<p> This thesis describes the isolation and investigation of a DNA-dependent RNA polymerase from the extreme halophile Halobacterium cutirubrum.</p> <p> The enzyme system was analyzed under conditions of very high ionic strengths which are characteristic of the internal salt concentrations of extreme halophiles and at much lower ionic strengths found in conventional bacterial systems. The enzyme was found to have activity in a wide range of salt concentrations when attached to its DNA template in the form of a DNA-Membrane-Protein complex. The enzyme, however, lost the ability to function at high ionic strengths when freed from this complex.</p> <p> The properties of the isolated DNA-dependent RNA polymerase from the halophile were then compared to the properties of the same enzyme isolated from the non-halophilic bacterium, Eschericia coli. Both enzymes were found to have the same approximate molecular weights and to share the same substrate requirements. The enzymes differed, however, in their response to inhibitors specific for RNA synthesis. </p> / Thesis / Master of Science (MSc)

No description available.

Marine vibrio MV-1 is a magnetotactic bacterium capable of aligning its cell in response to the Earth’s magnetic field. This ability is due to the presence of chainlike structures comprising magnetosomes, magnetite particles enclosed in a lipid membrane with associated proteins. Strain MV-1 differs from other, bettercharacterized strains of magnetotactic bacteria as the cells produce higher amounts of biomagnetite per litre of culture and its magnetosomes are unique in shape. This study investigates the presence and organisation of a gene cluster termed a “magnetosome island” within the genome of MV-1. In other magnetotactic bacteria this genomic region has been shown to contain many of the genes associated with magnetosome formation but has not been previously investigated for MV-1. One of the conserved fragments of this region was amplified using degenerate primers followed by extension of the known sequence using inverse PCR based technique and constructing plasmid libraries. Sequencing of the genome of strain MV-1 was accomplished as a part of this study. Significant work was done on comparison of the sequence quality obtained from SOLEXA, 454 and Sanger sequencing technologies. A number of obtained contigs were joined manually and the resulting sequence was automatically annotated using RAST. The obtained genome sequence of 3.6 Mb with a G+C content of 54.3 % was preliminarily analysed and used to search for magnetosome related genes. This study also analysed proteins associated with the magnetosomes of strain MV-1 using MALDI-TOF, LC-MS and Orbitrap mass spectrometry. These approaches allowed the identification of a number of proteins in the isolated magnetosome membrane fraction. Some of these proteins have very low similarity with other characterized proteins (either in magnetotactic bacteria or in other organisms). Another significant point is that genes that code for proteins such as MamR, MamK and MmsF were found to be present in several homologous copies within the “magnetosome island” of MV-1. Interestingly, this study shows that all homologous copies of these proteins were identified in the magnetosome membrane fraction. Generation of knock-out mutants of several specific genes from the “magnetosome island” of strain MV-1 was attempted; constructs were made based on suicide plasmids carrying the cre-lox or I-SceI systems. Despite altering numerous experimental conditions it was not possible to obtain conclusive evidence of the isolation of MV-1 transconjugants containing the integrated constructs. In order to investigate the cell localization of the magnetosome associated protein CAV30779.1, an enhanced green fluorescent protein (EGFP) fusion based construct was generated and transferred into MV-1 cells. The EGFP fluorescent protein fusions within the cells were detected by microscopy. This study reveals novel information about magnetosome formation in marine vibrio MV-1. The obtained results provide an important foundation for further investigation of this organism and contribute towards broadening the knowledge of the complex process of magnetosome formation in bacteria.

A unique undosciribed gramnegative rod is extensively characterized in this study. The cells of this unusual water isolate measure 1.2 X 6.5 microns, The most distinguishing characteristic of the bacterium is a polar tuft of 35-40 flagella that aggregate to function as a single organelle which is visible under phase contrast. Aging cells deposit poly- -hydroxybutyric acid granules which are bound by an inclusion membrane made up of four distinct layers. It also possesses an unusual exterior membrane outside the cell wall which contains large fibrils of protein running at a slight angle to the longitudinal axis of the cell. The guanosine-cytosine ratio was found to be 62.2$. The organism's taxonomic position was further investigated by immunological, morphological, and biochemical methods. It was found to be most closely akin to members of the genus Pseudo onas, although somewhat divergent from other species classified in this genus. After careful evaluation of the findings obtained during this study, the new bacterium was subsequently named Pseudomonas multiflagella.

