Le mercure (Hg) est un métal lourd ubiquiste et très toxique. Présent à l’état de traces dans la colonne d’eau des milieux aquatiques, il peut cependant atteindre des concentrations très élevées en fin de chaine alimentaire car il a la particularité d’être bioaccumulé et bioamplifié dans les organismes sous forme de méthylmercure (MeHg). L’identification et la caractérisation des transformations amenant à la formation de MeHg (méthylation) ou à sa dégradation (déméthylation) sont donc de première importance pour évaluer son devenir dans les milieux aquatiques. L’utilisation du comportement des isotopes stables du Hg, à la fois en laboratoire mais aussi dans des échantillons environnementaux, a permis d’évaluer l’influence des processus biotiques et abiotiques mis en jeu dans les système aquatiques sur les transformations et donc la spéciation du Hg dans de tels environnements. Le fractionnement isotopique du Hg, pouvant être dépendant et/ou indépendant de la masse, s’est également avéré être un outil performant pour étudier sa bioaccumulation dans plusieurs membres de la chaîne alimentaire endémique du Lac Baikal (Russie). Les signatures isotopiques mesurées dans ces échantillons ont permi d’améliorer la connaissance sur la distribution, les sources et les transformations affectant les espèces du Hg dans l’écosystème de ce grand lac faisant partie du patrimoine mondial de l’UNESCO depuis 1996 et étant la plus grande réserve d’eau douce liquide de surface mondiale. / Mercury (Hg) is a toxic and ubiquitous heavy metal. Only present at trace levels in the water column of aquatic systems, it can reach high amounts in food web end-members because of its ability to bioaccumulate and biomagnify in organisms as methylmercury (MeHg). Hence, the characterization of the transformations leading to the formation (methylation) and the degradation (demethylation) of MeHg is of great concern to evaluate its fate in aquatic environments. The use of the Hg stable isotopes, during laboratory experiments or in environmental samples, allowed to identify and characterize several biotic and biotic pathways involved in Hg transformations and speciation in aquatic systems. The study of Hg mass-dependent and/or mass-independent fractionation was also a competitive tool to assess its bioaccumulation process in several members of the Lake Baikal endemic food chain (Russia). Measured Hg isotopic signatures in such samples provided insight about Hg species fate and sources within the ecosystem of this lake, which has been nominated as a world heritage site by UNESCO in 1996 and constitutes the world’s largest freshwater lake in terms of volume.

Bactérias endofíticas e epifíticas podem conferir ao seu hospedeiro características como maior resistência a condições de estresse, alterações nas condições fisiológicas, fixação de nitrogênio atmosférico, suprimento de nutrientes, produção de reguladores de crescimento vegetal, entre outros. Desta forma, o presente trabalho teve por objetivos estudar a composição da comunidade bacteriana associada à soja e avaliar diferentes mecanismos de interação bactéria-planta hospedeira. Para isso, bactérias endofíticas e epifíticas de folhas, caules e raízes de duas cultivares de soja, crescidas em solo com e sem aplicação pré-plantio do herbicida glifosato, foram amostradas em três estádios de desenvolvimento do hospedeiro, durante as safras de 2000/01 e 2001/02. Foram observadas diferenças significativas na diversidade e densidade bacterianas em relação às fases de crescimento da soja e tecidos da planta. Os principais grupos foram identificados como pertencentes aos gêneros Pseudomonas, Burkholderia, Ralstonia, Enterobacter, Pantoea, Acinetobacter, Agrobacterium e Methylobacterium. Além da avaliação de populações cultiváveis, análise por DGGE revelou que a comunidade bacteriana endofítica de raiz de soja pode ser influenciada pelo tratamento do solo com o herbicida glifosato. O potencial destas bactérias para a promoção de crescimento vegetal por bactérias associadas à soja foi avaliado, sendo possível observar que populações endofíticas e epifíticas de soja apresentam características relacionadas à promoção de crescimento vegetal e que fatores como cultivar e estádio fenológico do hospedeiro podem influenciar as freqüências destas populações. A análise da variabilidade genética destas populações bacterianas revelou que diferentes fatores ambientais também podem influenciar a diversidade de grupos bacterianos. Além disso, populações endofíticas com capacidade de crescer na presença do herbicida glifosato foram caracterizadas e identificadas como pertencentes às espécies Burkholderia gladioli e Pseudomonas oryzihabitans, enquanto que Methylobacterium spp. colonizam ativamente a superfície e os tecidos internos de soja após inoculação via semente. Os resultados obtidos podem oferecer uma contribuição para a melhor compreensão da interação microrganismossoja e, conseqüentemente, de sua aplicação na cultura deste vegetal. / Endophytic and epiphytic bacteria may increase the fitness of the plant host by increasing resistance to stress conditions, alterations in the physiologic conditions, fixation of atmospheric nitrogen, nutrient supplying and plant growth regulators production. The aims of the present work were to study the composition of soybean-associated bacterial community and to evaluate different mechanisms for bacteria-host plant interaction. For that, endophytic and epiphytic bacteria from leaves, stems and roots of two soybean cultivars, planted in soil with and without pre-planting application of the glyphosate herbicide, they were colleted in three development stages of the host, during two crops. Significant differences were observed in the bacterial diversity and population density in relation to the soybean growth stages and plant tissues. The principal groups were identified as belonging to the genera Pseudomonas, Burkholderia, Ralstonia, Enterobacter, Pantoea, Acinetobacter, Agrobacterium and Methylobacterium. Besides the evaluation of cultivable populations, analyses by DGGE revealed that the endophytic bacterial community from soybean roots may be influenced by the treatment of the soil with the glyphosate herbicide. Other analyzed aspect was the potential for plant growth promotion by soybean-associated bacteria; revealing that soybean's endophytic and epiphytic populations presented characteristics related to the plant growth promotion; factors such as cultivar and developmental stage of the host may influence the frequency of these populations. Environmental factors may affect the genetic variability of these bacterial populations. Besides, endophytic populations able to growth in medium containing glyphosate were characterized and identified as belonging to Burkholderia gladioli and Pseudomonas oryzihabitans species. Methylobacterium spp. were reintroduced in soybean seeds and superficial and endophytic colonization were evaluated by scanning electronic microscopy. The obtained results could offer a contribution for a better understanding of the interaction microorganism-soybean and, consequently, their possible use to improve soybean productivity.

