Tesis sobre el tema "Pili (Microbiology)"

Les pili de type IV (PT4) sont des filaments micrométriques qui exercent de multiples fonctions à la surface de nombreux procaryotes. Chez Neisseria meningitidis, les PT4 sont des homopolymères de la piline majeure PilE. Leur implication dans l'agrégation interbactérienne et l'adhésion aux cellules humaines les rend centraux dans la virulence du méningocoque. Cependant, les mécanismes permettant aux PT4 d'exercer ces diverses fonctions restent trop élusifs. Durant ce doctorat, nous avons simultanément déterminé les régions de PilE impliquées dans l'assemblage des pili, l'auto-agrégation, l'adhésion aux cellules humaines et la compétence à la transformation en utilisant la technique de deep mutational scanning. L'analyse approfondie de cette carte fonctionnelle de la séquence de la piline offre de nouvelles perspectives sur les mécanismes de fonctionnement des PT4 : tout d'abord, le domaine hyperconservé 1 de PilE est impliqué dans la régulation de la balance entre la longueur et le nombre des pili ; par ailleurs, nous avons identifié un groupe d'acides aminés électropositifs autour de la lysine 140 requis pour l'agrégation ; enfin, nous montrons l'importance de l'extrémité distale des PT4 dans l'adhésion. En résumé, ces résultats sont en faveur d'un rôle direct de PilE dans l'agrégation et l'adhésion bactérienne et identifient les domaines spécifiquement impliqués dans ces fonctions. Ces travaux ouvrent aussi de nouvelles perspectives sur la pathogénicité de Neisseria meningitidis et pourraient participer au développement de nouvelles thérapies pour combattre les pathologies provoquées par le méningocoque<br>Type IV pili (TFP) are multifunctional micrometer-long filaments expressed at the surface of many prokaryotes. In Neisseria meningitidis, TFP are homopolymers of the major pilin PilE. They are crucial for virulence as they mediate interbacterial aggregation and adhesion to host cells although the mechanisms behind these functions remain unclear. During this doctoral work, we simultaneously determined the regions of PilE involved in pili display, auto-aggregation and adhesion to human cells by using deep mutational scanning. Mining of this extensive functional map of the pilin sequence provides new mechanistic insights: first, the hyperconserved 1-domain of PilE was found to be involved in the balance between pili length and number; moreover, we identified an electropositive cluster of residues centered around Lysine 140 necessary for aggregation; finally, we show the importance of the tip of TFP in adhesion. Overall, these results support a direct role of PilE in aggregation and adhesion to host cells and identify these specific functional domains. This doctoral work opens up new perspectives on the pathogenicity mechanisms of Neisseria meningitidis and could help design new therapies to fight meningococcal disease

Binding of K88ab pili by brush border membrane and mucus from pig small intestine was characterized by inhibition assay and Western blot. In Western blot, K88ab pili were bound by two major brush border membrane polypeptides with molecular weight of 61,500 and 57,000 in addition to numerous minor polypeptides and a major mucus polypeptide with molecular weight of 27,500. The results from Western blot assays with periodate oxidized and carbamylated brush border membrane and inhibition assay with brush border membrane glycopeptide suggest that amino groups (rather than carbohydrate) present on the protein moiety are a part of the recognition site for K88ab pili of receptor polypeptides in brush border membrane. Differences were obtained in the binding patterns of K88ab pili when brush border membranes were prepared from small intestines obtained from 2-, 21-, and 42-day-old piglets as well as adult hogs. Binding of K88ab pili by mucus polypeptides was greater when prepared from small intestines obtained from 2-day-old piglets than from piglets of other ages and adult hogs. In inhibition assay, most fractions from sow milk and colostrum inhibited binding of K88ab pili. After gel filtration of colostral whey, fractions which contained IgG, IgA, and IgM produced the strongest inhibition of K88ab binding. Among fractions prepared from cow milk, casein and skim milk significantly inhibited binding of K88ab pili. In Western blot, α_s1-casein, immunoglobulin chains, and MFGM polypeptides in sow milk and colostrum were shown to be able to bind K88ab pili. Additionally, α_s1-casein was the major protein in bovine milk responsible for binding K88ab pili. In dot blot assay, IgG as well as brush border membrane could strongly bind K88ab pili. However, bovine α_s1-casein showed only weak binding of K88ab pili. Binding of K88ab pili to these proteins and brush border membrane was inhibited by carbamylation and by addition of 100 mM D-galactosamine. The results suggest that the K88ab-binding proteins in milk and colostrum compete to bind K88ab pili with the receptors in the brush border membrane and that mechanisms involved in binding of K88ab pili by these proteins is similar to that by brush border membrane.<br>Ph. D.

Neisseria meningitidis est une bactérie diderme qui colonise le nasopharynx humain de façon commensale. Occasionnellement, elle franchit la barrière nasopharyngée et accède à la circulation sanguine où elle peut provoquer un choc septique et/ou une méningite Le pouvoir pathogène de N. meningitidis est lié à sa capacité à interagir avec les cellules endothéliales humaines. Après avoir adhéré aux cellules grâce à des organelles filamenteux, les pili de type IV, les bactéries induisent une déformation de la membrane plasmique de la cellule hôte sous la forme de protrusions riches en actine ressemblant à des filopodes. Ces protrusions permettent aux bactéries de résister aux forces de cisaillements générées par le flux sanguin et de proliférer à la surface des cellules. Contrairement à de nombreuses autres bactéries pathogènes, cette déformation de la membrane plasmique ne nécessite pas de polymérisation d’actine. Cependant, les mécanismes cellulaires et moléculaires de cette déformation sont inconnus. Dans cette étude, nous montrons que lorsque des bactéries individuelles adhèrent à la cellule hôte, la membrane plasmique se déforme en adhérant le long des fibres de pili de type IV de façon similaire au mouillage d’un liquide sur un solide. Les pili de type IV agissent donc comme un échafaudage extracellulaire qui guide les protrusions de membrane plasmique indépendamment du cytosquelette d’actine. Nous montrons également que la capacité de la membrane plasmique à se déformer le long de structures adhésives nanométriques est une propriété intrinsèque des cellules endothéliales. Ces travaux décrivent le mécanisme d’une étape importante de la pathophysiologie de N. meningitidis et mettent en évidence des propriétés nouvelles de la membrane plasmique des cellules humaines qui pourraient être impliquées dans d’autres processus fondamentaux de biologie cellulaire<br>Neisseria meningitidis is a diderm bacterium that is naturally found in the human nasopharynx as a commensal. Occasionally, it can cross the mucosa and reach the underlying blood vessels where it enters the circulation. Once in the bloodstream, it can cause severe septic shock and/or meningitis. The ability of N. meningitidis to cause disease is tightly linked to its ability to interact with human endothelial cells. In particular, upon bacterial adhesion via filamentous organelles called type IV pili, bacteria remodel the host cell plasma membrane in the form of actin-rich, filopodia-like protrusions. These protrusions allow bacteria to resist blood flow-generated shear stress and proliferate on top of the host cells. Unlike many other bacterial pathogens, plasma membrane remodeling induced by N. meningitidis does not require actin polymerization. Yet, the cellular and molecular mechanisms of this process are unknown. Here, we show that upon adhesion of individual bacteria, the host cell plasma membrane deforms by adhering along type IV pili fibers in a wetting-like fashion. Therefore, type IV pili act as an extracellular scaffold that guide plasma membrane protrusions in an F-actin-independent manner. We further show that the ability of the plasma membrane to deform along nanoscale adhesive structures is an intrinsic property of endothelial cells. Therefore, this study uncovers the mechanism of a key step of N. meningitidis pathophysiology and reveals novel properties of human cell plasma membrane that could be at play in other fundamental cellular processes

