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The ubiquitous nature of antibiotic resistance and antibiotic resistance genes (ARGs) among environmental pathogens from a variety of wastewater effluents, suggests that the aquatic environment, and specifically alternative wastewater treatment systems, may act as reservoirs for drug resistant bacteria and ARGs, thereby contributing to the widespread dissemination of antibiotic resistance. More research is necessary to contribute to our understanding of the occurrence, fate, and mobility of antibiotic resistance and ARGs among bacterial indicators of faecal contamination as well as pathogenic bacteria within Canadian wastewater treatment systems. The primary objective of this research was to determine the prevalence, fate, and potential transfer of bacterial resistance and ARGs among selected environmental pathogens exposed to alternative wastewater treatment systems, while considering the impact of treatment strategies on the expression of antibiotic resistance. A detailed analysis was initially conducted with respect to the characterization and quantification of microbial populations (including antibiotic resistant bacteria) in a variety of treatment systems and waste effluent sources. Traditional culture-based screening techniques in combination with molecular characterization (through colony or multiplex PCR), and molecular quantification using real-time quantitative PCR were utilized in order to help establish a preliminary environmental assessment of selected pathogens (Escherichia coli, Enterococcus spp., Salmonella spp.) and ARGs (tetA, blaSHV, & ampC) within a variety of wastewater treatment systems (lab-scale mesocosms, constructed wetland, constructed lagoon system, and pilot-scale biological nutrient removal (BNR) system). Overall, the level of multiple antibiotic resistance (MAR) among culturable indicator (E. coli & Enterococcus spp.) and environmental bacteria was high (reaching 100% in several instances) within different types of wastewater treatment systems and effluent sources (poultry waste effluent, municipal wastewater, aquaculture wastewater). Common antibiotic resistance profiles among E. coli isolates included simultaneous resistance to between three and five antimicrobials, whereas common MAR profiles among Enterococcus spp. isolates showed resistance to ten or more antibiotics. Real time quantitative PCR was used to determine the concentration of three bacterial pathogens; E. coli, Enterococcus faecalis, and Salmonella spp., and three ARGs; tetA, ampC, and blaSHV, within a variety of wastewater samples. Based on the results, it was concluded that high concentrations of ARGs were present in the treated effluent (10⁴- 10⁶ target gene copies/100 mL), regardless of system type (i.e. constructed lagoon, pilot-scale BNR, or constructed wetland), which may ultimately serve as a potential route for entry of ARGs and antibiotic resistant bacteria into the natural environment. Water is considered an important medium for transfer of resistance genes and resistant bacteria to the broader environment. Few studies have examined the transferability via conjugation of ARGs in E. coli and Salmonella spp. isolated from wastewater. Identification of three resistance determinants (tetA, strA, strB) conferring resistance to tetracycline and streptomycin was performed on selected multi-drug resistant Salmonella spp. and E. coli isolates. The potential for transfer of tetracycline and streptomycin resistance genes was demonstrated through broth conjugation experiments using multi-drug resistant Salmonella spp. and E. coli isolates as donors, and E. coli K12 as the recipient. Conjugation was successfully observed in 75% (9/12) of donor isolates, occurring in both Salmonella spp. and E. coli isolates. Six strains (50%) were capable of transferring their tetA, strA, and strB genes to the recipient strain, resulting in 58.5% (38/65) of total transconjugant strains acquiring all three resistance determinants. The results confirm the role of environmental bacteria (isolated from wastewater treatment utilities) as a reservoir of antibiotic resistance and ARGs, containing mobile genetic elements, which are capable of disseminating and transferring ARGs. As concerns about water quality and environmental contamination by human and agricultural effluents have increased, it has become increasingly more important to consider the prevalence and transferability of ARGs to opportunistic and human pathogens. As observed in this research, the ubiquitous nature of multi-drug resistant bacteria in water and wastewater effluents, the presence of diverse ARGs of human and veterinary health significance, as well as the transfer of resistance determinants through conjugative plasmids to recipient bacteria, suggests that environmental exposure through contact or consumption with contaminated water is probable. However, a lack of critical information still exists regarding the movement of resistance genes within and between microbial populations in the environment. In addition, the extent of human exposure to ARGs and antibiotic resistant bacteria is still not well understood, and future studies on human exposure to these resistant contaminants are necessary.