A novel Gram negative, capsule-forming bacterium was previously isolated in Dr. G. Roland Vela's laboratory. The distribution of this bacterium in soils from various locations was investigated. Soil samples from 188 locations around the world were examined. Isolates of the bacterium were obtained from 50 of these soils, with 48 of the isolates found in soils from the southwestern United States and northern Mexico. This suggests that this region is the natural habitat of the bacterium. The other two isolates were obtained from Madrid, Spain and Taipei, Taiwan. None were found in soils from South America or Australia. A lack of variation in morphology and physiological properties in the isolates suggests that a homogeneous population exists, even from widespread geographical locations.

The plant growth-promoting bacterium (PGPB) Pseudomonas putida UW4, previously isolated from the rhizosphere of common reeds growing on the campus of University of Waterloo, promotes plant growth in the presence of different environmental stresses, such as flooding, high concentration of salt, cold, heavy metals, drought and phytopathogens. The known mechanisms used by P. putida UW4 to promote plant growth include 1-aminocyclopropane-1-carboxylate (ACC) deaminase, indole-3-acetic acid (IAA) synthesis and siderophore production. In this work, the genome sequence of UW4 was obtained by pyrosequencing and the gaps between the contigs were closed by directed PCR. The P. putida UW4 genome contains a single circular chromosome that is 6,183,388 bp with a 60.05% G+C content. The bacterial genome contains 5,431 predicted protein-coding sequences that occupy 87.4% of the genome. Nineteen genomic islands were predicted and thirty one complete putative insertion sequences were identified. Genome analyses were conducted in order to better characterize the general features of the UW4 genome. Genes potentially involved in plant growth promotion such as IAA biosynthesis, trehalose production, siderophore production, and acetoin synthesis were identified, which will facilitate a better understanding of the mechanisms of plant-microbe interactions. Moreover, genes that contribute to the environmental fitness of UW4 were also determined including genes responsible for heavy metal resistance such as nickel, copper, cadmium, zinc, molybdate, cobalt, arsenate, and chromate. Central metabolic pathways helped elucidate the physiological roles of diverse metabolites of UW4. Unexpectedly, whole-genome comparison with other completely sequenced Pseudomonas sp. revealed that UW4 is more similar to the fluorescens group rather than to the putida group. More surprisingly, a putative type III secretion system (T3SS) was found in the UW4 genome, and T3SS was thought to be essential for bacterial pathogenesis. Although putative T3SS was observed in other non-pathogenic Pseudomonas spp. previously, this is the first report indicating that a T3SS in a Pseudomonas sp. is highly similar to the one from Salmonella spp.

The plant growth-promoting bacterium (PGPB) Pseudomonas putida UW4, previously isolated from the rhizosphere of common reeds growing on the campus of University of Waterloo, promotes plant growth in the presence of different environmental stresses, such as flooding, high concentration of salt, cold, heavy metals, drought and phytopathogens. The known mechanisms used by P. putida UW4 to promote plant growth include 1-aminocyclopropane-1-carboxylate (ACC) deaminase, indole-3-acetic acid (IAA) synthesis and siderophore production. In this work, the genome sequence of UW4 was obtained by pyrosequencing and the gaps between the contigs were closed by directed PCR. The P. putida UW4 genome contains a single circular chromosome that is 6,183,388 bp with a 60.05% G+C content. The bacterial genome contains 5,431 predicted protein-coding sequences that occupy 87.4% of the genome. Nineteen genomic islands were predicted and thirty one complete putative insertion sequences were identified. Genome analyses were conducted in order to better characterize the general features of the UW4 genome. Genes potentially involved in plant growth promotion such as IAA biosynthesis, trehalose production, siderophore production, and acetoin synthesis were identified, which will facilitate a better understanding of the mechanisms of plant-microbe interactions. Moreover, genes that contribute to the environmental fitness of UW4 were also determined including genes responsible for heavy metal resistance such as nickel, copper, cadmium, zinc, molybdate, cobalt, arsenate, and chromate. Central metabolic pathways helped elucidate the physiological roles of diverse metabolites of UW4. Unexpectedly, whole-genome comparison with other completely sequenced Pseudomonas sp. revealed that UW4 is more similar to the fluorescens group rather than to the putida group. More surprisingly, a putative type III secretion system (T3SS) was found in the UW4 genome, and T3SS was thought to be essential for bacterial pathogenesis. Although putative T3SS was observed in other non-pathogenic Pseudomonas spp. previously, this is the first report indicating that a T3SS in a Pseudomonas sp. is highly similar to the one from Salmonella spp.

Thesis (doctoral)--Swedish University of Agricultural Sciences, 2006. / Includes bibliographical references. Also available on the World Wide Web in PDF format.