The bacterium, Bagteroides nogosu , is the principal causative organism of footrot in sheep. a disease which causes major economic losses in Australia. Current footrot vaccines contain strains from each of the eight serogroups present in Australia. However, they are expensive and difficult to produce because B.nodosus an anaerobe has complex growth requirements and expression of the pilus, a major protective antigen is variable in liquid culture. The pilus (plural, pili) is composed essentially of one subunit protein called pilin and is the antigen which forms the basis for serotyping the organism. Purified pilus vaccines protect sheep against footrot challenge. It is therefore important to clone and characterise the genes reponsible for piliation in §.godosus. so that its regulation may be understood. With this knowledge, it should be possible to produce a cheap, reliable footrot vaccine based on genetically engineered strains that express high levels of pilus antigen. This work describes the cloning of the pilin gene from §.nodosu§ strain 215, which belongs to serogroup B. During the course of this project, three distinct b. nodosus loci have been cloned and expressed in Escherichia coli, an easily grown and well characterised bacterium. The first locus is represented by two clones pDWl and pDW3. carrying DNA inserts of 7.2 and 4.5 kilobases (kb). respectively. Analysis in maxicells shows that they both encode a protein which co—migrates with b. nodosus pilin and reacts weakly with pilus antiserum in Western blotting. It appears not to be the same pilin locus as that isolated from b. nodosus strain 198 (serogroup A: Anderson et al., 1984; Elleman et al., 1984). because the DNA shows no restriction homology with the strain 198 pilin clones. It is suggested that pDWl and pDW3 may encode an alternative pilin locus. The second locus is represented by pDW4, a clone carrying an insert of 10.2 kb. Western blotting shows that pDW4 encodes three proteins that are present in the pilus preparation used to raise antisera in rabbits. It does not encode pilin. These first two loci may encode proteins associated with pilus production, as the clones were detected using antibodies that had been raised against purified pili. The third locus is represented by pDWS and pDW6, carrying inserts of 10.0 and 5.6 kb repectively. These clones show strong expression of pilin in Western blotting experiments, and the DNA restriction maps exhibit homology with DNA from strain 198 pilin clones. Suggestions for further work, to determine the genes and conditions necessary for full pilus expression. are presented.

<p>This thesis focuses on host and pathogen specific interactions during invasive disease. We have investigated the role and impact of different virulence factors of <i>Neisseria gonorrhoeae, N. meningitidis</i> and <i>Streptococcus pyogenes</i> on host epithelial cells and <i>in vivo</i>. </p><p><i>N. gonorrhoeae</i> cause the sexually transmitted disease gonorrhoea and <i>N. meningitidis</i> is the most common cause of bacterial meningitis and may be leathal to the host within hours of infection. The neisserial type IV pili were shown to have an important impact on host cells for the induction of pro-inflammatory and other cellular defence transcriptional responses. Furthermore, <i>N. meningitidis</i> generally induced an earlier response compared to <i>N. gonorrhoeae</i>, probably as a result of the meningococcal capsule. The role of <i>N. meningitidis</i> serogroup B lipooliogsaccharide was investigated during invasive disease. Bacterial invasion of host cells and blood survival as well as virulence in vivo was dependent on the integrity of the LOS structure. </p><p><i>S. pyogenes</i> may cause a variety of diseases ranging from uncomplicated diseases such as 'strep-throat' to more severe invasive diseases such as necrotizing fasciitis and streptococcal toxic shock syndrome. <i>S. pyogenes</i> ScpC protease degrade interleukin 8 during necrotizing fasciitis. We investigated the role of ScpC in systemic disease and observed enhanced virulence by bacteria unable to degrade IL-8. Following an intravenous infection of mice pro-inflammatory cytokines and complement activation was induced by the ScpC negative mutant compared to the wild-type and correlated with higher bacteremia. These data indicate that the precense of the ScpC protease has an important impact on the host for the outcome of streptococcal sepsis. Another phagocytic escape mechanism of <i>S. pyogenes</i> is their ability to coat themselves with host proteins. We observed that released complement control protein, CD46, bound to the streptococcal cell surface. CD46 has been shown to interact with the streptococcal M protein and have now been found to bind to the surface of the bacteria in a growth phase dependent manner. We observed a more aggressive disease development in CD46 transgenic mice after an intravenous infection with an M6 serotype, resulting in higher mortality of CD46 transgenic mice compared with control mice. These data indicate that CD46 may confer a protection to the streptococci during early stage of systemic infection and contributes to the understanding of immune evsion of <i>S. pyogenes</i>.</p>

Pseudomonas aeruginosa is an important opportunistic pathogen. Many P. aeruginosa virulence factors are regulated by the AlgZ/R two component system. AlgZ is the sensor histidine kinase which phosphorylates AlgR, the response regulator. AlgR activates transcription of different gene targets based upon its phosphorylation state. The genes that encode AlgZ and AlgR are transcribed in an operon. While regulation of algR expression has been well studied, regulation of algZ expression has not. Using a pilW mutant in concert with algZTF-lacZ transcriptional fusion, we conducted a transposon mutagenesis to identify algZ regulators. We identified an unknown autoregulatory loop. The type IV pilus minor pilins prevent the phosphorylation of AlgR by AlgZ . This inhibition of the AlgZ/R system subsequently down-regulates both the expression of the fimU operon and the algZ/R operon. Because AlgR regulates virulence, it is possible that virulence can also be reduced by targeting activation of the AlgZ/R system.

