No description available.

No description available.

Industrial waste pollution is a problem of considerable magnitude and of great importance to modern industry. One critical aspect of waste pollution is reduction of dissolved oxygen in natural waters resulting from the oxygen uptake of bacteria while digesting organic wastes. Fundamental information concerning the effect of various physical and chemical factors on the rate of oxygen uptake by bacteria was sought by investigating the rate of oxygen uptake by Pseudomonas fluorescens, a common water organism, as a function of the concentration of substrate and the temperature of environment. The substrates were D-glucose, D-xylose, L-arabinose and acetate, and the temperatures investigated ranged from 15 to 37 ºC. Oxygen uptakes were determined manometrically using the direct Warburg method in conjunction with resting cell techniques. In all tests 3.0 milligrams or Pseudomonas fluorescens (stated as dry bacterial protoplasm) were suspended in 2.5 milliliters of 0.05 molar phosphate buffer of pH 6.8. At 25 °C, for all substrates with the exception of acetate, the rate of oxygen uptake is dependent on concentration in the lower concentration ranges, and follows the Michaelis-Menten equation. Saturation concentrations stated as millimoles per test were 0.0500 for glucose and 0.600 for xylose. Saturation had not been reached in the case or arabinose at the highest concentration tested of 0.8000 millimoles per test. The rate or oxygen uptake increased with acetate concentration up to 0.1000 millimoles per test, and at concentrations above this value a decrease in rate of uptake was observed. A special equation was developed to cover the latter case. When substrate concentrations were held constant and temperature of the environment was varied from 15.0 to 37.0 °C the rate of oxygen uptake increased with temperature in all cases. For glucose the rate of change of the rate of oxygen uptake was 15.8 ± 1.6 microliters per hour per degree centigrade between 15.0 and 20.0 °C, 2.4 ± 0.2 microliters per hour per degree centrigrade between 20.0 and 25.0 °C, and 11.7 ± 1.2 microliters per hour per degree centigrade between 25.0 and 37.0 °C. When glucose concentration was held constant at 0.1000 millimole. For xylose the rate of change of the rate of oxygen uptake was 18.0 ± 1.8 microliters per hour per degree centigrade between 15.0 and 18.0 ºC, 3.9 ± 0.4 microliters per hour per degree centigrade between 18.0 and 35.0 °C, and 16.5 ± 1.6 microliters per hour per degree centigrade between 35.0 and 37.0 ºC when xylose concentration was held constant at 0.400 millimole. For arabinose the rate of change of the rate of oxygen uptake was 10.4 ± 1.0 microliters per hour per degree centigrade between 15.0 and 17.5 °C, 0.4 ± 0.0 microliters per hour per degree centigrade between 17.5 and 30.0 ºC, and 2.4 ± 0.2 microliters per hour per degree centigrade between 30.0 and 37.0 ºC when arabinose concentration was held constant at 0.400 millimole. For acetate the rate of change of the rate of oxygen uptake was 19.7 ± 2.0 microliters per hour per degree centigrade between 15.0 and 18.0 ºC, 2.8 ± 0.3 microliters per hour per degree centigrade between 18.0 and 28.0 °C, and 12.5 ± 1.5 microliters per hour per degree centigrade between 28.0 and 37.0 °C when the acetate concentration was held constant at 0.0500 millimole. / Ph. D.