L’objectif de ce projet était l’étude systématique des interactions, de la microstructure et de la dynamique de suspensions de colloïdes qui imitent les mouvements de systèmes auto-propulsés (actifs), au moyen de techniques de diffusion : diffusion des rayons X aux (ultra) petits angles (USAXS/SAXS) et spectroscopie de corrélation de photons X (XPCS).À la différence des colloïdes passifs conventionnels (particules browniennes), les colloïdes actifs sont des systèmes hors équilibre faits d’objets auto-propulsés. Ces systèmes montrent une dynamique fascinante qui s’apparente aux mouvements de volée d'oiseaux, d’essaim d’insectes, etc.Les micro-organismes mobiles sont des exemples types de colloïdes actifs, notamment certaines variétés de bactéries, ou les colloïdes de type Janus caractérisé par une composition asymétrique de leur surface qui peux engendrer une propulsion, l’auto-diffusiophorèse. Les thèmes principaux de cette thèse sont les interactions inter-particule, les interactions particule-solvant et les dynamiques phorétiques des systèmes actifs.En premier lieu, la structure et la mobilité de la bactérie Escherichia coli ont été étudiées au moyen de SAXS/USAXS . Comme projet secondaire, l’obtention de données couvrant une très large gamme de vecteur de diffusion (q) a permis de dériver un modèle structural multi-échelle de la bactérie, en combinant les caractéristiques de colloïdes (corps cellulaire), de membranes (enveloppe cellulaire) et de polymères (flagelles). Ce modèle a été affiné au moyen de mesures complémentaires de diffusion de neutrons aux petits angles (SANS) sur des suspensions de la bactérie E. coli en variant le contraste externe (remplacement isotopique partielle du solvant) afin d’aboutir à une détermination quantitative de la densité électronique des membranes et des distances entre membranes.Ces bactéries ont ensuite été utilisées comme éléments actifs en mélange avec des colloïdes passifs de silice de taille sub-micrométrique, pour comprendre comment la présence de bactéries actives mobiles affecte les interactions effectives et la dynamique des colloïdes passifs. Des mesures USAXS et XPCS simultanées ont permis de déduire les propriétés statiques et hydrodynamiques de ces colloïdes passifs. Les données suggèrent que les bactéries actives se comportent comme un fluidifiant pour les colloïdes passifs, en réduisant leurs interactions attractives et en augmentant leur dynamique ; réciproquement, ces derniers sont affectés par la solution tampon et par la présence de bactéries augmente la viscosité environnante effective.Enfin, les mouvements phorétiques de colloïdes de silice et de type Janus (silice partiellement recouvert de nickel) suspendus dans un mélange de 3-methylpyridine (3MP) + eau/eau lourde pendant la séparation de phase liquide-liquide ont été investigués par USAXS et XPCS. Les mouvements des colloïdes sont fortement corrélés à la dynamique de la séparation de phase du fait de l’absorption préférentielle de 3MP à la surface de silice. Les colloïdes de silice montrent une dynamique advective avec une diffusion amélioré en direction des microdomaines riches en 3MP, évoquant la dynamique des systèmes auto-propulsés, jusqu’à l’aboutissement de la séparation de phase. Les suspensions de colloïdes de type Janus ont un comportement beaucoup plus complexe, la dynamique étant fortement corrélée aux interactions asymétriques avec le solvant. Cette dynamique est soit augmentée soit supprimée en fonction de la concentration en 3MP qui modifie aussi la micro-structure du système. Au lieu que les colloïdes de silice migrent vers la phase riche en 3MP, les colloïdes de type Janus agissent comme des tensioactifs en se plaçant à l’interface.Cette thèse démontre l’intérêt des techniques de diffusion pour explorer les propriétés des systèmes actifs et examiner leur comportement en thermodynamique hors équilibre afin de compléter les informations obtenues par observations microscopique. / This project aimed to systematically investigate the interactions, microstructure and dynamics in suspensions of colloidal particles that mimic active motions, using (Ultra) Small- Angle X-ray Scattering (USAXS/SAXS) and X-ray Photon Correlation Spectroscopy (XPCS). As opposed to the conventional passive colloids (Brownian particles), active colloids are non-equilibrium systems consisting of self-propelled particles that display many fascinating dynamics, such as streaming, swarming, flocking, etc. in appropriate media. Practical examples of active systems are motile microorganisms, such as some species of bacteria, or synthetic Janus colloids – characterized by an asymmetric chemical composition of their surface – that can induce a propulsion mechanisms, like self-diffusiophoresis. The foci of this thesis are on interparticle interactions, particle-medium interactions and the phoretic dynamics in active systems.Firstly, the structure and motility of Escherichia coli bacteria were investigated by combined USAXS and SAXS methods. As an offshoot, the scattering data spanning a broad scattering vector (q)-range permitted the derivation of a multiscale structural model by combining colloidal (cell-body), membrane (cell-envelope) and polymer (flagella) features. This model was further refined by contrast-variation Small Angle Neutron Scattering (SANS) measurements on E. coli suspensions at three match points and the full contrast which allowed the determination of the membrane electron-density and the inter-membrane distances on a quantitative scale.These bacteria were then used as active component in a mixture with micron-sized passive silica colloids, with the aim of investigating how the effective interactions and dynamics of passive colloids are affected by the presence and the motility of active E. coli. Both static and hydrodynamic information were obtained via the simultaneous use of USAXS and XPCS techniques. Data suggested active bacteria act as a fluidizing agent in such systems, reducing attractive interactions and enhancing the dynamics of passive colloids, which, at the same time, are affected by the buffer and more viscous environment due to the bacterial presence.Finally, the phoretic motions of micron-sized silica colloids and half-coated silica/nickel Janus colloids suspended in a mixture of 3-methylpyridine (3MP) and water/heavy water undergoing liquid-liquid phase separation were investigated using USAXS and XPCS. Due to the preferential wetting of 3MP on the silica surface, the motion of the colloids is strongly correlated to the dynamics of phase separation.Silica colloids displayed advective motion with enhanced diffusion toward the 3MP-rich phase reminiscent of self-propelled motion until the phase separation is completed. Suspensions of Janus colloids showed a much richer scenario, where colloid dynamics are strongly influenced by the asymmetric interactions with the solvent. The dynamics of Janus colloids were either enhanced or suppressed depending on the 3MP concentration, which, concurrently, affected the microstructure of the system. As opposed to the partitioning in 3MP-rich phase in the case of silica colloids, Janus particles behave like surfactants at the interface.The thesis demonstrates the ability of studying active systems by means of scattering methods and probe their behaviour in the thermodynamic limit and complement the information derived from direct microscopy observations.

