The expression of biodegradative lysine decarboxylase of E. coli is induced in cells grown at low pH, and the maximum induction was observed in the presence of excess lysine under anaerobic conditions. The cadA gene encoding the lysine decarboxylase resides in the cadBA operon. The sequence of about 5 kb of the cadBA operon (3.7 kb) and its flanking regions has been determined. The cadB gene encodes a protein very similar to the ArcD protein, the arginine: ornithine antiporter of Pseudomonas aeruginosa. Both cadA and cadB genes have been subcloned into different expression vectors and the protein products have been expressed and observed in maxicell experiments. The cadB gene has been found to facilitate the excretion of cadaverine, one of the end products of the enzymatic reaction of CadA (lysine decarboxylase), and therefore CadB probably encodes a lysine: cadaverine antiporter. A model for detoxification of extracellular high H$\sp+$ concentration based on the functions of CadA and CadB is proposed. Studies on regulation of the cad operon were performed using lac fusion techniques. A high affinity pH-responsive site(s) was found to be located over 100 bp upstream from the cad promoter by a series of deletion and mutation experiments, and the involvement of a positive pH regulator was confirmed in protein titration experiments. Random mutagenesis and in vivo footprinting were conducted to determine the importance of specific residues in the cad upstream region. The residues detected in these studies of the cad promoter region suggest a complex regulatory mechanism. The possible routes of regulation by the availability of lysine and oxygen were also explored. Hypothetical models account for experimental results are presented.

Genetic Regulation in Clostridium acetobutylicum was studied to understand what signals caused this bacterium to switch between different growth stages. A biochemical approach was employed to study DNA supercoiling, while a molecular approach was employed to study sigma factors. Cells treated with novobiocin, a gyrase inhibitor, produced higher butyrate levels and lower butanol and acetone levels. Seven enzyme activities involved in acid and solvent production were tested; CoA transferase, which catalyzes acetone formation and acid uptake, experienced a 50% decrease in activity. However, Northern analysis showed that CoA transferase mRNA levels did not decrease to a higher extent than mRNA levels of other enzymes involved in acid and solvent production, suggesting that the regulation was at the post-transcriptional level. The data also suggest that acetone and alcohol production may be regulated by different mechanisms. Three genes were cloned from C. acetobutylicum into Escherichia coli and sequenced. Two of them encoded proteins that show high homology with two Bacillus subtilis sporulation-specific sigma factors, $\rm\sigma\sp{E}$ and $\rm\sigma\sp{G}$ and the third one encoded a putative $\sigma\sp{E}$-processing enzyme. The gene arrangement of these three genes was conserved in two Bacillus species. Duplication and inactivation of these genes were then attempted. While the wild-type strain eventually ceased to grow and produce solvents in batch cultures (in test tubes) over time, the mutant strains showed sustained growth for a much longer time and produced a threefold increase in acetone and butanol. By regulating sporulation, $\rm\sigma\sp{E}$ and additional sigma factors may play a role in both solvent induction and termination. A model was proposed suggesting an overlap between signals causing cells to produce solvents and to sporulate. Loss of such signals may lead to degenerate cells. By understanding more about the genetic regulation of this organism, it is possible to perform metabolic engineering to increase its potential to be used in biotechnology.

Both neoplastic and Neisseria meningitidis cells can obtain iron from transferrin in vivo. Previous findings (Holbein, 1980) showed that the murine hypoferremic response inhibits Neisseria meningitidis infection by iron deprivation. The present studies demonstrated that hypoferremia does not limit Fe acquisition by or growth of murine lymphoma cells. Transferrin binding sites on lymphoma cells and N. meningitidis were enumerated and the transferrin binding affinities were determined. Iron deprivation increased the number of transferrin binding sites on N. meningitidis cells. N. subflava and Escherichia coli were unable to bind transferrin. Competition binding studies demonstrated that lymphoma cells bound human transferrin over murine and bovine transferrin, conalbumin, and lactoferrin and suggested that neisserial transferrin binding sites had lower specificity. Further competition studies indicated that lymphoma transferrin binding sites had higher affinity for ferri-transferrin than for apo-transferrin while neisserial sites bound these two ligands to the same extent. This can explain why hypoferremia inhibits iron acquisition by N. meningitidis but not by lymphoma cells.