Streptococcus gallolyticus est une bactérie à Gram positif qui colonise de façon asymptomatique le système gastrointestinal de 2,5 à 15 % de la population humaine. Cette bactérie est également responsable d'endocardites infectieuses et il existe une forte association entre une infection à S. Gallolyticus et le cancer du colon. La séquence du génome de la souche UCN34 révèle l'existence de trois loci pilus (pil1,pil2 et pil3). Nous avons caractérisé l’ operon pil1 et montré que le pilus Pil1 permet la colonisationet la formation de biofilm sur tissus exposant du collagène. Le rôle de Pill dans le développement d'endocardites a été montré in vivo chez le rat. De façon surprenante, Pil1 est exprimé de façon hétérogène à la surface de la souche UCN34, générant deux sous-populations cellulaires distinctes, une majorité de cellules faiblement piliées (Pil1low) et une minorité de cellules hyper-piliées (Pil1high). Dans les cellules Pil1high traduction d'un peptide leader contrôle la transcription des gènes pi1l en aval en empêchant la formation d'un terminateur transcriptionnel. De plus, un outil génétique permettant de construire des mutants définis chez S. Gallolyticus a été développé. Cette étude identifie le pilus Pil1comme le premier facteur de virulence caractérisé chez S. Gallolyticus. Nous décrivons un mécanisme original de régulation de l'expression de Pil1, combinant de la variation de phase dans une séquence leader et de l'atténuation transcriptionnelle. L'hétérogénéité de Pil1 apparaît comme essentielle pour l'échappement de la bactérie au système immunitaire, et pour assurer des capacités optimales de colonisation et de dissémination au sein de l'hôte<br>The Gram-positive bacterium Streptococcus gallolyticus asymptomatically colonizes the gastrointestinal tract of 2. 5-15% of humans. It is also an emerging cause of endocarditis and has been epidemiologically linked to colonic malignancies. Genome sequence of strain UCN34 revealed the existence of three pilus loci (pill,pil2 and/7/73). Pil1 are long filamentous structures playing a key role as adhesive organelles in many pathogens. We first characterized pill operon and demonstrated that Pil1 pilus mediates colonization and biofilm formation on host tissues exposing collagen. Moreover, Pill role in endocarditis development has been demonstrated in vivo in a rat model. By studying Pil1 pilus by microscopy, we observed that Pill is heterogeneously expressed in S. Gallolyticus UCN34, giving rise to two distinct cellular subpopulations consisting of a majority of weakly piliated (Pil1low) cells and a minority of hyper piliated (Pil1high) cells- This population heterogeneity is dependent on the promoter region of the pill operon. In Pil1high cells translation of the leader peptide controls the transcription of the downstream pill genes by preventing formation of the transcriptional terminator. We also developed a genetic tool to construct defined mutants in S. Gallolyticus strains. This study identifies Pil1 as the first virulence factor characterized in S. Gallolyticus. Moreover, it describes an original regulatory mechanism of Pill expression combining phase variation in a leader sequence and transcription attenuation. Pill heterogeneity appears crucial for evading the host immune System and to ensure optimal colonization versus dissemination in host tissues

Pseudomonas aeruginosa is a central nosocomial pathogen that can infect any tissue in the human body. A two-component system in P. aeruginosa that regulates many virulence factors is the AlgZ/R system. A previously unidentified regulator of algZ, that does not affect algR, has been identified via transposon mutagenesis, ‘PA2771’. The mechanism of regulation has not been previously studied, and novel evidence of PA2771 functioning as a diguanyalate cyclase was observed. When PA2771 is active, cyclic di-GMP levels are high, promoting the upregulation of the fimU operon and Type VI pili. In the PA2771 mutant, an upregulation in the expression of the flagellar genes and swarming phenotype was observed, and restored via complementation. PA2771's function in regulating algZ expression, is likely indirect and alters virulence gene regulation and phenotypic outputs in P. aeruginosa in the switch between twitching and swimming motility, and appears to be specific to PA2771.

Les pili de type IV (PT4), certainement les organelles les plus répandues des bactéries à Gram-négatif, sont des machineries à multiples fonctions qui jouent un rôle crucial dans la pathogenèse de nombreux pathogènes humains, notamment notre modèle Neisseria meningitidis. L’assemblage des PT4 nécessite une machinerie complexe incluant au moins vingt protéines localisées dans la membrane interne, le périplasme et la membrane externe. Certaines de ces protéines ne sont pas nécessaires pour la biosynthèse des PT4, mais supportent les fonctions qui leur sont associées. Ces protéines, appelées pilines mineures, sont au nombre de trois. Par l’analyse phénotypique des mutants dans les gènes codant pour les pilines mineures, le rôle de chacune a pu être déterminée. Ainsi la piline mineure ComP est nécessaire pour la compétence pour la transformation d’ADN, PilV est requise pour la déformation de la membrane plasmique de la cellule hôte et PilX est essentielle pour l’adhésion des bactéries sur les cellules épithéliales et endothéliales, la formation d’agrégats bactériens et la déformation de la membrane plasmique de la cellule hôte. De nombreuses similarités avec la piline majoritaire laissent penser que les pilines mineures s’insèrent dans la fibre des PT4 pour exercer leurs fonctions, bien que ceci n’a jamais été démontré. Si on connait bien les fonctions des pilines mineures, leur mode d’action n’est toujours pas compris. L’objectif global de ce travail de thèse a été de comprendre comment une fibre protéique peut assurer une diversité de fonctions aussi importante. Pour y parvenir, l’étude du mode d’action des pilines mineures a été entreprise. Contrairement à ce qui prévalait dans le modèle dominant, les pilines mineures PilV et PilX exercent leur fonction à partir de l’espace périplasmique pour moduler la quantité de pili exprimés en surface. En effet, les mutants pilV et pilX présentent respectivement des défauts de piliation de l’ordre de 39% et de 63% par rapport à la souche sauvage. Ces défauts expliquent cependant les phénotypes des mutants. En effet, l’ensemble des fonctions dépendantes des PT4 nécessite une forte quantité de PT4, soit au moins 40% pour l’agrégation et l’adhésion et 70% pour le déclenchement de la réponse cellulaire. Ces résultats révèlent que les pilines mineures sont impliquées dans la biogenèse des PT4 plutôt que dans le support biochimique direct de leurs propriétés. Le défaut de piliation de ces mutants est restauré par l’absence de rétraction, indiquant que les pilines mineures PilV et PilX jouent un rôle dans la stabilité des PT4. Nous avons également montré que la piline mineure ComP est nécessaire pour la piliation et qu’elle présente une fonction redondante avec la piline mineure PilV. Afin de comprendre comment les pilines mineures PilV et PilX exercent leur rôle sur la quantité de pili exprimés en surface, nous avons réalisé une étude structure/fonction de ces deux protéines. Nous avons observé une absence de piliation, en bloquant les pilines PilV et PilX dans la membrane interne, indiquant une interaction directe avec la machinerie des PT4 probablement via la piline majeure PilE. Nous avons également montré qu’il existe une interaction entre les pilines mineures et PilE au niveau de la membrane interne et en amont de l’assemblage des pili. Ces résultats, obtenus par une technique de pontage disulfure, ont cependant besoin d’être confirmés par des contrôles supplémentaires. Par une stratégie de mutagenèse, nous avons enfin mis en évidence que la région D de PilV et les boucles α/β et β2/β3 de PilX sont nécessaires à leur fonctionnement. Ces travaux ont permis de montrer que la quantité de pili exprimés par la bactérie est un facteur déterminant pour définir les propriétés des PT4. Les pilines mineures agissent au niveau du périplasme pour promouvoir la biosynthèse des pili, ce qui met en avant le rôle direct de la piline majeure PilE dans les fonctions associées aux PT4<br>Type IV Pili (TFP) are widespread filamentous organelles extending from the surface of many Gram-negative bacteria that mediate multiple functions and play a key role in the pathogenesis of several important human pathogens, including our model, Neisseria meningitidis. The assembly of TFP requires a complex machinery composed by at least twenty proteins that are localized in the inner membrane, the outer membrane and the periplasm. Three of these proteins, called minor pilins, are not required for the biosynthesis of the TFP, but support their functions. Based on the phenotypes associated with the mutants, their role on TFP functions has been determined. The minor pilin Comp is required for natural competence for DNA transformation, PilV is required for the deformation of the host cell plasma membrane and PilX is essential for the adhesion of bacteria to epithelial and endothelial cells, the bacterial aggregation and the deformation of the host cell plasma membrane. Many similarities with the major pilin PilE suggests that minor pilin are inserted into the fiber of TFP to exert their functions, although it has never been demonstrated. How these proteins carry out their functions mechanistically is not elucidated. The general objective of this thesis was to understand how a single fiber can provide such a variety of functions. To achieve this, the study of the mode of action of minor pilins was undertaken. Contrarily to what has been previously proposed, the PilV and PilX minor pilins seem to exert their functions from the periplasmic space to modulate the amount of surface exposed pili. Indeed, pilV and pilX strains show piliation defects of 39 % and 63 % respectively compared to the wild type. Besides, we have shown that TFP functions require a large amount of TFP, at least 40 % for the aggregation and adhesion and 70% to induce the reorganization of the plasma membrane. Thus these modest decreases in the amount of pili explain the phenotypes of these mutants. These results indicate that the minor pilins are involved in the biogenesis of TFP rather than in the direct support of their biochemical properties. Moreover, the piliation defect of these mutants is restored in the absence of retraction, indicating that the PilV and PilX minor pilins play a role in the stability of TFP. To understand how PilV and PilX minor pilins modulate surface exposed pili level, we performed a structure/ function analysis of these two proteins. Blocking the PilV and PilX minor pilins in the inner membrane abolishes piliation, indicating a direct interaction with the machinery of TFP, probably via the major pilin PilE. We have also shown that an interaction between the minor pilins and the major pilin occurs in the inner membrane and upstream of the pilus assembly. However, these results, obtained by biochemical techniques, need to be confirmed by additional controls. By a mutagenesis strategy, we finally demonstrated that the D region of PilV and the α/β and β2/β3 loops of PilX are necessary for their functions. This study has shown that a relatively modest decrease in the amount of pili displayed on the bacterial surface leads to a strong effect on the functions carried by TFP. Minor pilins act in the periplasm to promote the biosynthesis of pili, which highlights the direct role of the major pilin in the TFP-dependent functions