Bactérias endofíticas e epifíticas podem conferir ao seu hospedeiro características como maior resistência a condições de estresse, alterações nas condições fisiológicas, fixação de nitrogênio atmosférico, suprimento de nutrientes, produção de reguladores de crescimento vegetal, entre outros. Desta forma, o presente trabalho teve por objetivos estudar a composição da comunidade bacteriana associada à soja e avaliar diferentes mecanismos de interação bactéria-planta hospedeira. Para isso, bactérias endofíticas e epifíticas de folhas, caules e raízes de duas cultivares de soja, crescidas em solo com e sem aplicação pré-plantio do herbicida glifosato, foram amostradas em três estádios de desenvolvimento do hospedeiro, durante as safras de 2000/01 e 2001/02. Foram observadas diferenças significativas na diversidade e densidade bacterianas em relação às fases de crescimento da soja e tecidos da planta. Os principais grupos foram identificados como pertencentes aos gêneros Pseudomonas, Burkholderia, Ralstonia, Enterobacter, Pantoea, Acinetobacter, Agrobacterium e Methylobacterium. Além da avaliação de populações cultiváveis, análise por DGGE revelou que a comunidade bacteriana endofítica de raiz de soja pode ser influenciada pelo tratamento do solo com o herbicida glifosato. O potencial destas bactérias para a promoção de crescimento vegetal por bactérias associadas à soja foi avaliado, sendo possível observar que populações endofíticas e epifíticas de soja apresentam características relacionadas à promoção de crescimento vegetal e que fatores como cultivar e estádio fenológico do hospedeiro podem influenciar as freqüências destas populações. A análise da variabilidade genética destas populações bacterianas revelou que diferentes fatores ambientais também podem influenciar a diversidade de grupos bacterianos. Além disso, populações endofíticas com capacidade de crescer na presença do herbicida glifosato foram caracterizadas e identificadas como pertencentes às espécies Burkholderia gladioli e Pseudomonas oryzihabitans, enquanto que Methylobacterium spp. colonizam ativamente a superfície e os tecidos internos de soja após inoculação via semente. Os resultados obtidos podem oferecer uma contribuição para a melhor compreensão da interação microrganismossoja e, conseqüentemente, de sua aplicação na cultura deste vegetal. / Endophytic and epiphytic bacteria may increase the fitness of the plant host by increasing resistance to stress conditions, alterations in the physiologic conditions, fixation of atmospheric nitrogen, nutrient supplying and plant growth regulators production. The aims of the present work were to study the composition of soybean-associated bacterial community and to evaluate different mechanisms for bacteria-host plant interaction. For that, endophytic and epiphytic bacteria from leaves, stems and roots of two soybean cultivars, planted in soil with and without pre-planting application of the glyphosate herbicide, they were colleted in three development stages of the host, during two crops. Significant differences were observed in the bacterial diversity and population density in relation to the soybean growth stages and plant tissues. The principal groups were identified as belonging to the genera Pseudomonas, Burkholderia, Ralstonia, Enterobacter, Pantoea, Acinetobacter, Agrobacterium and Methylobacterium. Besides the evaluation of cultivable populations, analyses by DGGE revealed that the endophytic bacterial community from soybean roots may be influenced by the treatment of the soil with the glyphosate herbicide. Other analyzed aspect was the potential for plant growth promotion by soybean-associated bacteria; revealing that soybean's endophytic and epiphytic populations presented characteristics related to the plant growth promotion; factors such as cultivar and developmental stage of the host may influence the frequency of these populations. Environmental factors may affect the genetic variability of these bacterial populations. Besides, endophytic populations able to growth in medium containing glyphosate were characterized and identified as belonging to Burkholderia gladioli and Pseudomonas oryzihabitans species. Methylobacterium spp. were reintroduced in soybean seeds and superficial and endophytic colonization were evaluated by scanning electronic microscopy. The obtained results could offer a contribution for a better understanding of the interaction microorganism-soybean and, consequently, their possible use to improve soybean productivity.