Plusieurs habitats de l'Océan profond sont caractérisés par la présence de composés chimiques réduits. Les communautés benthiques prospèrent dans ces "habitats réducteurs", en raison de liens trophiques avec des bactéries chimiosynthétiques, qui tirent l'énergie de donneurs (sulfures, hydrocarbures) et accepteurs (O2) d'électrons. Les moules bathymodiolines sont un taxon clé dans ces habitats. À l'âge adulte, presque toutes possèdent des bactéries symbiotiques, mais les données sont rares concernant leur cycle de vie. Cette recherche explore la biologie de la reproduction, le développement et la nutrition dans le cycle de vie des espèces Idas modiolaeformis et "I." simpsoni. Les analyses anatomiques, histologiques et moléculaires à divers stades du développement sont associées à des observations in vivo. Une comparaison est proposée entre ces espèces. Chez les deux, les post-larves n'ont pas de symbiotes, suggérant des larves strictement hétérotrophes. L'infection par les symbiotes est environnementale, extracellulaire et initialement non-spécifique, se restreignant aux surfaces latéro-abfrontales des filaments branchiaux chez les adultes. La maturation est rapide, de même que la transition de l'hétérotrophie à la mixotrophie chimiosymbiotique: les symbioses, l'alimentation par filtration, et la rétention d'un système digestif complet coïncident. Les différences entre espèces et habitats sont discutées dans le contexte de leur évidente réussite évolutive à s'adapter aux habitats réduits et éphémères de l'Océan profond. / An array of deep-sea habitats are characterised by thermo- and/or biogenic production of chemically reduced compounds. Benthic communities thrive at these ‘reducing habitats’, due to trophic links to free-living and symbiotic chemosynthetic bacteria which derive energy from electron donors (sulphides, hydrocarbons) and acceptors (O2), at reduction-oxidation boundaries. The bathymodiolin mytilids (sensu lato) are a keystone taxon in these habitats; all-but-one species host gill-associated symbiotic Gammaproteobacteria as adults, but data are scarce on the remaining lifecycle, particularly at organic falls. To understand how mussels are adapted to these habitats, this research characterises the reproductive, developmental and nutritional lifecycle biology of two species, Idas modiolaeformis and I. simpsoni. Anatomical, histological and molecular analyses on post-larval-to-adult size spectra are complemented with live observations. Contrasting and converging aspects of their biology and symbioses are presented. In both species, aposymbiotic post-larvae confirm strict, larval heterotrophy. While still very small, environmental symbiont infection is extracellular and initially non-specific, becoming progressively isolated to latero-abfrontal gill surfaces in adults. Maturation is rapid, in parallel with a transition from heterotrophy to chemosymbiotic mixotrophy: symbioses, filter-feeding, and the retention of a complete gut are observed simultaneously. Interspecific and habitat differences are discussed in the context of both species’ evident evolutionary success in adapting to ephemeral, chemically reduced habitats in the deep sea.