Many diverse microbes have been detected in or isolated from glaciers, including novel taxa exhibiting previously unrecognized physiological properties with significant biotechnological potential. Of 29 unique phylotypes isolated from Victoria Upper Glacier, Antarctica (VUG), 12 were related to the poorly studied bacterial genus Hymenobacter including several only distantly related to previously described taxa. Further study of these microorganisms revealed genotypic, phenotypic, morphological and chemotaxonomic divergence from named species and suggested that they likely represent novel Hymenobacter species. These studies also indicated, however, that the systematics of Hymenobacter and related microorganisms is more complex than previously realized, and may exhibit poorly defined species boundaries due to cosmopolitan dispersal, significant rates of horizontal gene transfer and reintroduction of archived genotypes, e.g., from glacial ice. These processes are reflected in the carotenoid composition of Hymenobacter and related organisms, which includes several novel methyl- and xylosyl-derivatives of 2'-hydroxyflexixanthin with distributions indicative of horizontal gene transfer or differential gain and/or loss of terminal biosynthetic pathway steps. These processes have been previously underappreciated in assessments of microbial carotenoid diversity and suggest the need for fine-scale phylogenetic study of carotenoid distribution in other microbial taxa. Further comparative genomics-based evaluation of microbial carotenoid biosynthesis indicated its wide phylogenetic distribution and diversification, controlled by several lineage-specific modes of evolution including horizontal transfer, de novo enzyme evolution followed by differential gene loss, co-evolution with biochemically associated structures and elevated mutation rates. The latter especially interacts with horizontal transfer depending on metabolic pathway topology, exemplified by the evolution of purple bacterial carotenoid biosynthesis. Exploration of VUG microbial diversity, therefore, not only revealed novel taxa and biotechnologically interesting compounds but also spurred broader evaluation of the mechanisms of metabolic pathway evolution applicable to many other taxa and biochemical pathways. / Microbiology and Cell Biotechnology

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 5 uppsatser.

Les progrès en biotechno]ogie ont permis le développement d’outils pour le transfert de gène intégratif en transgénèse, bioproduction et thérapie génique. Cependant, trois challenges majeurs doivent être relevés pour garantir un système sécurisé : l’innocuité et l’efficacité du transfert, l’intégration ciblée et contrôlée dans le génome, le niveau et la durée d’expression du transgène au cours du temps. Dans ce but, mes travaux de thèse ont consisté à tester des solutions pour améliorer la biosécurité du transposon piggyBac qui nécessite un plasmide porteur du gène d’intérêt à insérer dans le génome et une source de transposase catalysant la réaction d’intégration du transgène. Une des stratégies de ma thèse repose sur l’apport de la source de transposase sous forme d’ARN messager au lieu d’ADN afin d’améliorer la stabilité de l’intégration et de réduire les effets génotoxiques en limitant la transposase dans les cellules. Pour la première fois, la biodisponibilité de l’ARNm de la transposase et les conditions optimales d’utilisation en cellules humaines ont été déterminées pour augmenter la biosêcurité du système. Le second objectif de mes travaux consiste à améliorer l’expression du transgène en ajoutant des insulateurs connus pour s’opposer à l’extinction de l’expression des gênes. En termes de biosécurité, cette stratégie permet de réduire le nombre de copies du transgène nécessaires pour obtenir une expression suffisante. Deux candidats ont été identifiés pour améliorer l’expression du transgène. La combinaison des approches ARNrn et insulateurs est prometteuse pour sécuriser le transfert de gène médié par piggyBac et pour maintenir l’expression du gène d’intérêt. / Advances in biotechnology have enabled the development of tools for gene transfer applicable to transgenesis, bioproduction and gene therapy. But, 3 major challenges must be met to ensure a secure system: the safety and effectiveness of the transfer. the targeted and controlled integration into the genome. and the level of transgene expression over time. In this aim, my thesis project was to validate solutions to improve the biosafety of the piggyBac transposon, which requires a plasmid carrying the gene of interest to be inserted in the genome, and a source of transposase which catalyzes the transgene integration. One approach of my thesis work is to deliver the source of piggyBac transposase as an mRNA molecule instead of DNA. This strategy aims to improve the stability of the integration and reduce the genotoxic effects by limiting the transposase in the cells. For the 1st time, the bioavailability of the transposase rnRNA and the optimal conditions for its use in human cells were determined to increase the biosafety of the transposon system. The 2nd objective ofmy project is to improve the expression of the transgene by adding insulators known to counteract the transgene silencing. This strategy reduces the number of integrations required ta get a sufficient expression of the transgene and thus, improve biosecurity. Two candidates have been identified to improve transgene expression. The combination of the mRNA and insulator strategies is promising to secure the piggyBac-mediated gene transfer and to maintain the expression of the gene of interest