O fungo Pleurotus ostreatus pertence ao grupo de basidiomicetos que degradam madeira. Este cogumelo cultivado em todo mundo apresenta grande rusticidade e produtividade, e pode ainda ser usado em processos de biorremediação e biopolpação. Devido a seu potencial biotecnológico, torna-se interessante a compreensão da interação deste com outros microrganismos. Neste sentido, recentemente foi observada a presença de bactérias associadas a P. ostreatus em culturas in vitro, que apresentavam grande pleomorfismo. A partir desta observação foram elaborados ensaios que visaram a confirmação da presença de bactérias. Para tanto, foi utilizada a estratégia do “Ciclo Completo de Análise do rRNA” (full-cycle rRNA analysis) empregada em microrganismos não cultiváveis ou de crescimento fastidioso, além do emprego de técnicas de microbiologia básica, e de estudos de ultraestrutura. Os estudos de microbiologia básica indicaram que se tratava de um microrganismo fastidioso e que se desenvolvia melhor na presença do fungo em sistema de co-cultivo em meios contendo Tween 80 ou Tween 20. Por sua vez, a análise de ultraestrutura demonstrou a presença de estruturas pleomórficas, tanto internamente como externamente à hifa. Em relação ao “Ciclo completo de Análise do rRNA” este se iniciou pela amplificação e seqüenciamento de parte do rDNA bacteriano, que revelou a proximidade desta bactéria com o Complexo Burkholderia cepacia (CBC). A partir desta seqüência, foi realizado um estudo de bioinformática que indicou sondas específicas para este grupo de bactérias. Completando o Ciclo completo de Análise do rRNA, foram realizados ensaios de hibridização in situ fluorescente (FISH) para a confirmar a relação entre as estruturas bacterianas e a seqüência obtida. Este método comprovou a presença das bactérias no interior das hifas de P. ostreatus. Este trabalho constitui o primeiro relato de bactérias pleomórficas pertencente ao complexo B. cepacia associados a P. ostreatus. / The fungus Pleurotus ostreatus, which belongs to white rot basidiomycete group, is a widely cultivated mushroom; this species has high productivity and rusticity, besides its use in biobleaching and bioremediation processes. This biotechnological potential justifies microbial interaction studies between this fungi and others microorganisms. In P. ostreatus mycelia, it has been observed pleomorphic bacteria growing on agar media. This research describes several assays to confirm bacterial presence in this sample. Therefore, the full-cycle rRNA analysis (described for unculturable or fastidious microorganism), ultrastructure and basic microbiology approaches were employed. Basic microbiology approaches indicated slow growing bacteria, which grown faster near to fungi colonies in solid media amended with Tween 80 or Tween 20 (co-culture system). Ultrastructure studies confirm the presence of intracellular and extracellular pleomorphic bacteria. The full-cycle rRNA analysis started with 16S rDNA amplification and sequencing. This approach demonstrated a relation between these bacteria with Burkholderia cepacia complex. By bioinformatics analysis was determinate which DNA probes can be use to identified this bacterial group. The last step for full-cycle rRNA analysis was applying fluorescent in situ hybridization (FISH). This technique confirmed the relationship between 16S rDNA bacterial sequence and bacterial forms. This is the first time that a pleomorphic bacteria from B. cepacia complex is found associated with P. ostreatus.

L’augmentation de température que subit la planète ces dernières décennies a de nombreuses conséquences dont la recrudescence de maladies infectieuses aussi bien chez l’homme que chez les animaux. Certaines populations de l’ormeau européen Haliotis tuberculata, vivant dans les zones les plus chaudes de Bretagne et de Normandie ont ainsi subi de très importantes mortalités depuis 1997, dues à la bactérie Vibrio harveyi. Cependant, certaines des populations les plus sévèrement touchées se sont aujourd’hui reconstruites et les mortalités semblent s’être arrêtées dans certaines de ces zones. La question se pose donc de l’apparition d’une résistance de l’ormeau face à cette maladie émergente. Pour répondre à cette question, les réponses à l’infection de plusieurs populations naturelles par cette bactérie ont été analysées. Une population présentant une forte résistance à la maladie a été identifiée.La voie d’entrée de la bactérie (ie. les branchies) a été identifiée comme jouant un rôle dans la résistance à l’infection. Par ailleurs, des infections successives ont permis de démontrer un effet d’amorçage immunitaire. Suite à une première exposition, une protection durant jusqu’à deux mois intervient contre l’effet d’inhibition de la phagocytose, provoquée normalement par une infection à V. harveyi. La différence d’expression de gènes des hémocytes d’ormeaux sensibles et résistants a été quantifiée par RNAseq pendant une infection expérimentale. Cette comparaison a montré une reconnaissance plus efficace du pathogène chez les résistants, par des récepteurs tels que les TLR ou les PGRP. La forte surexpression chez la population résistante, d’un gène impliqué dans la synthèse de mucine qui est l’un des composants principaux du mucus renforce l’hypothèse d’une forte implication des branchies dans la résistance. Enfin, une analyse in silico des séquences obtenues en RNAseq a permis d’apporter des preuves de l’existence d’un système de méthylation de l’ADN chez H. tuberculata ainsi qu’une possible implication de ce système dans l’adaptation de l’ormeau à son milieu. / Increasing global temperatures have numerous consequences for marine ecosystems, including the rise of infectious diseases. Certain populations of the European abalone Haliotis tucerculata have suffered from severe and recurrent mortality since 1997 due to infection caused by the bacterium Vibrio harveyi, particularly in areas with higher average summer temperatures. Given the spatial heterogeneity in mortalities, and the observation that the historically most severely impacted populations have recovered in recent years, the question of the emergence of resistance to the disease was addressed. The mortality rate in response to infection by V. harveyi was quantified experimentally in abalone originating from three natural populations, and one population exhibiting resistance to the disease was identified. In a subsequent experiment, the immune response of abalone was compared between infected individuals from a resistant and from a susceptible population. The portal of entry of the bacterium (ie. gills) was identified as playing a role in resistance. Furthermore, successive exposures of abalone to the bacterium demonstrated an immune priming effect, such that following a first exposure, phagocytosis was no longer inhibited by infection with V. harveyi, and that this improved protection against the disease lasted for at least two months. Differences in gene expression was quantified by RNAseq in the hemocytes of resistant and susceptible abalone following exposure to the pathogen. This comparison showed that resistant abalone had more effective recognition of the bacterium by receptors as the TLR or PGRP. The substantial over-expression of a gene involved in the synthesis of mucin, the main component of mucus, (UDP-GalNAC) in the resistant population, supports the interpretation of a strong involvement of gills in the resistance. Finally, an in-silico analysis of the sequences obtained from RNAseq indicate the existence of a DNA methylation system in H. tuberculata and suggested an involvement of epigenetic mechanisms in the adaptation of abalone to its environment.