Répondre aux besoins énergétiques croissants de nos sociétés et limiter leur impact sur l’environnement est un enjeu actuel majeur. De nouvelles technologies alternatives comptent tirer profit de sources d’énergie négligées. Le potentiel énergétique des eaux usées peut être exploité par de nouvelles technologies telles que les piles à combustible microbiennes (PACM). Ces piles, pouvant produire de l’énergie électrique à partir d’eaux usées, montrent une diminution de leur rendement énergétique lorsque leur taille augmente, ce qui ne permet pas encore leur application industrielle. Ces travaux de thèse visent à identifier certains verrous de ce changement d’échelle et à proposer de nouvelles directions pour leur optimisation. Une première partie s’est intéressée à l’influence des collecteurs de courant anodiques sur les performances électriques et sur le développement du biofilm électro-actif. Nous avons émis l’hypothèse qu’à grande échelle, les collecteurs de courant peuvent être un élément limitant à la production d’électricité. Pour vérifier cette hypothèse, quatre PACM avec une anode de 490 cm² connectée de différentes manières ont été étudiées. L’augmentation du nombre de collecteurs a permis une hausse de la puissance produite par les PACM. La disposition des collecteurs affecte la répartition du potentiel sur la surface d’anode et peut engendrer dans certains cas, des gradients de potentiel qui influencent la structure microbiologique du biofilm, en particulier Geobacter. Par ailleurs, des mesures d’impédance ont montré que multiplier les collecteurs augmente la capacité de double couche de l’anode et engendre un courant capacitif dont l’importance pour les performances de fonctionnement en cycles de charge/décharge est non négligeable. La suite du travail s’est attachée à prendre en compte différents aspects physiques, notamment l’aspect hydrodynamique, afin de modéliser leur fonctionnement. Pour cela, trois PACM de volumes différents ont été mises en œuvre et testées à différents débits. Les données de configuration, d’opération et de performances de ces piles ont permis de construire des modèles statistiques de régression linéaire multiple prédisant la valeur de puissance maximale. Ces deux modèles ont montré que la puissance maximale produite était principalement corrélée à la vitesse de l’électrolyte circulant dans la pile et à la contrainte de cisaillement appliquée à l’anode par le mouvement du fluide. Ces deux parties ont également montré que l’abondance dans le biofilm de Geobacter, une bactérie électro-active très répandue dans les PACM, n’était pas corrélée avec la puissance maximale. Tout en étant très abondante, son seul nombre n’explique donc pas entièrement les performances électriques d’une PACM<br>Facing increasing energy needs and limiting their impact on the environment are current and major issues for society. Renewable energy development is needed and new alternative technologies could benefit from exploiting neglected energy sources, such as microbial fuel cells (MFC), for energy production. MFCs can be operated with wastewater and produce a reasonable quantity of energy at the small laboratory-scale. Unfortunately, when their size is increased, their efficiency dramatically decreases, which prevents their industrial use. This thesis aims at identifying some obstacles to scale-up of MFC and proposing new directions for its optimization. The first part of the study was focused on the influence of anodic current collectors on electrical performance and on electroactive biofilm development. Our hypothesis was that they could be a limiting factor for electricity production at large scales. To test this hypothesis, four MFCs were operated with a 490 cm² anode connected to the external circuit in a different ways. Increasing the number of collectors improved the power. Collector’s layout influenced electrical potential on the anode surface and created an electrical potential gradient on the anode and this gradient shaped the microbiological structure of the biofilm. This effect especially concerns Geobacter, whose clade G. metallireducens is favored at strongly negative potentials. In addition, impedance measurements showed that multiplying collectors increased the double layer capacitance and, thus, generated a capacitive current that was important for MFC functioning in cycles of charge/discharge and that would improve its performance. Then, MFCs were considered as bioreactors and their different aspects, notably hydrodynamics, were taken into account to model their power output. Three MFCs of different volumes were operated under continuous-flow conditions and tested at four different flow rates. Configuration, operation and performance data were used to build two multiple linear regression statistical models: the first with variables selection through LASSO, the second with dimensionless numbers created with the Vaschy-Buckingham theorem. These two data-driven models showed that the maximal power was mostly correlated to electrolyte transfer rates inside MFC chamber and to shear stress at the anode generated by fluid movement. These two major experimental projects also showed that the abundance of Geobacter, an electroactive bacteria, inside the biofilm was widespread in MFCs, but it was not correlated to maximal power. Despite its large abundance, its quantity alone does not entirely explain the performance of a MFC. In order to succeed at MFC scale-up, fundamental research on electroactive biofilms, process engineering and modeling need to be associated and generalized as empirical results and their explanation