Le système de sécrétion de type VI (SST6) est un complexe multi-protéique permettant l’export d’effecteurs. Ce mécanisme est impliqué à la fois dans la virulence envers les cellules eucaryotes, dans l’activité antibactérienne mais également dans l’acquisition d’ions présents dans ’environnement. Ainsi, le SST6 joue un rôle important dans l’adaptation et la compétition, éléments essentiels dans la colonisation et la persistance au sein d’une niche écologique. Actuellement, très peu d’études portent sur l’importance du SST6 chez des souches environnementales, contrairement aux nombreuses études portant sur des pathogènes tels que Pseudomonas aeruginosa, Burkholderia thailandensis, Vibrio cholerae ou Escherichia coli. Mon sujet de recherche avait pour objectif de caractériser le ou les rôles du SST6 de la souche environnementale Pseudomonas fluorescens MFE01. Ces travaux ont permis d’appréhender certaines fonctions du SST6 de cette souche. Le génome de MFE01 ne comporte qu’un seul cluster de gènes du SST6 où sont regroupés les gènes codant pour la machinerie du SST6 (le « core-component ») à l’exception des gènes hcp. Les protéines Hcp sont des éléments structuraux du SST6 dont elles forment le tube interne qui permet le transfert des effecteurs. Différents gènes hcp sont disséminés sur le chromosome et parmi ces « hcp » orphelins, hcp2 et hcp3 codent respectivement pour les protéines Hcp2 et Hcp3. Ces deux Hcp sécrétées par le SST6, sont associées à l’activité antibactérienne de MFE01 sur différentes souches pathogènes et environnementales, tels que P. aeruginosa, P. fluorescens MFN1032 et Pectobacterium atrosepticum. La protéine Hcp1, codée par le gène orphelin hcp1, est impliquée dans l’inhibition de mobilité de souche compétitrice. Hcp1 permettrait la sécrétion d’au moins deux toxines qui perturberaient l’assemblage du flagelle. Chez MFE01Δhcp1 et MFE01ΔtssC (TssC est un élément de la gaine contractile du SST6), ces toxines seraient accumulées dans le cytoplasme, inhibant ainsi ’assemblage de leur propre flagelle. La surproduction du régulateur FliA, qui contrôle notamment l’assemblage du filament flagellaire, restaure la mobilité chez ces deux mutants. En parallèle, le SST6 de la souche MFE01 est essentiel à la formation et la maturation de biofilm mais également à la compétition bactérienne en biofilm mixte. Ce système interviendrait dans la communication bactérienne indispensable au comportement social, requis lors de l’élaboration des biofilms. / Type VI secretion system (T6SS) is a multiproteic apparatus that secreted proteinaceous effectors. T6SS participate in a variety of functions, whose eukaryote virulence, antibacterial activity or metal ion uptake. These capacities conferring an advantage in adaptation and competition, crucial to colonization or persistence within ecological niche. As well, only a few studies have focused on the T6SS functions of environmental strains, contrary to numerous studies dealing with pathogens as Pseudomonas aeruginosa, Burkholderia thailandensis, Vibrio cholerae or Escherichia coli. The purpose of my research project was to characterize the T6SS function(s) of the environmental strain Pseudomonas fluorescens MFE01. This work had led to understand the various functions of T6SS of MFE01 strain. This strain has a single T6SS cluster where all the core component proteins were gathered, except hcp genes. Three orphan hcp genes where found and are scattered in genome. Hcp proteins form the inner tube allowing effectors secretion. Both Hcp2 and Hcp3 proteins were involved in antibacterial activity on pathogens or environmental strains like P. aeruginosa, P. fluorescens or Pectobacterium atrosepticum. Characterization of Hcp1 proteins role constituted a major focus of this project. Hcp1 proteins participate to motility inhibition of competitive strains through T6SS. Hcp1 may be associated with secretion of at least two toxins perturbing the flagellar filament assembly. In MFE01Δhcp1 and MFE01ΔtssC mutants (Tss is a contractile sheath constituent), these toxins may be accumulated into cytoplasm and perturb assembly of their own flagella. Interestingly, overproduction of FliA flagellar regulator, which controls assembly of flagellar filament, restores motility of both mutants. Simultaneously, T6SS of MFE01 strain contributes to maturation and biofilm formation but also in bacterial competition within mixed biofilm. T6SS may be a mean of bacterial communication and thus coordinate a social behavior, primordial for biofilm formation.