H. pylori has a panmictic population structure due to high genetic diversity. The homoplasy index for H. pylori is 0.85 (where 0 represents a completely clonal organism and 1.0 indicates a freely recombining organism) which is much higher than homoplasy index for E. coli (0.26) or naturally competent Neisseria meningitides (0.34). It undergoes both inter as well as intra strain transformation. Intergenomic recombination is subject to strain specific restriction in H. pylori. Hence, a high homoplasy index means that competence predominates over restriction in H. pylori. Annotation of the genomes of H. pylori strains 26695 and J99 show the presence of nearly two dozen R-M systems out of which 16 were postulated to be Type II for J99. H. pylori has been described to be an ideal model system for understanding the equilibrium between competing tension of genomic integrity and diversity (42). R-M systems allow some degree of sexual isolation in a population of competent cells by acting as a barrier to transformation. The mixed colonizing population of H. pylori has a polyploidy nature where each H. pylori strain adds to ‘ploidy’ of the colonizing population. Maintenance of polyploidy nature of mixed colonizing population in a selective niche of stomach needs a barrier to free gene flow. Restriction barrier maintains a polyploidy nature of H. pylori population which is considered as yet another form of genetic diversity helping in persistence of infection. Thus, according to the model proposed by Kang and Blaser, where H. pylori are considered as perfect gases like bacterial population, transformation and restriction both add to genetic diversity of the organism. Again, restriction barriers are not completely effective, which could be due to cellular regulation of restriction system. Thus, a perfect balance between restriction and transformation in turn regulates the gene flow to equilibrate competition and cooperation between various H. pylori strains in a mixed population. RecA, DprA and DprB have been shown to be involved in the presynaptic pathway for recombination substrates brought in through the Com system. Biochemical characterization of HpDprA, during this study revealed its ability to bind to ssDNA and dsDNA. Binding of HpDprA to both ssDNA as well as dsDNA results in large nucleoprotein complex that does not enter the native PAGE. However, DNA trapped in the wells could be released by the addition of excess of competitor DNA, illustrating that the complex are formed reversibly and do not represent dead-end reaction products. Transmission electron microscopy for SpDprA interaction with ssDNA established that a large nucleoprotein complex consisting of a network of several DNA molecules bridged by DprA is formed which is retained in the well. A large DNA-protein complex that sits in the well has also been observed with other DNA binding proteins like RecA. It has been observed for ssDNA binding protein (SSB) that they bind non-specifically to dsDNA under low salt condition (20 mM NaCl) in the absence of Mg2+. The non specific binding of SSB to dsDNA was prevented under high salt conditions (200 mM NaCl) or in the presence of Mg2+. HpDprA interaction with both ssDNA and dsDNA was stable under high salt condition (200 mM NaCl) and in the presence of Mg2+ indicating that these interactions are specific. The interaction of HpDprA with dsDNA is significant since dsDNA plays an important role in natural transformation of H. pylori. The pathway of transformation by dsDNA is highly facilitated (nearly 1000 fold) as compared to ssDNA. However, dsDNA is a preferred substrate for REases which are a barrier to horizontal gene transfer. This implies that the decision of ‘restriction’ or ‘facilitation for recombination’ of incoming DNA might be taken before the conversion of dsDNA into ssDNA. The incoming DNA has been shown to be in the double-stranded form in periplasm and in single-stranded form in cytoplasm. Hence, the temporal and spatial events surrounding endonuclease cleavage remain to be understood. Taken together, these results suggest a very important role of dsDNA in natural transformation in H. pylori. Hence, binding and protection of dsDNA by HpDprA is possibly of crucial importance in the success of natural transformation process of the organism. DprA is characterized by presence of a conserved DNA binding domain. The DNA binding domain adopts a Rossman fold like topology spanning most region of the protein. Rossman fold consists of alternating alpha helix and beta strands in the topological order of β-α-β-α-β. It generally binds to a dinucleotide in a pair as a single Rossman fold can bind to a mononucleotide only. All homologous DprA proteins characterized till date show that in addition of the prominent Rossman fold domain they consist one or more smaller domains. RpDprA consists two more domains other than the Rossman fold domain i.e., N- terminal SAM (sterile alpha motif) domain and a C-terminal DML-1 like domain. SpDprA consist of an N-terminal SAM domain other than Rossman fold domain. While the main function of Rossman fold is to bind DNA, the supplementary domains are highly variable in sequences and functions. For example, the SAM domain in S. pneumoniae plays a key role in shut-off of competence by directly interacting with ComE~P. HpDprA consist of an N-terminal Rossman fold domain and a C-terminal DML-1 like domain. Both these domains are found to be prominently α-helical in nature. Amino acid sequence analysis of the protein suggests that NTD is basic and CTD is acidic in nature. NTD is sufficient for binding with ssDNA and dsDNA, while CTD plays an important role in formation of higher order polymeric complex with DNA. For HpDprA and SpDprA, dimerization site was mapped in Rossman fold domain. Gel filtration data revealed an important observation that HpDprA can exist as a monomer (dominant species at lower concentration) as well as a dimer (dominant species at higher concentration) in solution. However, the exchange between these two forms is very fast resulting in a single peak of elution. Since, HpDprA binds to DNA in dimeric form, the dimer species will be favoured in presence of DNA. Hence, even at lower concentrations HpDprA will be mainly a dimer in presence of DNA. Interestingly, both domains of HpDprA i.e., NTD and CTD were able to form dimers but no higher oligomeric form. On the other hand, HpDprA was seen to form oligomeric forms higher than dimer in gluteraldehyde cross linking assay. The strength of CTD dimer was much lower that NTD dimer, therefore it could be proposed that there are two sites of interaction present in HpDprA - a primary interaction site (N-N interaction) and a secondary interaction site (C-C interaction). The N-N interaction is responsible for dimer formation but further oligomerization of HpDprA necessitates the interaction of two dimers using C-C interaction site. It was shown that NTD binds to ssDNA but forms lower molecular weight complex. SPR analysis of DprA and NTD – DNA interaction pointed out that deletion of CTD leads to faster dissociation of the protein from DNA. Concomitantly, reduction in binding affinity was observed for both ss and ds DNA upon deletion of CTD from full length protein. These results suggest that CTD does play an important role in interaction of full length HpDprA with DNA. Two possible roles of CTD were proposed by Wang et al (2014) group to explain their observation of formation of lower molecular weight complex in absence of CTD. (i) CTD possesses a second DNA binding site but much weaker than site present in NTD. (ii) CTD is not involved in