Pseudomonas aeruginosa est un pathogène opportuniste responsable d’infections nosocomiales sévères associées à un taux élevé de mortalité. Le système de sécrétion de Type III (SST3) et les effecteurs qu’il injecte sont considérés comme des facteurs de virulence prépondérants de P. aeruginosa. Récemment nous avons caractérisé, un groupe de souches ne possédant pas les gènes du SST3, mais dont la virulence repose sur la sécrétion d’une nouvelle toxine de 172 kDa, nommée Exolysine (ExlA) qui provoque la perméabilisation de la membrane des cellules hôtes. ExlA est sécrétée dans le milieu par une porine de la membrane externe, nommée ExlB, formant ainsi un nouveau système de sécrétion à deux partenaires (TPS), ExlBA. Outre le domaine TPS du coté N-terminal de la protéine, impliqué dans sa sécrétion, ExlA possède différents domaines ; des répétitions hémagglutinines, cinq motifs Arginine-Glycine-Acide Aspartique (RGD) et un domaine C-Terminal faiblement conservé. Des tests de cytotoxicité sur des cellules eucaryotes ont montrés que la délétion du domaine C-terminal abolissait l’activité toxique d’ExlA. En utilisant un modèle de liposomes et différents types de cellules eucaryotes, comme les globules rouges, nous avons démontré qu’ExlA forme des pores membranaires de 1.6 nm. De plus, par un criblage cellulaire à haut-débit d’une banque de mutants obtenus par une mutagenèse de transposition, nous avons montré qu’un facteur bactérien additionnel était requis dans la toxicité d’ExlA. En effet, parmi les 7 400 mutants, nous avons identifiés 3 transposons insérés dans des gènes codant pour le pili de type IV, démontrant ainsi que cet appendice impliqué dans l’adhésion des bactéries participe à la toxicité d’ExlA, en permettant un contact rapproché entre la bactérie et les cellules hôtes. Un criblage de macrophages primaires de souris KO pour différentes protéines impliquées dans la voie de l’activation de l’inflammasome, nous a permis de démontrer que le pore formé par ExlA est responsable de l’activation de la Caspase-1 par l’inflammasome NLRP3 conduisant à la maturation de l’interleukine-1ß. Une étude bio-informatique a révélé la présence de gènes homologues à exlA chez d’autres espèces de Pseudomonas non pathogènes, comme P. putida, P. protegens, P. entomophila. Nous avons montré que ces bactéries environnementales sont aussi capables de provoquer une mort cellulaire dépendante de la Caspase-1. Finalement, un criblage d’une banque de macrophages dont les gènes ont été invalidés par la technologie CRISPR/cas9 a révélé que plusieurs protéines du système immunitaire, indirectement liées à l’activation de la Caspase-1 sont impliquées dans la mort cellulaire médiée par ExlA. De plus, nous avons montré que plusieurs sgRNAs ciblant un microARN, mir-741, était grandement enrichi dans les macrophages ayant résisté à une infection avec ExlA. Mir-741 régule l’expression d’enzymes (St8sIa1 et Agpat5) impliquées dans la voie de biosynthèse des sphingolipides et des glycérophospholipides, suggérant ainsi que l’activité d’ExlA requiert un environnement lipidique particulier<br>Pseudomonas aeruginosa is a human opportunistic pathogen responsible for nosocomial infections associated with high mortality. The type III secretion system (T3SS) and T3SS-exported toxins have been considered as key infectivity virulence factors. Our team recently characterized a group of strains lacking T3SS, but employing a new pore-forming toxin of 172 kDa, named Exolysin (ExlA) that provokes cell membrane disruption. In this work we demonstrated that the ExlA secretion requires ExlB, a predicted outer membrane protein encoded in the same operon, showing that ExlA-ExlB define a new active Two-Partner Secretion (TPS) system. In addition to the TPS secretion signals, ExlA harbors several distinct domains, which comprise hemagglutinin domains, five Arginine-Glycine-Aspartic acid (RGD) motifs and a non-conserved C-terminal region lacking any identifiable sequence motifs. Cytotoxic assays showed that the deletion of the C-terminal region abolishes host-cell cytolysis. Using liposomes and eukaryotic cells, including red blood cells, we demonstrated that ExlA forms membrane pores of 1.6 nm. Based on a transposon mutagenesis strategy and a high throughput cellular live-dead screen, we identified additional bacterial factors required for ExlA-mediated cell lysis. Among 7 400 mutants, we identified three transposons inserted in genes encoding components of the Type IV pili, which are adhesive extracellular appendices. Type IV pili probably mediate close contact between bacteria and host cells and facilitate ExlA cytotoxic activity. These findings represent the first example of cooperation between a pore-forming toxin of the TPS family and surface appendages to achieve host cell intoxication. Using mice primary bone marrow macrophages we showed that ExlA pores provoke activation of Caspase-1 via the NLRP3-inflamasomme followed by the maturation of the pro-interleukin-1ß. Mining of microbial genomic databases revealed the presence of exlA-like genes in other Pseudomonas species rarely associated with human infections P. putida, P. protegens and P. entomophila. Interestingly, we showed that these environmental bacteria are also able to provoke Caspase-1 cleavage and pro-inflammatory cell death of macrophages. Finally, genome-wide loss-of-function CRISPR/cas9 RAW library screen revealed that several components of the immune system response, indirectly linked to Caspase-1 are involved in the ExlA-mediated cell lysis. Moreover, we found at least three sgRNAs targeting miRNA, mir-741 were highly enriched in resistant macrophages challenged by ExlA. This miRNA regulates enzymes (St8sIa1 and Agpat5) in the sphingolipids and glycerophololipids biosynthesis pathways, suggesting that ExlA activity may require proper lipid environment