Des études récentes ont démontré que les activités des agents de contraste ultrasonoresous l’effet des ultrasons modulent transitoirement la perméabilité de la membraneplasmique des cellules. Ce procédé aussi appelé sonoporation est étudié pour incorporer desmolécules extracellulaires telles que des gènes thérapeutiques. Le but de la thèse fut d’étudierl’effet des paramètres acoustiques et expérimentaux pour une transfection efficace des cellulesin vitro et in vivo. Parallèlement, afin de clarifier le mécanisme de perméabilisation induit lorsde la sonoporation, nous avons, d’une part, étudié l’effet des ultrasons et des microbulles surla libération de molécules intracellulaires et d’autre part, observé en microscopie électroniqueles effets des ultrasons et des microbulles sur la membrane plasmique et sur l’incorporation denanoparticules d’or. / Future applications of ultrasound and microbubbles extend beyond imaging applications.Over the last few years, reports have shown that the activation of contrast microbubblesunder ultrasound waves, transiently modulates the cell membrane permeability. This process,named sonoporation has been studied to incorporate extracellular molecules such as therapeuticgenes. The aim of this work was to study the effect of various interrogation parameters toachieve an efficient cell transfection in vitro as well as in vivo. In parallel, and in order to clarifythe mechanism of permeabilization induced by sonoporation, we investigated the effect ofultrasound and microbubbles on release of intracellular molecules from fluorescent cells. Moreover,using electron microscopy, we examined the effect of the sonoporation process on thecell membrane and on incorporation of gold nanoparticles.

Orientador: Maria do Carmo Bittencourt de Oliveira / Banca: Luiz Henrique Zanini Branco / Banca: Mariana Cabral de Oliveira / Resumo: As cianobactérias apresentam uma ampla variabilidade fenotípica e ecológica. Porém, esta variabilidade, muitas vezes, não corresponde à sua diversidade genética. Assim, o uso de marcadores moleculares é fundamental para os estudos de filogenia neste grupo. Entretanto, a filogenia molecular enfrenta um desafio na seleção dos marcadores devido à ocorrência relativamente frequente da transferência de genes de forma lateral entre os procariotos. Em cianobactérias os marcadores dos espaçadores dos genes ribossomais (16S-23S-ITS rDNA) e do operon da ficocianina (PC-IGS) estão entre os mais utilizados nestes estudos. Contudo, alguns trabalhos sugerem que o PC-IGS possa ter sito transferido lateralmente em sua história evolutiva. A identificação de morfoespécies dos gêneros Microcystis e Geitlerinema é baseada em caracteres morfológicos que em geral não correspondem à sua variabilidade genética. Com o objetivo de investigar a transferência lateral do operon da ficocianina em Geitlerinema e Microcystis, foram obtidas e comparadas árvores filogenéticas de ambas espécies baseadas nos marcadores PC-IGS e 16S-23S-ITS rDNA. As topologias das árvores obtidas para ambos os marcadores foram muito semelhantes e indicaram que o PC-IGS é estável e indicado para os estudos de taxonomia e filogenia de linhagens de Geitlerinema e Microcystis. Assim, hipótese inicial de transferência lateral foi refutada. Algumas linhagens tiveram seu posicionamento divergente entre um marcador e outro, o que ressalta a importância do uso de mais de um marcador em estudos de filogenia. O marcador PC-IGS apresentou melhor desempenho que 16S-23S-ITS rDNA. As árvores filogenéticas de Geitlerinema baseadas em ambos os marcadores indicaram a ocorrência de espécies crípticas dentre as linhagens estudadas e corroboraram que G. amphibium e G. unigranulatum devem ser consideradas sinonímias... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Cyanobacteria show a wide phenotypic and ecological variability, but frequently this variability does not correspond to their genetic variation. Therefore, the use of molecular markes is critical for phylogenetic studies in this group. At the same time, the selection of molecular markers represents a challenge for the molecular phylogeny due to the horizontal gene transfer, witch is a relatively common process among the prokaryotes. In cyanobacteria, makers for the ribosomal genes spacer (16S-23S-ITS rDNA) and for the phycocyanin operon spacer (PC-IGS) are among of the most used for phylogeny. However, some studies suggest that the PC-IGS marker may have been horizontally transferred during its evolutionary history. The identification of the morphospecies from the genus Microcystis and Geitlerinema is based in their morphological characters, but they generally do not correspond to genetic variability. In order to investigate the possibility of horizontal transfer of the phycocyanin operon in Microcystis and Geitlerinema, phylogenetic trees based on the PC-IGS and 16S- 23S-ITS rDNA were generated and compared. The topologies obtained for both markers were very similar, indicating that the PC-IGS marker is stable and suitable for taxonomical and phylogenetic studies in Microcystis and Geitlerinema. Therefore, the initial hypothesis of horizontal transfer was rejected. Some strains were found to have divergent positions between the trees based on the two molecular markes, witch highlights the importance of using more than one marker in phylogenetic studies. The PC-IGS marker performed better than 16S-23SITS rDNA. The Geitlerinema phylogenetic trees based on both markers indicated the occurrence of cryptic species among the strains and corroborated that G. amphibium and G. unigranulatum should be treated as synonyms. The phylogenetic tree based on PC-IGS formed a monophyletic clade... (Complete abstract click electronic access below) / Mestre