No description available.

Plasmacytoid dendritic cells (pDCs) are especially suited for initiating immune responses against viruses because they are the most potent producers of type I interferon (IFN). This property of controlling viral infections is accomplished by their selective expression of toll-like receptor (TLR)-7 and TLR-9, which recognize viral single-stranded RNA (ssRNA) and unmethylated cytosine-phosphate-guanine (CpG)-containing double-stranded DNA (dsDNA), respectively. Through the secretion of Type I IFN, pDCs can modulate natural killer (NK) cell cytotoxicity, myeloid DC (mDC) activation, B cell maturation and T cell priming. Mouse pDCs express Ly49Q, a C-type lectin-like immunoreceptor tyrosine-based inhibitory motif (ITIM) -containing receptor that binds to the classical major histocompatibility complex class I molecule (MHC-Ia), H-2K(b). Prior to our studies, both the ligand and function of Ly49Q on pDCs was unknown. / We used reporter cell analysis to demonstrate that a high-affinity ligand for Ly49Q is present on H-2(b), but not H-2(d), H-2(k), H-2(q), or H-2(a)-derived tumour cells and normal cells ex vivo. The ligand is peptide-dependent and MHC-Ia-like, as revealed by its functional absence on cells deficient in TAP-1, beta-2m, or H-2K(b)/D(b) expression. Furthermore, Ly49Q is specific for H-2K(b), as the receptor binds peptide-loaded H-2K(b) but not H-2D(b) complexes, and Ly49Q recognition can be blocked using anti-K(b) but not anti-D(b) mAb. These results demonstrate that Ly49Q efficiently binds H-2K(b) and suggest that pDC function is regulated by classical MHC-Ia molecules. / To characterize the implications of Ly49Q: H-2K(b) interaction, we utilized the Ly49Q-deficient mice previously generated in our lab. Ex vivo pDCs stimulated with CpG resulted in enhanced IFN-alpha and IL-12 production. Blocking with soluble Ly49Q or H-2K(b) mAb abrogated cytokine production, while receptor cross-linking with platebound anti-Ly49Q and recombinant H-2K(b) increased cytokine production. Sera obtained from CpG injected Ly49Q-deficient mice and supernatant obtained from CpG stimulated Ly49Q-deficient pDCs displayed reduced cytokine production. / Taken together, we conclude that Ly49Q recognition of H-2K(b) is crucial for pDC cytokine production. Insight into human pDC immune response against transformed and pathogen infected cells can be acquired from characterizing the Ly49Q ligand and receptor in the mouse model. / Les cellules dendritiques plasmacytoïdes (pDCs) sont très efficaces pour initier une réponse immunitaire contre les virus parce qu'elles sont des productrices efficaces d'interféron (IFN) de type I. Cette habileté à contrôler les infections virales est accompagnée par une expression sélective des récepteurs Toll-like (TLR)-7 et TLR-9 qui reconnaissent, en ordre, l'ARN viral à simple brin et l'ADN à double brin contenant des séquences cytosine-phosphate-guanosine (CpG) non-méthylées. Par la sécrétion d'IFN de type I, les pDCs peuvent moduler la cytotoxicité des cellules natural killer (NK), l'activation des DC myéloïdes (mDCs), la maturation des lymphocytes B et l'activation des lymphocytes T. Les pDCs murines expriment le récepteur Ly49Q, une lectine de type C contenant une séquence de type immunoreceptor tyrosine-based inhibitory motif (ITIM) qui reconnaît une molécule classique du complexe majeur d'histocompatibilité (CMH de classe Ia), H-2K(b). Avant nos études, le ligand et la fonction de Ly49Q chez les pDCs étaient inconnus. / Nous avons utilisé un test de cellules ayant un gène rapporteur pour démontrer qu'un ligand pour Ly49Q est présent sur des cellules tumorales et des cellules isolées ex vivo d'origine H-2(b), mais pas d'origine H-2(d), H-2(k), H-2(q), ou H-2(a). Le ligand dépend de la présence de peptides et est une molécule CMH de type Ia puisque le ligand est absent de cellules déficientes pour l'expression de TAP-1, beta-2m, ou d'H-2K(b)/D(b). De plus, Ly49Q est spécifique pour H-2K(b), puisque le récepteur se lie à H-2K(b) couplé à un peptide et non à un complexe de peptide et d'H-2D(b) et la