<p><italic>Kingella kingae</italic> is an emerging bacterial pathogen that is being recognized increasingly as an important etiology of septic arthritis, osteomyelitis, and bacteremia, especially in young children. The pathogenesis of <italic>K. kingae</italic> disease begins with bacterial adherence to respiratory epithelium in the posterior pharynx. Previous work identified type IV pili as a critical factor for adherence to human epithelial cells. However, the finding that a significant percentage of pharyngeal isolates are non-piliated suggests that <italic>K. kingae</italic> expresses additional surface factors that modulate interactions with host cells and likely play key roles in the pathogenesis of <italic>K. kingae</italic> disease. The purpose of this work was to increase our understanding of <italic>K. kingae</italic> virulence determinants, specifically focused on defining the surface factors and the mechanism involved in <italic>K. kingae</italic> adhesive interactions with epithelial cells. Additionally, this work aimed to further characterize components of the <italic>K. kingae</italic> type IV pilus system, namely the PilC proteins and PilA2. </p><p>We first set out to identify non-pilus factors that influence <italic>K. kingae</italic> interactions with human epithelial cells. Using targeted genetic approaches, we found that insertional inactivation of the gene encoding a predicted trimeric autotransporter protein called Knh (Kingella NhhA homolog) resulted in reduced adherence to human epithelial cells. In addition, using a variety of techniques, including morphological analysis, cationic ferritin staining, and thin section transmission electron microscopy, we established that <italic>K. kingae</italic> elaborates a surface-associated polysaccharide capsule that requires a predicted ABC-type transporter export operon called <italic>ctrABCD for surface presentation. Furthermore, using quantitative human epithelial cell adherence assays, we discovered that the presence of surface capsule interferes with Knh-mediated adherence by non-piliated organisms and that maximal adherence in the presence of capsule requires the predicted type IV pilus retraction machinery, PilT/PilU. Based on the data presented here, we propose a novel adherence mechanism that allows <italic>K. kingae</italic> to adhere efficiently to human epithelial cells while remaining encapsulated and more resistant to immune clearance. </p><p>Having established that <italic>K. kingae</italic> produces a capsule, a large-scale polysaccharide purification technique was developed for capsule analysis of strain 269-492. Biochemical assays determined that the purified material contained thiobarbituric and phenol-sulfuric acid reactive glycosyl residues. In collaboration with the University of Georgia Complex Carbohydrate Research Center (CCRC), mass spectrometry identified galactose, N-acetyl-galactosamine, and Kdo as the major glycosyl components of the polysaccharide preparation. NMR spectroscopy revealed that the purified material contained two distinct polysaccharides with the structures of &rarr;5)&ndash;&beta;&ndash;Gal<italic>f</italic>&ndash;(1&rarr; and &rarr;3)&ndash;&beta;&ndash;GalNAc<italic>p</italic>&ndash;(1&rarr;5)&ndash;&beta;&ndash;Kdo<italic>p</italic>&ndash;(2&rarr;. Further characterization of the polysaccharides expressed by <italic>K. kingae</italic> may have implications for disease prevention strategies. </p><p>Previous work in our lab found that two PilC-like proteins called PilC1 and PilC2 influence type IV pili expression and pilus-mediated adherence. Production of either PilC1 or PilC2 is necessary for <italic>K. kingae</italic> piliation and bacterial adherence. We set out to further investigate the role of PilC1 and PilC2 in type IV pilus-associated phenotypes. We found that PilC1 contains a functional nine amino acid calcium-binding (Ca-binding) site with homology to the <italic>Pseudomonas aeruginosa</italic> PilY1 Ca-binding site and that PilC2 contains a functional 12 amino acid Ca-binding site with homology to the human calmodulin Ca-binding site. Using targeted mutagenesis to disrupt the Ca-binding sites, we demonstrated that the PilC1 and PilC2 Ca-binding sites are dispensable for piliation. Interestingly, we show that the PilC1 site is necessary for twitching motility and adherence to Chang epithelial cells, while the PilC2 site has only a minor influence on twitching motility and no influence on adherence. These findings establish key differences in PilC1 and PilC2 function in <italic>K. kingae</italic> and provide insights into the biology of the PilC-like family of proteins.</p><p>Lastly, we set out to define the role of the PilA2 minor pilin in <italic>K. kingae</italic> strain 269-492. While previous studies indicated that PilA2 is not essential for pilus expression or adherence to epithelial cells, analysis of the pilin locus in a diverse set of clinical isolates revealed that the <italic>pilA2</italic> gene sequence is highly conserved, suggesting it serves an important function. Using targeted mutagenesis we showed that PilA2 is not essential for twitching motility and may or may not be involved in natural competence. Western blot analysis was unable to detect PilA2 in wild type pilus preparations, indicating that it is expressed at a level beneath the assay detection limit or does not localize to the pilus. Additionally, site-directed mutagenesis was used to place <italic>pilA2</italic> under control of the highly active <italic>pilA1</italic> promoter and showed that PilA2 is able to be assembled into fibers that mediate intermediate adherence to epithelial cells. </p><p>Taken together, this work expands our knowledge of the <italic>K. kingae</italic> surface factor repertoire and provides insights into the roles of type IV pilus components. The mechanism of<italic> K. kingae</italic> adherence to epithelial cells is beginning to emerge. These contributions may lead to novel strategies for the prevention of invasive <italic>K. kingae</italic> disease in young children.</p><br>Dissertation

Vibrio parahaemolyticus is a Gram-negative, halophilic, human pathogenic bacterium ubiquitous in the marine environment. Like many Vibrio species, V. parahaemolyticus commonly associates with shellfish, particularly oysters. Ingestion of a raw or under cooked oysters contaminated with V. parahaemolyticus can cause gastroenteritis, which is typically self-limiting and rarely causes death. Globally, oyster production is highly lucrative, especially on the West Coast of the United States where approximately 60% of oyster production occurs each year. Outbreaks of V. parahaemolyticus can result in a significant public health problem as well as an economic burden for the oyster farms implicated in the outbreak. With the increase in overall V. parahaemolyticus outbreaks, improved post-harvest processing strategies have been developed to reduce this natural contaminant. Depuration was developed to allow shellfish to purge contaminants from their tissues into the clean, flowing seawater where they are held. This post-harvest processing technique can typically reduce fecal contaminants from the oyster tissues but is relatively ineffective at eliminating V. parahaemolyticus and other Vibrio species.. Thus, improved methods for reducing this and other human pathogenic Vibrio are needed to effectively produce safer oysters for the consumer. To develop more effective and novel V. parahaemolyticus intervention strategies, first we must identify the factors that are involved in V. parahaemolyticus colonization of the oyster, allowing them toresist depuration. This study sought to investigate specific factors utilized by V. parahaemolyticus and, in the process, determined that various strains of V. parahaemolyticus have different alleles of the Type IV pili, mannose-sensitive hemagglutinin (MSHA)and chitin-regulated pilus (PilA). In addition, we expanded our investigations into the allelic diversity of MSHA and PilA from Vibrio cholerae and Vibrio vulnificus and found that V. cholerae strains that possess the Type IV toxin co-regulated pilus (TCP) maintained highly conserved MSHA and PilA sequences while strains of V. cholerae without TCP, and all of the V. vulnificus and V. parahaemolyticus strains examined, had highly divergent sequences with no discernable connection to isolation source or observed phenotype. Following that discovery, we determined that Type I, and Type IV pili, as well as polar and lateral flagellar systems contribute to V. parahaemolyticus persistence in the Pacific oyster during depuration, while Type III secretion systems and phase variation do not. Overall, we have identified factors involved in colonization of the Pacific oyster by V. parahaemolyticus. Future studies investigating conditions that affect pili and flagella production in V. parahaemolyticus may provide novel depuration conditions that could easily and effectively increase the efficiency of oyster depuration, ultimately reducing the risk of seafood-borne illness by V. parahaemolyticus associated with oysters.<br>Graduation date: 2013

