procedure to investigate the use of established clostridial group II intron biology for constructing chromosomal gene knockout mutants of C. pasteurianum. Through methylome analysis of C. pasteurianum genome sequencing data and transformation assays of various vector deletion constructs, we identified a new Type I restriction-modification system that inhibits transfer of vectors harboring group II intron gene knockout machinery. We designated the new restriction system CpaAII and proposed a recognition sequence of 5’-AAGNNNNNCTCC-3’. Overcoming restriction by CpaAII, in addition to low intron retrohoming efficiency, allowed the isolation of a gene knockout mutant of C. pasteurianum with a disrupted CpaAI Type II restriction system. The resulting mutant strain should be efficienty transformed with plasmid DNA lacking M.FnuDII methylation. Lastly, we investigated the use of plasmid-based gene overexpression and chromosomal gene downregulation to alter gene expression in C. pasteurianum. Using a β-galactosidase reporter gene, we characterized promoters corresponding to the ferredoxin and thiolase genes of C. pasteurianum and show that both promoters permitted high-level, constitutive gene expression. The thiolase promoter was then utilized to drive transcription of an antisense RNA molecule possessing complementarity to mRNA of our β-galactosidase reporter gene. Our antisense RNA system demonstrated 52-58% downregulation of plasmid encoded β-galactosidase activity throughout the duration of growth. In an attempt to perturb the central fermentative metabolism of C. pasteurianum and enhance butanol titers, we prepared several antisense RNA constructs for downregulation of 1,3-propanediol, butyrate, and hydrogen production pathways. The resulting downregulation strains are expected to exhibit drastically altered central fermentative metabolism and product distribution. Taken together, we have demonstrated that C. pasteurianum is amendable to genetic manipulation through the development of methods for plasmid DNA transfer and gene overexpression, knockdown, and knockout. Further, our genome sequence should provide valuable nucleotide sequence information for the application of our genetic tools. Thus, the genome sequence, electrotransformation method, and associated genetic tools and techniques reported here should promote extensive genetic manipulation and metabolic engineering of this biotechnologically important bacterium.

Pour diversifier leur matériel génétique, s’adapter aux perturbations environnementales et coloniser de nouvelles niches, les bactéries utilisent plusieurs processus évolutifs dont l’acquisition de matériel génétique par transfert horizontal de gènes comme la conjugaison, la transduction et la transformation. À ces trois mécanismes naturels s’ajoute l’électrotransformation due aux phénomènes électriques liés à la décharge de foudre. La présence dans les nuages de bactéries aérosolisées capables de former des noyaux de glace à l’origine des précipitations et impliquées dans le déclenchement de la foudre, telles que la bactérie phytopathogène à répartition mondiale Pseudomonas syringae, nous a conduit à proposer que l’électrotransformation naturelle dans les nuages pouvait affecter les bactéries, contribuant ainsi à augmenter leur potentiel adaptatif. Dans un premier temps, nous avons déterminé si la bactérie glaçogène P. syringae pouvait survivre à des électroporations simulant des décharges de foudre et acquérir du matériel génétique exogène dans les nuages. Comparée à deux autres bactéries, P. syringae se révèle être mieux adaptée pour la survie et l’électrotransformation génétique, ce qui suggère qu’elle serait capable de survivre et d’évoluer durant son transport dans les nuages. Nous avons ensuite évalué l’impact d’électroporations simulant les décharges de foudre sur la survie, le potentiel d’électrotransformation et la diversité de bactéries présentes dans des échantillons de pluie comme substitut des communautés bactériennes des nuages. Ces dernières étaient plus résistantes que les souches de laboratoire et certaines étaient capables d’acquérir de l’ADN exogène par électrotransformation. Les bactéries de la pluie isolées provenaient de différentes origines et présentaient différents modes de vie, représentatifs des sources probables d’émissions de bactéries terrestres. Cette étude montre que les bactéries aérosolisées de divers écosystèmes terrestres sont susceptibles de se disséminer dans de nouveaux habitats grâce aux nuages tout en étant capable d’acquérir de nouveaux gènes par éléctrotransformation, et d’augmenter ainsi potentiellement leur diversité génétique. La dernière partie de mon travail a évalué si l’électrotransformation appliquée aux bactéries indigènes du sol pouvait être employée pour dépolluer les sols contaminés par un pesticide largement utilisé autrefois, le lindane. L’optimisation des expériences met en évidence l’incorporation par les bactéries indigènes d’un plasmide contenant le gène codant les premières déchlorinations du lindane au travers d’une combinaison de transformation naturelle et d’électrotransformation. En conclusion, nous avons montré que l’électrotransformation naturelle liée aux décharges électriques, comme celles se produisant dans les nuages ou atteignant le sol, peut être impliquée dans le transfert horizontal de gènes chez les bactéries et, considérant l’importance de la foudre à travers le monde, pourrait jouer un rôle dans l’adaptation et l’évolution de ces organismes. / To diversify their genetic material, allowing adaptation to environmental disturbances and colonization of new ecological niches, bacteria use various evolutionary processes, including the acquisition of new genetic material by horizontal transfer mechanisms such as conjugation, transduction and transformation. Electrotransformation mediated by lightningrelated electrical phenomena may constitute an additional gene transfer mechanism occurring in nature. The presence in clouds of bacteria capable of forming ice nuclei that lead to precipitations and are involved in the triggering of lightning, such as the global phytopathogen Pseudomonas syringae, led us to postulate that natural electrotransformation in clouds may affect bacteria, by contributing to increase their adaptive potential. We first determined if the ice nucleator bacterium P. syringae could survive when in clouds and acquire exogenous genetic material through lightning shock-simulating in vitro electroporation. In comparison to two other bacteria, P. syringae appears to be best adapted for survival and for genetic electrotransformation under these conditions, which suggests that this bacterium would be able to survive and evolve whilst being transported in clouds. Secondly, we evaluated the impact of lightning shock-simulating in vitro electroporation on the survival, the electrotransformation potential and the diversity of bacteria collected from rain samples. These isolates better resisted lightning than the laboratory strains and some were able to electrotransform exogenous DNA. The rain bacteria we isolated were of different origins and were representative of life modes of the various sources of bacterial emissions on Earth. Our study suggests that bacteria aerosolized from diverse terrestrial ecosystems can spread to new habitats through clouds whilst also being able to acquire new genetic material via lightning-based electrotransformation, thereby potentially enhancing their genetic diversity. The final part of our work consisted of evaluating whether electrotransformation could be applied to the engineering of indigenous soil bacteria in order to develop a tool for the bioremediation of lindane, a once widely used pesticide. Optimized experiments revealed that both natural and electrotransformation contributed to the incorporation of a plasmid harboring a gene encoding the first lindane dechlorination steps by indigenous soil bacteria. In conclusion, we showed that natural electrotransformation mediated by electrical discharges such as those occurring in clouds or reaching soils can be involved in the horizontal gene transfer process among bacteria and, considering the importance of lightning worldwide, may play a role in the adaptation and evolution of these organisms.