reconnaissance par Ly49Q peut être bloquée par un anticorps pour H-2K(b) mais pas pour H-2D(b). Ces résultats démontrent que Ly49Q se lie efficacement à H-2K(b) et suggèrent que la fonction des pDCs est contrôlée par des molécules classiques CMH de type Ia. / Afin de caractériser les implications de l'interaction entre Ly49Q et H-2K(b), nous avons utilisé la souris déficiente pour Ly49Q précédemment générée par notre laboratoire. Des pDCs isolés ex vivo stimulées avec CpG produisent des quantités élevées d'IFN-alpha et d'IL-12. La production de cytokines peut être bloquée à l'aide d'anticorps pour Ly49Q ou H-2K(b) tandis que la ligation de Ly49Q à l'aide d'un anticorps ou d'H-2K(b) recombinant augmente la production de cytokines. Du sérum isolé de souris déficientes en Ly49Q injectées avec du CpG et du milieu de culture de pDCs déficients en Ly49Q stimulés avec du CpG démontrent une production diminuée de cytokines. / Pour résumer, nous concluons que la reconnaissance d'H-2K(b) par Ly49Q est cruciale pour la production de cytokines par les pDCs. Une meilleure compréhension de la réponse immunitaire des pDCs humains contre les cellules transformées et infectées par des pathogènes peut être obtenue par l'étude du récepteur Ly49Q et de son ligand dans le modèle murin.

The cell envelope of Gram-negative bacteria such as Escherichia coli is composed of an outer (OM) and cytoplasmic membrane (CM), which enclose a space known as the periplasm. For acquisition of essential nutrients, bacteria must transport molecules across both membranes. The import of certain scarce nutrients, such as iron and vitamin B12, requires the input of energy; however, the OM is devoid of any source of energy. To drive the transport of scarce nutrients across the OM, bacteria use the proton motive force of the CM. The TonB–ExbB–ExbD protein complex located within the CM is responsible for harnessing the energy of this proton gradient and transducing it to OM receptors. While the existence and the role of this complex has been investigated many years ago by genetic means and by in vivo crosslinking experiments, structural and functional aspects remain largely unknown. To date, only partial structures of periplasmic domains of TonB and ExbD are known. Additionally, information on the structure of the complex as well as stoichiometry has not been deduced. We have therefore designed a strategy for the co-expression and co-purification of ExbB and ExbD in complex. Here we produced milligram amounts of pure ExbB–ExbD; size-exclusion chromatography revealed that our samples were homogenous and monodisperse. Observation of our samples by negative-staining electron microscopy revealed uniform particles from which 2D reconstructions were generated. These reconstructions suggested a pentameric and perhaps cylindrical arrangement for the complex. In parallel, crystallization trials have identified promising leads. Our optimization efforts have led to the successful detergent exchange of the protein complex from dodecyl maltoside to decyl maltoside, to GNG-12 and to MNG-28; the latter two amphiphiles are proprietary and of undisclosed structure. We also adopted an NMR detergent quantitation method to standardize amounts of detergent in samples bound for crystalliz / L'enveloppe cellulaire des bactéries à Gram négatif tel Escherichia coli est constituée d'une membrane externe (ME) et d'une membrane cytoplasmique (MC), entre lesquelles se trouve un espace nommé périplasme. L'acquisition de nutriments essentiels par les bactéries nécessite le transport de molécules à travers chacune des deux membranes. Le transport de certains nutriments rares, tel le fer et la vitamine B12, requiert un apport en énergie. Cependant, la ME est dénuée de toute source d'énergie. Ainsi, pour alimenter en énergie le transport de nutriments rares à la ME, les bactéries font appel à la force proton motrice de la MC. Le complexe protéique TonB–ExbB–ExbD, situé à la MC, arnache l'énergie de ce gradient de protons et la transduit aux récepteurs de la ME. Bien que l'existence et le rôle de ce complexe aient