DNA binding but mediates nucleoprotein complex formation through protein – protein interaction. EMSA and SPR analysis with purified CTD protein confirmed that there is no secondary DNA binding site present in CTD. As discussed above, it was observed that CTD can mediate interaction between two HpDprA through C-C interaction. Since the interaction is weaker it is lesser likely to be responsible for dimer formation but in trimer or higher oligomeric form of HpDprA, the presence of N-N interaction will facilitate and stabilize C-C interaction. These observations together bring forward an interesting model for HpDprA – DNA interaction. HpDprA forms dimer through N-N interaction (favourably in presence of DNA) and many HpDprA dimers bind to DNA owing to their high affinity and sequence independent nature of binding. These dimers interact with each other through C-C interaction resulting in higher molecular weight nucleoprotein complex. HpDprA - DNA complex formation is slower than NTD – DNA complex but the former one is more stable (Fig. 2). According to the above proposed model there are two binding events (DNA – protein and protein – protein) in case of HpDprA – DNA complex formation and hence it would take longer time than NTD-DNA complex formation which involves only one binding event. But the resulting higher order complex with HpDprA – DNA would be much more stable. NTD is able to offer equally efficient protection from nuclease to ssDNA and dsDNA (Fig. 7). This shows that NTD alone is sufficient to completely coat single molecule DNA. AFM images confirm the difference in binding pattern of HpDprA full length protein and NTD. As can be seen in Fig. 8F, NTD binds a DNA molecule by entirely occupying all the available space but forms nucleoprotein filaments isolated from each other. In contrast to full length HpDprA, which forms tightly packed, condensed, extensively cross linked polynucleoprotein complexes, NTD forms much thinner complexes with DNA. In the electron micrographs of SpDprA – DNA complex, extensive cross filament interaction was observed resulting in a dense molecular aggregate. Similar kinds of complexes with DNA were also observed for Bacillus subtilis DprA in atomic force microscope images. Thus, it could be proposed that HpDprA binds to a single DNA molecule (single strand or double strand) mainly as a dimer formed through N-N interaction. Such multiple individual nucleoprotein filaments come together and interact with each other through C- C interaction resulting in dense and intricate poly – nucleoprotein complex. HpDprA is proposed to undergo conformational changes from closed state to open state in presence of ssDNA. In agreement with this, structural transition (resulting in reduction of α-helicity of the protein) was observed in presence of ssDNA. Similar structural transitions were observed for dsDNA indicating possibly a common mode of interaction for both forms of DNA. Further, mutation of the residues shown to be involved in binding ssDNA from crystallographic data, resulted in decrease of binding affinity with dsDNA as well. The fold reduction in binding affinity of dsDNA was lower than that for ssDNA despite that it is obvious that the same positively charged pocket which is primarily involved in ssDNA interaction is also responsible (atleast partially) for binding with dsDNA. However, the residues crucial for interaction with these two forms of DNA may be different. Both DprA and R-M systems have been shown to have presynaptic role in natural transformation process. While DprA has a protective role, R-M systems have an inhibitory role for incoming DNA suggesting a functional interaction between them. Results of this study show that HpDprA interacts with dsDNA, inhibits Type II restriction enzymes from acting on it and at the same time stimulates the activity of MTases resulting in increased methylation of bound DNA. This observation is of significance from the view of genetic diversity as the only way a bacterial cell discriminates between self and nonself DNA is through the pattern of methylation. Binding of HpDprA to incoming DNA inhibits its access to restriction endonucleases but not to methyltransferases. As a result DNA will be methylated with the same pattern as that of the host cell. Hence, it no longer remains a substrate for restriction enzymes. HpDprA thus, effectively alleviates the restriction barrier. However, it remains to be understood as to how DNA in complex with HpDprA, while not accessible to REases or other cellular nucleases, is accessible to a MTase? A possible explanation could be that HpDprA interacts with MTase and recruits it on DNA. It has been shown that there is a overlap between DprA dimerization and RecA interaction interfaces and in presence of RecA, DprA-DprA homodimer is replaced with DprA-RecA heterodimer allowing RecA nucleation and polymerization on DNA followed by homology search and synapsis with the chromosome. A similar scenario can be thought for interaction of HpDprA with the MTase. R-M systems play an important role in protection of genomic DNA from bacteriophage DNA. Hence, downregulation of restriction barrier by HpDprA may not be desirable by host during the entire life cycle. Therefore, the expression of HpDprA, which is ComK dependent and that which takes place only when competence is achieved is noteworthy. In H. pylori, DNA damage induces genetic exchange via natural competence. Direct DNA damage leads to significant increase in intergenomic recombination. Taken together it can be proposed that when genetic competence is induced, R-M systems are down regulated to allow increased genetic exchange and thus, increasing adaptive capacity in a selective environment of stomach. There is an evolutionary arms race between bacterial genomes and invading DNA molecules. R-M systems and anti-restriction systems have co-evolved to maintain an evolutionary balance between prey and predator. Phages and plasmids employ anti-restriction strategies to avoid restriction barrier by a) DNA sequence alteration, b) transient occlusion of restriction sites and c) subversion of restriction-modification activities. DNA binding proteins have been shown to bind and occlude restriction sites. On the other hand, λ Ral protein alleviates restriction by stimulating the activity of Type IA MTases. The observations of MTase stimulation and site occlusion of restriction sites by HpDprA appears to be analogous to anti restriction strategies, otherwise employed by bacteriophages. Thus, DprA could be a unique bacterial anti-restriction protein used by H. pylori for downregulating its own R-M systems to maintain the balance between fidelity and diversity. In conclusion, HpDprA has unique ability to bind to dsDNA in addition ssDNA but displays higher affinity towards ssDNA. Binding of HpDprA to DNA results in a compact complex that is inert to the activity of nucleases. A novel site of oligomerization for HpDprA was observed which suggests the role of C-C interaction in inter-nucleoprotein filament interaction. It would be interesting to further study the effects of CTD deletion on the transformation efficiency of H. pylori, to understand these mechanisms better. It has been well demonstrated that R-M systems offer a barrier to incoming DNA, but our understanding of the regulation of R-M systems has been poor. While other factors like regulation of cellular concentration of restriction enzymes and conversion of dsDNA into ssDNA might play crucial roles in striking the perfect balance between genome diversity and integrity, one of the factors that regulate R-M systems could be DprA.