Les fimbriae sont des structures protéiques extracellulaires retrouvées chez une vaste diversité de bactéries. Ces structures ont fait l’objet de nombreuses études et sont maintenant reconnus pour leur implication dans l’adhésion et l’invasion aux cellules eucaryotes, mais aussi dans la production de biofilms. Ils sont groupés selon leur voie de sécrétion. Certains utilisent une machinerie spécifique et individuelle, c’est le cas des pili de type IV, tandis que d’autres utilisent la voie de sécrétion générale suivit d’une voie spécifique telle que la voie du chaperon-placier (« Chaperon Usher Pathway ») (fimbriae CUP) ou la voie de nucléation précipitation (« nucleation precipitation pathway ») (Curli). Malgré toutes les connaissances actuelles concernant les fimbriae, très peu d’informations sont disponibles quant aux fimbriae de Salmonella enterica sérovar Typhi (S. Typhi). Ce pathogène unique à l’homme est l’agent étiologique de la fièvre typhoïde. Puisque les fimbriae sont reconnus pour être impliqués dans l’adaptation à l’hôte, nous avons décidé d’étudier davantage l’arsenal fimbriaire de S. Typhi, dans l’espoir d’identifier des facteurs de virulence uniques à S. Typhi et impliqués dans la ségrégation de l’hôte. La souche S. Typhi ISP1820 possède 14 opérons codant pour des systèmes d’adhésion, mais plusieurs contiennent des pseudogènes et leur expression n’a jamais été observée in vitro. Afin d’étudier les systèmes d’adhésion de S. Typhi, nous avons supprimé chaque opéron du génome individuellement et cumulativement à l’aide une technique de mutagénèse par échange allélique. Ainsi, nous avons testé chaque mutant individuel et la souche mutante pour tous les systèmes d’adhésion dans plusieurs essais tels que des infections de cellules épithéliales et de macrophages, de mobilité et de formation de biofilm. Nous avons aussi évalué l’expression des fimbriae lors de différentes conditions de croissance en laboratoire par RT-PCR. Tous les tests réalisés nous ont permis de découvrir que plusieurs opérons fimbriaires de S. Typhi sont opérationnels et utilisés pour différentes fonctions par la bactérie.<br>Fimbriae are extracellular proteinaceous appendages found in many bacteria. They are widely studied and believe to be implicated in several cellular functions such as adhesion, invasion of eukaryotic cells, and biofilm production. They are classified depending on their pathway of secretion: some, like type IV pili, use self-specific machinery, while others use the general secretory pathway followed by their own assembly pathway such as the Chaperon Usher Pathway (CUP fimbriae) and the nucleation precipitation pathway (curli). Despite everything that is known about these structures, little has been discovered regarding fimbrial systems of Salmonella enterica serovar Typhi (S. Typhi). This pathogen is a human restricted serovar and the etiological agent of typhoid fever. Since fimbriae have been implicated in host adaptation, we have decided to further study S. Typhi fimbrial arsenal in the hope of uncovering virulence factors unique to S. Typhi and implicated in host specificity. The S. Typhi ISP1820 strain carries 14 operons encoding for fimbrial structures, but many are believed pseudogenes or are not expressed in vitro. In order to study these different adhesion systems in S. Typhi, we have deleted each one individually and cumulatively by allelic exchange mutagenesis. Hence, we have tested every individual mutation and the mutant strain deprived of all 14 operons in many different assays including epithelial cell and macrophage infection, mobility, and biofilm formation. We also evaluate expression during growth under laboratory conditions by RT-PCR. These experiments have allowed us to discover that many of S. Typhi fimbriae are functional, expressed, and used by the bacteria in many different processes.

Cystic fibrosis (CF) is a fatal, incurable genetic disease that affects over 30,000 people in the United States alone. People with this disease have a homozygous mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) which causes defects in chloride transport and leads to build up of mucus in the lungs and disruption of function in various organs. CF patients often suffer from chronic bacterial infections within the lungs, wherein the bacteria persist as a biofilm, leading to poor prognosis. Two of these pathogens, <i>Stenotrophomonas maltophilia</i> and <i>Pseudomonas aeruginosa</i>, are often found in the lungs of patients with CF and are an increasing medical concerns due to their intrinsic antimicrobial resistance. Both species can readily form biofilms on biotic and abiotic surfaces such as intravascular devices, glass, plastic, and host tissue. Biofilm formation starts with bacterial attachment to a surface and/or adjacent cells, initiating the acute infection stage. Chronic, long-term infection involves subsequent or concurrent altered genetic regulation, including a downregulation of virulence factors, resulting in the bacteria committing to a sessile lifestyle, markedly different from the planktonic one. Many of these genetic switches from an acute to chronic lifestyle are due to pressures from the host immune system and lead to permanently mutated strains, most likely an adaptive strategy to evade host immune responses. Biofilms are extremely problematic in a clinical setting because they lead to nosocomial infections and persist inside the host causing long-term chronic infections due to their heightened tolerance to almost all antibiotics. Understanding the genetic networks governing biofilm initiation and maintenance would greatly reduce consequences for CF and other biofilm-related infections and could lead to the development of treatments and cures for affected patients. This study showed that in<i> S. maltophilia</i>, isogenic deletion of phosphoglycerate mutase (<i>gpmA</i>) and two chaperone-usher pilin subunits, <i>S. maltophilia</i> fimbrae-1 (<i>smf-1</i>) and<i> cblA</i>, lead to defects in attachment on abiotic surfaces and cystic fibrosis derived bronchial epithelial cells (CFBE). Furthermore, Δ<i>smf-1</i> and Δ<i>cblA</i> showed defects in long-term biofilm formation, mimicking that of a chronic infection lifestyle, on abiotic surfaces and CFBE as well as stimulating less of an immune response through TNF-α production. This study also showed that in <i>P. aeruginosa</i>, the Type III secretion system (T3SS), an important virulence factor activated during the acute stage of infection, is downregulated when <i>polB</i>, a stress-induced alternate DNA polymerase, is overexpressed. This downregulation is due to post-transcriptional inhibition of the master regulatory protein, ExsA. Taken together, this project highlights important genes involved in the acute and chronic infection lifestyle and biofilm formation in <i>S. maltophilia</i> and genetic switches during the acute infection lifestyle in <i>P. aeruginosa</i>.