été sondés il y a plusieurs années par des voies génétiques et des expériences de réticulation in vivo, plusieurs aspects de la structure et de la fonction du complexe restent inconnus. Jusqu'à maintenant, seule la structure des domaines périplasmiques de TonB et ExbD sont connus. Qui plus est, le structure du complexe et sa stoechiometrie n'ont pas été résolus. Nous avons donc élaborée une stratégie pour la co-expression et la co-purification d'ExbB et d'ExbD en complexe. Nous produisons plusieurs milligrammes d'ExbB–ExbD pur et la chromatographie par filtration sur gel révèle que nos échantillons sont homogènes et monodispersés. L'observation de nos échantillons par microscopie électronique en coloration négative révèle des particules de taille uniformes à partir desquelles des reconstructions 2D ont été générées. Ces reconstructions semblent suggérer un arrangement pentamérique et peut-être cylindrique. En outre, nos essais de cristallisation nous ont permis d'identifier des pistes prometteuses. Nos efforts d'optimisation nous ont amenés à échanger avec succès notre protéine depuis l

To investigate the functionality of the Sinorhizobium meliloti dihydrolipoamide dehydrogenases, the lpdA1, lpdA2, and lpdA3 genes were mutated through the site-directed, single cross-over recombination, using modified pVIK112 suicide plasmids. The lpdA1 mutant failed to grow on pyruvate, and was significantly delayed on other tested carbon sources. The activity of the pyruvate dehydrogenase complex was not detected in this mutant, and the regulation of the lpdA1 was not dependent on the presence of pyruvate. This mutant was Nod+, but appeared Fix-. Although the lpdA2 mutant was able to grow on all of the tested carbon sources, it was significantly delayed growing on pyruvate, arabinose, glutamate, and leucine. The activity of α-ketglutarate dehydrogenase was almost abolished, but the mutant was able to fix atmospheric nitrogen. The expression of the lpdA2 was upregulated during growth on arabinose, malate, and succinate. The lpdA3 mutant, otherwise indistinguishable from the wild-type RmG212, was significantly delayed growing on leucine, which also upregulated the expression of the lpdA3. Although the activity of the branched-chain α-ketoacid dehydrogenase was not detectable in this mutant, it was able to fix the atmospheric nitrogen. This study demonstrated that the lpdA genes encode functional proteins that are constitutive elements of the three different enzyme complexes. / Afin d'étudier la fonctionnalité des déshydrogénases du dihydrolipoamide de Sinorhizobium meliloti, les gènes lpdA1, lpdA2 et lpdA3 ont été mutés par la méthode de mutagenèse dirigée par le recombinaison cross-over unique, au moyen de plasmides suicides modifiés pVIK112. Le mutant lpdA1n'a pas été capable de croître sur le pyruvate, et sa croissance a été considérablement retardée sur les autres sources de carbone qui furent essayées. L'activité du complexe pyruvate déshydrogénase n'a pas été décelée dans ce mutant, et la régulation du lpdA1 ne dépendait pas de la présence de pyruvate. Ce mutant était Nod +, mais il paraissait être Fix-. Bien que le mutant  lpdA2 s'avéra capable de pousser sur toutes les sources de carbone qui furent essayées, sa croissance fut ralentie de façon appréciable sur les milieux pyruvate, arabinose , glutamate et leucine. L'activité de la déshydrogénase α-ketglutarate était presque abolie, mais le mutant a été capable de fixer l'azote atmosphérique. L'expression du mutant lpdA2 a été augmentée au cours de la croissance sur l'arabinose, le malate et le succinate. L'expression du mutant lpdA3, autrement impossible à distinguer de la souche mère RMG212, a été considérablement retardée sur la leucine, qui a aussi augmenté l'expression du lpdA3. L'activité de la déshydrogénase à chaîne ramifiée α-cétoacide n'était pas détectable dans ce mutant, mais il a été capable de fixer l'azote atmosphérique. Cette étude a démontré que les gènes lpdA codent pour des protéines fonctionnelles qui sont des éléments constitutifs de trois complexes enzymatiques différents.