Les Streptomyces sont des bactéries communes des écosystèmes forestiers tempérés. Elles sont présentes au sein de différentes niches écologiques telles que la rhizosphère des plantes, la mycorhizosphère ou encore la minéralosphère. Dans ces niches, elles interagissent avec de nombreux autres genres bactériens et différents champignons symbiotiques, pathogènes et saprophytes. Nous avons entrepris une étude sur l'interaction entre deux souches bactériennes modèles du sol Streptomyces ambofaciens ATCC23877 et Pseudomonas fluorescens BBc6R8. Des expériences de cocultures sur différents milieux gélosés ont révélé que chaque souche bactérienne affecte la production de métabolites secondaires chez l'autre partenaire. Ainsi, P. fluorescens inhibe la capacité de S. ambofaciens à produire la kinamycine, un « effet ping-pong » a été mis en évidence. D'autre part, sur un milieu déficient en fer, P. fluorescens est capable d'utiliser les desferrioxamines et la coelicheline produites par S. ambofaciens en tant que xénosiderophores et ne produit alors plus ses propres sidérophores, la pyoverdine et l’énantio-pyochéline. Enfin, au cours des études d’interactions, P. fluorescens BBc6R8 s’est révélée capable d’inhiber la production du pigment bleu diffusible γ-actinorhodine chez Streptomyces coelicolor A3(2) M145 en acidifiant le milieu par la production d’acide gluconique. L’ensemble de ces résultats nous permet de proposer différentes hypothèses sur l’importance écologique de ces trois interactions en les replaçant au sein du contexte de l’écosystème sol / Streptomyces are common bacteria in temperate forest ecosystems, which inhabit various ecological niches such as plant rhizosphere and mycorrhizosphere, as well as mineralosphere. In these niches, they interact with many other bacterial genera and different symbiotic, pathogenic and saprotrophic fungi. We have initiated a study on interaction between two soil model bacteria strains Streptomyces ambofaciens ATCC23877 and Pseudomonas fluorescens BBc6R8. Cocultures experiments on different agar media have revealed that each bacteria affects the production of secondary metabolites in the other partner. Thus, P. fluorescens inhibits the ability of S. ambofaciens to produce kanamycin, a "ping-pong effect" was highlighted. On the other hand, on iron deficient medium, P. fluorescens is able to use the desferrioxamines and coelichelin produced by S. ambofaciens as xenosiderophores and does no longer produce its own siderophores, pyoverdin and enantio-pyochelin. In another way, during pairwise co-culture experiments, Pseudomonas fluorescens BBc6R8 was shown to prevent the production of the diffusible blue pigment antibiotic γ-actinorhodin by Streptomyces coelicolor A3(2) M145 by acidifying the medium through the production of gluconic acid. Other fluorescent Pseudomonas strains and the opportunistic pathogen Pseudomonas aeruginosa PAO1 also prevented the γ-actinorhodin production in a similar way. Altogether these results prompt us to propose some hypotheses on the ecological significance of these three interactions by replacing them in the soil ecosystem context

The pyrimidine salvage enzyme, nucleoside hydrolase, is catalyzes the irreversible hydrolysis of nucleosides into the free nucleic acid base and D-ribose. Nucleoside hydrolases have varying degrees of specificity towards purine and pyrimidine nucleosides. In E. coli, three genes were found that encode homologues of several known nucleoside hydrolases in protozoa. All three genes (designated yaaF, yeiK, and ybeK) were amplified by PCR and cloned. Two of the gene products (yeiK and ybeK) encode pyrimidine-specific nucleoside hydrolases, while the third (yaaF) encodes a nonspecific nucleoside hydrolase. All three were expressed at low levels and had different modes of regulation. As a comparative analysis, the homologous genes of Pseudomonas aeruginosa and P. fluorescens (designated nuh) were cloned. Both were determined to encode nonspecific nucleoside hydrolases. The nucleoside hydrolases of the pseudomonads exhibited markedly different modes of regulation. Both have unique promoter structures and genetic organization. Furthermore, both pseudomonad nucleoside hydrolase were found to contain an N-terminal extension of 30-35 amino acids that is shown to act as a periplasmic-signaling sequence. These are the first two nucleoside hydrolases, to date,that have been conclusively demonstrated to be exported to the periplasmic space. The physiological relevance of this is explained.