L’utilisation d’inoculants de champignon mycorhizien arbusculaire (CMA) et de bactéries promotrices de la croissance de la plante (PGPR) comme biofertilisants pour améliorer la croissance de la plante et augmenter les rendements des cultures connait de plus en plus un intérêt remarquable. Ces inoculants peuvent influencer les relations trophiques à travers les changements induits au niveau de la plante. Cependant, en contexte agroécologique, il est difficile de prédire les effets de leur application sur les niveaux trophiques supérieurs, notamment sur les insectes phytophages et leurs ennemis naturels. Ainsi, l’objectif de ce projet de thèse était d’évaluer les effets des inoculants de champignon mycorhizien et bactériens sur les insectes du soja. Spécifiquement il est question : 1) d’évaluer l’influence sur le puceron du soja Aphis glycines (2e niveau trophique), de la symbiose tripartite entre un CMA Rhizophagus irregularis, une bactérie rhizobium Bradyrhizobium japonicum et le soja Glycine max ; 2) de déterminer l’impact des inoculants de R. irregularis et de B. japonicum associés au soja sur les ennemis naturels du puceron de soja, la coccinelle Coleomegilla maculata et le parasitoïde Aphelinus certus (3e niveau trophique) ; 3) d’évaluer les effets de la co-inoculation des inoculants de R. irregularis, B. japonicum et de Bacillus pumilus sur les insectes phytophages et leurs ennemis naturels associés au soja au champ. Les expériences en chambre de croissance (objectifs 1 et 2) ont montré une altération des traits de performance des plantes en présence des inoculants comparées aux plantes témoins. Les différences de colonisation mycorhizienne et de nodulation, observées entre les traitements sont une preuve du fonctionnement de l’inoculation. Avec le double inoculant, j’ai observé une augmentation de la biomasse de la plante, des concentrations en azote et carbone, et une diminution de la concentration en phosphore. Avec le rhizobium seul, j’ai obtenu les mêmes effets, sauf pour la biomasse racinaire qui n’a pas été affectée. Avec le CMA seul, une augmentation de la concentration en phosphore a été observée, mais aucun des autres paramètres de la plante n'a été affecté. Au deuxième niveau trophique sur le puceron du soja, le soja inoculé avec le double inoculant CMA+rhizobium, suivi du rhizobium seul, ont augmenté significativement la densité de la population de pucerons, alors qu’aucun des inoculants n’a eu un effet sur le fitness du puceron. En revanche, aucun des traits de performance du puceron (fécondité et taille des individus) n’a été affecté par la présence de l’inoculant CMA seul. D’autre part, j’ai noté une corrélation positive entre la concentration en azote et le taux de reproduction des pucerons, alors qu’avec la concentration en phosphore, la corrélation avec la colonie des pucerons est négative. Quant au troisième niveau trophique, une réduction significative du taux de parasitisme chez A. certus a été observée en présence du rhizobium seul, mais aucun autre paramètre n’a été affecté comparativement aux autres traitements : contrôle, CMA et double inoculant (CMA+rhizobium). Avec le prédateur C. maculata, aucun paramètre mesuré n’a été affecté par les effets indirects des inoculants. Il ressort de l’évaluation que j’ai faite au champ (objectif 3), une augmentation de l'abondance des insectes piqueurs-suceurs avec le triple inoculant (CMA+rhizobium+Bacillus), mais aucune différence entre les traitements pour les autres groupes d'insectes (broyeurs, et ennemis naturels des pucerons). Aucun groupe fonctionnel n’a été affecté en présence du double inoculant CMA+rhizobium sauf le puceron du soja, A. glycines, qui a vu sa population décroître mais seulement dans les parcelles fertilisées en potassium. J’ai également montré que les abondances des insectes piqueurs-suceurs et des insectes broyeurs, et la diversité alpha des insectes phytophages étaient toutes corrélées négativement avec la colonisation mycorhizienne globale. Ces résultats confirment que la co-inoculation de deux symbiotes peut non seulement améliorer les performances des plantes mais aussi celles des insectes phytophages au-delà de ce que chaque symbiote peut apporter seul. Au moins dans notre système d'étude en chambre de croissance, il semble que les avantages que les symbioses microbe-plante confèrent au deuxième niveau trophique avec une augmentation de la population de pucerons sont peu transférés au troisième niveau sur les ennemis naturels du puceron. Dans un système agricole où les agriculteurs veulent bénéficier des retombées de ces inoculants, notamment dans la gestion des insectes associés aux cultures, les CMA semblent favorables s’ils sont utilisés seuls et du fait de leur potentiel à réduire les insectes ravageurs. / The use of arbuscular mycorrhizal fungi (AMF) and plant growth promoting bacteria (PGPR) inoculants, as biofertilizers to boost plant growth and increase crop yields, is gaining interest. These inoculants can also influence trophic relationships through changes on plant. However, in an agroecological context, it is difficult to predict the effects of their application on higher trophic levels, namely phytophagous insects, and their natural enemies. The objective of this thesis research is to evaluate the effects of mycorrhizal fungi and bacterial inoculants on soybean-associated insects. Specifically, it is to: 1) evaluate the influence on the soybean aphid, Aphis glycines (2nd trophic level), of the tripartite symbiosis between an AMF, Rhizophagus irregularis, a rhizobium bacterium, Bradyrhizobium japonicum, and the soybean, Glycine max; 2) determinate the impact of R. irregularis and B. japonicum inoculants associated with soybean on the natural enemies of soybean aphid, the ladybug Coleomegilla maculata and the parasitoid wasp Aphelinus certus (3rd trophic level); 3) evaluate the effects of co-inoculation of R. irregularis, B. japonicum and Bacillus pumilus inoculants on soybean-associated phytophagous insects and their natural enemies in an agricultural field conditions. The walk-in growth room experiments (objectives 1 and 2) showed an alteration of the performance traits of the plants in the presence of the inoculants compared to control plants. The differences in mycorrhizal colonization and nodulation, observed between treatments, are evidence that the inoculation works. With the double inoculant, I observed an increase in plant biomass, nitrogen and carbon concentrations, and a decrease in phosphorus concentration. With rhizobium inoculation alone, without AMF, I obtained the same effects, except the root biomass that was not affected. With AMF alone, an increase in phosphorus concentration was observed, but none of the other plant parameters were affected. At second trophic level, inoculation of soybeans with the dual inoculant AMF-rhizobium, followed by rhizobium alone, significantly increased aphid population density, while none of inoculant influenced the fitness of the aphid. However, AMF alone did not affect any parameter traits (reproduction and fitness) of the aphid. I noted a positive correlation between nitrogen concentration and aphid reproductive