Caulobacter crescentus provides an accessible system for investigating the regulation of chromosome replication and cellular development. The Caulobacter cell cycle produces a free-swimming swarmer cell and a sessile stalk cell. In swarmer cells, chromosome replication is selectively repressed while stalk cells are committed to chromosome replication. / In Caulobacter, chromosome replication is repressed, in part, by the binding of the response regulator CtrA to five binding sites (a-e) within the Caulobacter origin of replication (Cori ). Periodic phosphorylation of CtrA stimulates binding to the consensus sequence TTAA-N7-TTAA (N= any nucleotide) found in Cori and many cell-cycle regulated genes. This thesis presents an alternate mode of CtrA binding, namely, that phosphorylation does not stimulate binding to a specific class of CtrA-regulated promoters. This work shows that CtrA and CtrA-phosphate bind to two ctrA promoters with equal and weak affinity. As well, in vivo binding assays reveal that a non-proteolyzable CtrA allele (CtrADelta3) can occupy the ctrA promoters continuously without altering the temporal regulation of these promoters. The data suggest phosphorylation, while not increasing affinity for weak CtrA binding sites, provides allosteric signals that permit the recruitment of components required for transcription. / The proposed allosteric mechanism of CtrA-regulated transcription may also be important for CtrA-mediated repression of chromosome replication. Chromatin Immunoprecipitation assays (ChIP) allow for the sensitive detection of specific protein/DNA complexes in vivo. ChIP reveals that CtrA binds to Cori in swarmers but not in stalk cells when chromosome replication commences. The protein chaperone, ClpX, was recruited to Cori prior to the start of S-phase and correlates with the loss of CtrA binding to Cori. Expression of a non-proteolyzable CtrADelta3 allele showed increased affinity for Cori DNA. The increase in CtrADelta3 binding stimulated a corresponding increase in C1pX binding to Cori. This evidence suggests that C1pX recruitment to Cori is likely CtrA-dependant. The absence of CtrA binding in stalk cells suggests other mechanisms may be required to prevent re-replication in stalk cells. / An analysis of the Caulobacter genome identifies two DnaA-like genes. The first, cdl-1, is a homolog of the E. coli hda gene, a protein essential for regulated inactivation of DnaA (RIDA). The second, cdl-2, is a novel gene restricted to the alpha-proteobacteria group and whose function is unknown. Overexpression of either gene in Caulobacter produced filamentous cells that could not divide. DNA synthesis in these cells is also impaired and suggests the intracellular concentrations of these two proteins are important for coordinating proper cell cycle progression.

The work in this thesis consisted of two studies: 1) analysis of the microbial biodiversity of multiple ground ice types from the Arctic and the High Arctic; 2)examination of the survival of Cryptococcus NP33 under simulated Martian conditions over 41 days. The first study involved culture-dependent and independent evaluations of the microbial communities found in a buried firnified snow bank, a buried glacier, a pingo and ice wedges. Direct and culturable counts in the various ground ice types differed from each other (104 – 108 cellsmL-1 direct counts; 0 - 105 CFUmL-1 culturable counts), and were only weakly correlated to increasing sample age. Culturable counts were consistently highest in ice wedge samples. All sample isolates were dominated by Actinobacteria. Bacterial pyrosequencing analysis for one ice wedge showed a dominance (<50% of sequences) by Gammaproteobacteria. In an Archaeal clone library of the buried glacier, no clones were closely related to sequenced isolates, but were similar (>90%) to uncharacterized clones from marine environments. The pingo Bacterial clone library clones matched closely to environmental isolates as well as clones from cryoenvironments as well as soil environments. For the survivability study, Cryptotoccus strain NP33 was selected as a candidate organism to undergo Martian simulations. After 41 simulation days, it had a half-life of 10.1 days in simulated sunlightand 16.1 days in darkness. The compiled results suggest that the organism traits most crucial to survival under simulated Martian conditions were desiccation, radiation and freeze-thaw resistance. / Cette thèse contient deux études : 1) la biodiversité de différents types de glacesde sol de l'Arctique et du Grand Arctique, de même que la survie