La fixation de l’azote diatomique est un processus très important à la vie, vu sa nécessité dans la biosynthèse de plusieurs molécules de base; acides aminés, acides nucléiques, etc. La réduction de l’azote en ammoniaque est catalysée par la nitrogénase, une enzyme consommatrice de beaucoup d’énergie étant donné qu’elle nécessite 20 à 30 moles d’ATP pour la réduction d’une mole d’azote. De ce fait une régulation rigoureuse est exigée afin de minimiser le gaspillage d’énergie. Plusieurs systèmes de contrôle sont connus, aussi bien au niveau post-traductionnel que traductionnel. Chez la bactérie photosynthétique pourpre non-sulfureuse R. capsulatus, la régulation de l’activité de la nitrogénase nécessite une panoplie de protéines dont la protéine membranaire AmtB, qui est impliquée dans le transport et la perception d’ammonium, et les protéines PII qui jouent plusieurs rôles clés dans la régulation de l’assimilation d’azote. Suite à l’ajout de l’ammonium dans le milieu, une inhibition réversible de l’activité de la nitrogénase est déclenchée via un mécanisme d’ADP-ribosylation de la nitrogénase. La séquestration de GlnK (une protéine PII) par l’AmtB permet à DraT, une ADP-ribosyltransférase, d’ajouter un groupement ADP-ribose sur la protéine-Fe de la nitrogénase l’empêchant ainsi de former un complexe avec la protéine-MoFe. Donc, le transfert d’électrons est bloqué, engendrant ainsi l’inhibition de l’activité de la nitrogénase qui dure aussi long que la concentration d’azote fixé reste élevé, phénomène appelé le « Switch-off/Switch-on » de la nitrogénase. Dans ce mémoire, pour mieux comprendre ce phénomène de régulation, des mutations ponctuelles au niveau de certains résidus conservés de la protéine AmtB, dont D338, G367, H193 et W237, étaient générées par mutagénèse dirigée, afin d’examiner d’avantage leur rôle dans le transport d’ammonium, la formation du complexe AmtB-GlnK, ainsi que dans le « Switch-off » et l’ADP-ribosylation. Les résultats permettent de conclure l’importance et la nécessité de certains résidus telle que le G367 dans la régulation de la nitrogénase et le transport d’ammonium, contrairement au résidu D338 qui ne semble pas être impliqué directement dans la régulation de l’activité de la nitrogénase. Ces résultats suggèrent d’autres hypothèses sur les rôles des acides aminés spécifiques d’AmtB dans ses fonctions comme transporteur et senseur d’ammonium.<br>The reduction of diatomic nitrogen is a very important biological process given the need of all organisms for fixed nitrogen for the biosynthesis of basic key molecules such as, amino acids, nucleic acids, etc.. The reduction of nitrogen to ammonia is catalyzed by nitrogenase, an enzyme with high energy demands since it requires 20 to 30 moles of ATP for the reduction of one mole of nitrogen. Therefore a strict control is required to minimize energy waste. Several systems of regulation are known, both at the translational and post-translational level. In the purple non-sulfur photosynthetic bacterium R. capsulatus, the post-translational regulation of nitrogenase activity requires an array of proteins, including; the membrane protein AmtB, implicated in the perception and transport of ammonium, and PII proteins, which play key roles in the regulation of nitrogen assimilation. Following the addition of ammonium to the medium nitrogenase activity is reversibly inhibited (nitrogenase switch-off) via a mechanism of ADP-ribosylation of nitrogenase. Sequestration of GlnK (PII protein) by AmtB allows DraT, an ADP-ribosyltransferase, to add an ADP-ribose group to the Fe protein preventing it from forming a complex with the MoFe protein and nitrogenase activity is consequently inhibited. To better understand this phenomenon, in this Master’s thesis point mutations were created by site-directed mutagenesis at specific conserved residues of the AmtB protein, namely, D338, G367, H193 and W237, in order to examine their role in ammonium transport, formation of an AmtB-GlnK complex, and the regulation of nitrogenase (Switch-off/ADP-ribosylation). Plasmid-borne mutant alleles were transferred to a ∆AmtB strain of R. capsulatus, and the resultant strains were subjected to a series of tests. These demonstrated the importance and necessity of certain residues, such as G367, in the regulation of nitrogenase and ammonium transport, in contrast to residue D338, which seems to have no direct role in the regulation of nitrogenase activity. These results suggest further hypotheses about the roles of specific amino acids of AmtB in its functions as a sensor and transporter for ammonium.

Geobacter sulfurreducens possesses type IV pili that are considered to be conductive nanowires and a crucial structural element in biofilm formation, enabling electron transfer to insoluble metal oxides in anaerobic sediments and to graphite anodes in microbial fuel cells. The molecular mechanism by which electrons are transferred through the nanowires to the electron acceptor is not fully understood. Prior to the work described in this thesis, the gene (pilA) encoding the structural pilus subunit had been identified, but little was known about the functional translation start codon, the length of the mature secreted protein, or what renders the pili conductive. Using mass spectrometry, I found that a tyrosine residue (Y32) near the carboxyl terminus of the mature PilA protein is posttranslationally modified by attachment of glycerophosphate. I studied the significance of Y32 for biofilm formation on various surfaces and for growth of G. sulfurreducens with insoluble electron acceptors. A mutant in which Y32 was replaced by phenylalanine lacked the glycerophosphate; biofilm formation on graphite surfaces was severely diminished and current production in microbial fuel cells was initiated only after a long lag phase. Moreover, cells with Y32F mutation in the pilA gene exhibited growth deficiency when Fe(III) oxide was the sole electron acceptor. My data confirm the role of G. sulfurreducens pili in biofilm formation and electron transfer to Fe(III) oxide and identify an amino acid in the PilA protein that is essential for these two processes. I also confirmed the existence of two functional translation start codons for the pilA gene and identified two isoforms (short and long) of the PilA preprotein by series of genetic complementation experiments. The short PilA isoform is found predominantly in an intracellular fraction, and seems to stabilize the long isoform and influence the secretion of several outer surface c-type cytochromes. The long PilA isoform, on the other hand, is required for secretion of PilA to the outer surface of the cell, a process that requires co-expression of pilA and the nine genes on its 3' side. The long isoform is essential for biofilm formation on various surfaces, for optimum current production in microbial fuel cells, and for growth on insoluble Fe(III) oxide. This study provides new insight concerning the function and biogenesis of Geobacter type IV PilA, as well as a foundation for further research that will be conducted on microbial nanowires.