rate, whereas the correlation between phosphorus concentration and aphid colony growth was negative. At the third trophic level, a significant reduction in the rate of parasitism of A. certus was observed in the presence of rhizobium inoculant alone, but no other parameters were affected with any other treatments, namely the control, the AMF inoculant, or the double inoculant (AMF+rhizobium). With the predator C. maculata, no measured parameters were affected by the indirect effects of any of the inoculant treatments. During my field assessment (objective 3), I observed an increase in the abundance of piercing-sucking insects with the triple inoculant (AMF+rhizobium+Bacillus), but no differences between treatments for other insect groups (chewing insects, or natural enemies of aphids). No functional group was affected by the presence of the double inoculant, CMA+rhizobium except the soybean aphid, A. glycines, which saw its population decrease in potassium-fertilized plots. I also showed that the abundance of piercing-sucking insects, chewing insects, and the alpha diversity of phytophagous insects were all negatively correlated with mycorrhizal colonization. The results of my studies confirm that co-inoculation of two symbionts can not only improve plant performance but also that of phytophagous insects beyond what each symbiont can provide alone. At least in our growth chamber study system, it appears that the benefits that microbe-plant symbioses confer to the second trophic level, with an increase in aphid population, are little transferred to the third level on the natural enemies of the soybean aphid. In agricultural system where farmers seek the benefits of these inoculants, especially in the management of insects associated with crops, AMF seem favorable when they are used alone and because of their potential to reduce insect pests.

Les composés organiques hydrophobes (HOC), une grande famille de molécules naturelles ou d’origine anthropique incluant les lipides et les hydrocarbures, constituent une part significative de la matière organique dans les écosystèmes marins. Du fait de leur faible solubilité dans l’eau, les bactéries qui les dégradent requièrent la mise en place de fonctions cellulaires spécifiques permettant d’augmenter la fraction assimilable de ces HOC. La formation de biofilms à l’interface eau-HOC est une de ces stratégies adaptatives. C’est le cas pour Marinobacter hydrocarbonoclasticus SP17, modèle d’étude utilisé au laboratoire, qui est capable de former des biofilms sur un large spectre de HOC métabolisables tels que les alcanes, les triglycérides et les alcools gras. Le but de mes recherches consistait à améliorer la compréhension du processus d’adhésion et de développement des biofilms sur les HOC, à travers la caractérisation fonctionnelle de 10 gènes candidats mis en évidence lors d’analyses d’expression en protéomique et en transcriptomique. Pour mener à bien ce projet, des outils génétiques et une caractérisation fonctionnelle propre à chaque gène ont dû être développés. L’étude fonctionnelle du gène MARHY2686 a relevé son implication dans la formation de biofilm sur les alcanes. La co-expression de MARHY2686 et des gènes adjacents MARHY2687 et MARHY2685 en transcriptomique, leur distribution phylogénétique et leur conservation de la synthénie suggèreraient que ces trois gènes soient impliqués dans le même processus biologique. D’après l’identité forte de 36 % qui existe entre la protéine MARHY2686 et une protéine périplasmique AdeT d’un système de pompe d’efflux tripartite d’Acinetobacter baumanii, cette protéine, en association avec MARHY2687 et MARHY2685, pourrait faire partie d’un système de ce type. Par ailleurs, des observations ont permis d’envisager une implication potentielle de ce gène dans l’assimilation des HOC ou dans l’accumulation des réserves lipidiques intracellulaires. M. hydrocarbonoclasticus SP17 utilise les pili de type IV lors de la formation de biofilm sur les HOC. Ces appendices interviennent lors de l’adhésion de cette souche à des HOC ainsi que dans un processus de détachement d’un support hydrophobe. Les pili pourraient soit intervenir directement pour permettre à la bactérie de se détacher de la surface à laquelle elle s’est adhérée, soit indirectement par l’action de bactériophages. La présence d’une mobilité de type twitching sur les HOC a pu être également envisagée. Enfin, le rôle du système de sécrétion de type VI (T6SS), connu pour permettre à la bactérie d’interagir avec une cellule hôte, lors de la formation de biofilm mono-spécifique sur HOC, où aucun autre microorganisme que M. hydrocarbonoclasticus SP17 n’est présent, a été étudié. / Hydrophobic organic compounds (HOC), a large family of naturally-produced or anthropogenic molecules including lipids and hydrocarbons, represent a significant part of organic matter in marine ecosystems. Because of their low solubility in water, bacteria that degrade those compounds require the establishment of specific cell functions to increase their biodisponibility. Biofilm formation in water-HOC interface is one of these adaptations. The model of bacteria used in our laboratory, Marinobacter hydrocarbonoclasticus SP17, is able to form a biofilm on a wide range of HOC, such as alkanes, fatty alcohols and triglycerides, in order to use them as a carbon and energy source. The main purpose of my work was to broaden the knowledge of how bacteria adhere to and from biofilms on HOC, through the functional characterization of 10 candidate genes highlighted during proteomic and transcriptomic studies. Genetic tools and a gene-specific functional characterization have been developed in order to carry out this project. Functional study conducted on MARHY2686 revealed its involvement in the formation of biofilm on alkanes. Co-expression of MARHY2686 and the adjacent genes MARHY2687 and MARHY2685 durnig transcriptomic analysis together with their phylogenetic distribution and synteny conservation suggest that these three genes are involved in the same biological process. According to the high peptide sequence identity between MARHY2686 and AdeT, a periplasmic protein of a tripartite efflux pump system of Acinetobacter baumanii, MARHY2686 in combination with MARHY2687 and MARHY2685 could be the components of such a system. Other phenotypic observations would consider the involvement of MARHY2686 either in the assimilation of HOC or in the accumulation of intracellular lipid reserves. M. hydrocarbonoclasticus SP17 uses type IV pili during biofilm formation on HOC. These appendages are involved in the adhesion of this strain to and in a detachment process from HOC. Type IV pili could either act directly to allow bacteria to detach from the surface to which it is adhered, or indirectly through the action of bacteriophages. The presence of twitching motility on HOC has also been suggested. Finally, the role of the type VI secretion system (T6SS), a well-known protein system which allows interactions between bacteria and host cells, during the formation of a mono-species biofilm on HOC where no other microorganism than M. hydrocarbonoclasticus SP17 is present, has been studied.