Sequenced diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum belong to the chromist algae harboring secondary plastids, which display distinct evolutionary history when compared to photosynthetic organelles from rhodophytes, green algae and plants. Via secondary endosymbiosis, heterotrophic eukaryotic ancestor of diatoms engulfed red alga, and in addition to the new organelle, it obtained fitness increasing peculiarities in the chimerical cell metabolism and lifestyle. We examined phylogeny and in silico localization of the nuclear-encoded but plastid located enzymes of tryptophan biosynthesis. We suggest that the diatom tryptophan pathway represents an extreme in the trend of plastid (cyanobacterial) enzymes to be replaced by eukaryotic isoforms. In addition, the gene napped during the endosymbiotic gene transfer from the diatom plastid genome to the diatom nucleus (psb28) was described.

Genes encoding proteins that carry out essential informational tasks in the cell, in particular where multiple interaction partners are involved, are less likely to be transferable to a foreign organism. Here, we investigated the constraints on transfer of a gene encoding a highly conserved informational protein, translation elongation factor Tu (EF-Tu), by systematically replacing the endogenous tufA gene in the Escherichia coli genome with its extant and ancestral homologs. The extant homologs represented tuf variants from both near and distant homologous organisms. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 billion years ago (bya). Our results demonstrate that all of the foreign tuf genes are transferable to the E. coli genome, provided that an additional copy of the EF-Tu gene, tufB, remains present in the E. coli genome. However, when the tufB gene was removed, only the variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth which demonstrates the limited functional interchangeability of E. coli tuf with its homologs. Relative bacterial fitness correlated with the evolutionary distance of the extant tuf homologs inserted into the E. coli genome. This reduced fitness was associated with reduced levels of EF-Tu and reduced rates of protein synthesis. Increasing the expression of tuf partially ameliorated these fitness costs. In summary, our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria. IMPORTANCE Horizontal gene transfer (HGT) is a fundamental driving force in bacterial evolution. However, whether essential genes can be acquired by HGT and whether they can be acquired from distant organisms are very poorly understood. By systematically replacing tuf with ancestral homologs and homologs from distantly related organisms, we investigated the constraints on HGT of a highly conserved gene with multiple interaction partners. The ancestral homologs represented phylogenetically resurrected tuf sequences dating from 0.7 to 3.6 bya. Only variants obtained from the gammaproteobacterial family (extant and ancestral) supported growth, demonstrating the limited functional interchangeability of E. coli tuf with its homologs. Our analysis suggests that the functional conservation of protein activity, the amount of protein expressed, and its network connectivity act to constrain the successful transfer of this essential gene into foreign bacteria.