de Cryptococcus NP33dans des conditions martiennes simulées pendant 41 jours. La première étude impliquait des analyses dépendantes et indépendantes des conditions de culture pour évaluer les communautés microbiennes dans une congère névée enterrée, un glacier enterré, un pingo et des coins de glace. Les nombres de cellules totales et les nombres de cellules culturées dans les différents types de glaces de sol variaient (104 – 108 cellulesmL-1 nombre total; 0- 105 CFUmL-1 cellules culturées), et étaient que très faiblement dépendants de l'âge du iispécimen. Les nombres de cellules culturées étaient constamment plus élevées dans les coins de glace. Actinobacteria dominait les isolats de chaque spécimen. Un pyroséquençage bactérien d'un coin de glace a révélé une dominance (>50% desséquences) de Gammaproteobacteria. Dans une librairie de clones d'Archées du glacier enterré, les clones avaient peu de similarité à des isolats environnementaux, mais étaient similaires (>90%) à des clones environnementaux non-caractérisés d'environnements marins. Dans une librairie de clones de Bactéries du pingo, les clones étaient très similaires à des isolats et des clones provenant de cryo-environnements et d'environnements de sol. Pour la simulation martienne, Cryptococcus NP33 a été choisicomme organisme candidat suite à des expériments pour sélectionner des organismes résistant à la dessiccation, au froid et aux concentrations élevées de sel. Au cours de 41 jours dans le simulateur, Cryptococcus NP33 avait une demi-vie de 10.1 jours dans le soleil simulé et 16.1 jours dans le noir. Halorubrum avait un taux de survie de 100%(demi-vie estimée de ~70 - ∞ jours), tandis que d'autres organismes avaient une demi-vie beaucoup moins élevée (~2 - ~8 jours). Les résultats combinés suggèrent que les caractéristiques nécessaires à la survie dans des conditions martiennes simulées étaient la résistance à la dessiccation, la radiation et aux cycles de gel-dégel.

Studies of extraintestinal infections caused by genetically related strains of Escherichia coli among unrelated people have demonstrated the epidemic potential of this group of bacteria. These related extraintestinal pathogenic E. coli (ExPEC) may have a common source. Our group recently described how retail meat, particularly chicken, may be a reservoir for ExPEC causing human urinary tract infections (UTIs). By moving upstream on the farm to fork continuum, this study tests whether the reservoir for ExPEC is in food animals themselves. A total of 824 geographically and temporally matched E. coli isolates from cecal contents of slaughtered food animals (n=349) and human UTI (n=475) sources were compared. Using 6 different typing methods, an evolutionary relationship was observed between E. coli isolates from the food animal reservoir and human UTI. Moreover, chicken was the predominant animal species from where the related isolates originated. Using an evolutionary model, chicken was determined to be the most likely source of the human UTI isolates. This study confirmed that an animal reservoir, principally in chicken, may exist for ExPEC causing community-acquired UTI. / Les études portant sur les infections extra-intestinales causées par des souches d'Escherichia coli génétiquement apparentées, chez des personnes non reliées entre elles, ont démontré le potentiel épidémique de ce groupe de bactéries. Ces souches d'E. coli pathogènes extra-intestinales (ExPEC) apparentées auraient possiblement une source commune. Notre groupe a récemment décrit comment la viande de détail, plus particulièrement le poulet, pourrait être un réservoir d'ExPEC responsables d'infections urinaires (IUs) chez les humains. En se déplaçant plus en amont dans le continuum de la ferme à la fourchette, cette étude teste si le réservoir d'ExPEC se trouve dans les animaux de production eux-mêmes. Un total de 824 isolats d'E. coli de provenances géographique et temporelle communes, prélevés dans le contenu caecal d'animaux abattus (n=349) et de cas d'IU humaine (n=475) ont été comparés. Par l'utilisation de 6 différentes méthodes de typage, une relation évolutionnaire a été observée entre les isolats d'E. coli provenant du réservoir animal et d'IU humaine. De plus, le poulet était l'espèce animale prédominante parmi les isolats parentés. L'utilisation d'un modèle évolutionnaire a permis de déterminer que le poulet est la source la plus probable des isolats d'IU humaine. Cette étude a confirmé qu'un réservoir animal, principalement chez le poulet, pourrait exister pour les ExPEC qui causent des IUs acquises en communauté.