A prevalent morphology in the microscopic world of artificial microswimmers, bacteria and viruses is that of a helix. The intriguingly different physics at play at the small scale level make it necessary for bacteria to employ swimming strategies different from our everyday experience, such as the rotation of a helical filament. Bio-inspired microswimmers that mimic bacterial locomotion achieve propulsion at the microscale level using magnetically actuated, rotating helical filaments. A promising application of these artificial microswimmers is in non-invasive medicine, for drug delivery to tumours or microsurgery. Two crucial features need to be addressed in the design of microswimmers. First, the ability to selectively control large ensembles and second, the adaptivity to move through complex conduit geometries, such as the constrictions and curves of the tortuous tumour microvasculature. In this dissertation, a mechanics-based selective control mechanism for magnetic microswimmers is proposed, and a model and simulation of an elastic helix passing through a constricted microchannel are developed. Thereafter, a theoretical framework is developed for the propulsion by stiff elastic filaments in viscous fluids. In order to address this fluid-structure problem, a pertubative, asymptotic, elastohydrodynamic approach is used to characterise the deformation that arises from and in turn affects the motion. This framework is applied to the helical filaments of bacteria and magnetically actuated microswimmers. The dissertation then turns to the sub-bacterial scale of bacteriophage viruses, 'phages' for short, that infect bacteria by ejecting their genetic material and replicating inside their host. The valuable insight that phages can offer in our fight against pathogenic bacteria and the possibility of phage therapy as an alternative to antibiotics, are of paramount importance to tackle antibiotics resistance. In contrast to typical phages, flagellotropic phages first attach to bacterial flagella, and have the striking ability to reach the cell body for infection, despite their lack of independent motion. The last part of the dissertation develops the first theoretical model for the nut-and-bolt mechanism (proposed by Berg and Anderson in 1973). A nut being rotated will move along a bolt. Similarly, a phage wraps itself around a flagellum possessing helical grooves, and exploits the rotation of the flagellum in order to passively travel along and towards the cell body, according to this mechanism. The predictions from the model agree with experimental observations with respect to directionality, speed and the requirements for succesful translocation.

År 2020 drabbades världen av Covid-19. Spridningen av viruset resulterade i att många länder upprättade en lockdown. Detta påverkade sjöfartsbranschen negativt och ställde till med problem när folk blev sjuka. Detta är på grund av den unika miljön som finns ombord på fartyg. Syftet med studien var att analysera och utvärdera hur olika fartyg och rederier agerade under covid-19-pandemin med fokus på effektivitet, framgång och problem när det kommer till smittskydd. Studiens frågeställningar berörde hur rederiers existerande smittskyddsplaner såg ut och huruvida de kan förbättras, vilka lärdomar som kan dras om smittspridning ombord på fartyg och huruvida en förbättrad smittskyddsplan kan konstrueras utifrån de som redan existerar. En kvalitativ metod i form av granskning och inläsning av studier i området smittspridning i allmänhet samt studier om utbrott av covid-19 ombord på fartyg. Smittskyddsplaner samlades in genom ett mailutskick till svenska rederier. Resultatet visar att rederierna efterlevde många av WHO:s riktlinjer där de även implementerade egna tillägg till den ombordhavna smittskyddsplanen. Vi kom även fram till att landbaserad assistans är en viktig komponent för att kunna bekämpa smittspridning ombord. / In 2020, the world was struck by Covid-19. The spread of the virus resulted with many countries establishing  lockdowns. This negatively affected the shipping industry and caused problems when people got sick. This is because of the unique environment that exists on board ships. The purpose of the study was to analyze and evaluate how different ships and shipping companies acted during the COVID-19 pandemic with a focus on efficiency, success and problems when it came to controlling the spread of infections. The study's questions concerned how shipping companies' infection control plans were complied with and whether they can be improved, what lessons can be learned about the spread of infections on board ships and whether an improved infection control plan can be constructed from those that already exist. A qualitative method of reviewing and reading studies in the field of infection control in general and studies on COVID-19 outbreaks on board ships. Infection control plans were collected through a mailing to Swedish shipping companies. The results showed that the shipping companies complied with many of the WHO's guidelines where they also implemented their own additions to the on-board infection control plan. We also came to the conclusion that land-based assistance is an important component for combating the spread of infections on board.