The loss of rice plants (Oryza sativa L.) due to damage caused by abiotic stresses (water deficit and allelopathy) are recurrent, the objective was to evaluate the behavior of rice plants through agronomic and morphological changes under different layers of water in the soil (LAS) of 100%, 70%, 50% and 30% of field capacity (CC), and to identify the anatomical and physiological changes in plant in LAS 100% and 50% of CC, Induced by PGPR (Burkholderia pyrrocinia BRM-32113 and Pseudomonas fluorescens BRM-32111), that indicate the mitigating effect of the damages caused by the water deficit. And in plantings with residue of rice plants in the soil was to identify and understand the effect of the application of rhizobacteria on highland rice plants in consecutive plantations. Test one, The PGPR were subjected to temperature abiotic stresses (30, 35, 40 oC), salinity (0.5%, 7.5%), and water deficit (0, -0.2, - 0.4, -0.6, -0.8, -1.0, -1.2 Mpa) simulated by PEG with 10 replicates and evaluated the growth. Test two, the rice plants inoculated with: BRM-32111, BRM-32113 and control, were submitted the LAS of: 100, 70, 50 and 30% of CC, with three pots per treatment and five plants per pot. were evaluated: evapotranspirated water and water potential (ψam), biomass and length of plant and root, leaf area and relative chlorophyll content. Test three, plants Control, inoculated with BRM-32111 and BRM-32113, submitted to 100% and 50% of CC, and maintained for 28 days, and evaluated growth, physiology and anatomy. Test four, were used four treatments consisting of seed rice inoculated with P. fluorescens BRM-32111 in soil with residue, B. pyrrocinia BRM-32113 in soil with residue, plants control in soil with residue (CR)and plants control on soil without residue (SR) roots of plants rice (residues of Allelochemicals), the essay were in (DIC), and results submitted to ANOVA, Duncan's test (p <0.05). Test one, It was verified that PGPR were tolerant to abiotic stresses of: salinity (80%), temperature (96%) and dry (96%), test two, plants have reached the critical LAS of up to 63% of CC, plants with BRM-32111 had the effect of reducing the damage caused by the water deficit, In biomass 30%, root length 88% in LAS of 30% CC when compared to control plants. Test three, The seeds with BRM-32111 differed from the control in germination, and with BRM 32113 differed from the control in IVG. The plants inoculated with BRM 32111 and BRM32113 had a smaller reduction in root diameter, number of protoxilema pores, cortex thickness, and increase in density of stomata, and increase in carbon assimilation rate (A), efficiency of water use (EUA), carboxylation efficiency rubisco (A / Ci), higher accumulation of chlorophyll a, proline and reduction in the concentration of malonic aldehyde (MDA), in relation to control plants submitted to the same LAS. Test four, the growth of seedlings and plants control rice (CR) was negatively affected by allelopathic compounds. In sowing with residue, plants inoculated with rhizobacteria P. fluorescens BRM-32111 and B. pyrrocinia BRM-32113 induced an increase of 88% in biomass, 3% in the leaf area, 40% and 67% in length and root biomass, respectively, 21% in chlorophyll a, 50% in A, and 63% in the EUA compared to CR control plants CR. These results, evidences that the rhizobacteria are tolerant to the temperature stresses, salinity and osmotic pressure and help to alleviate the harmful effect caused by the water deficit, and increase tolerance of rice plants to stress with allelochemicals in upland rice. / A perda na produtividade de plantas de arroz (Oryza sativa L.) devido aos danos causados por estresses abioticos (déficit hídrico e alelopatia) são recorrentes, o objetico foi avaliar o comportamento de plantas de arroz através das alterações agronômicas e morfológicas sob diferentes lâminas de água no solo (LAS) de 100%, 70 %, 50% e 30 % da capacidade de campo (CC), e identificar as modificações anatômicas e fisiológicos em planta em LAS de 100 % e 50 % da CC induzidas por PGPR (Burkholderia pyrrocinia BRM-32113 e Pseudomonas fluorescens BRM-32111), que indiquem o efeito mitigador dos danos causados pelo déficit hídrico. E em plantios com resíduo de plantas do arroz no solo foi identificar e compreender o efeito da aplicação de rizobactérias em plantas de arroz de terras altas em plantios consecutivos. No primeiro ensaio, as PGPR foram submetidas aos estresses abióticos de temperatura (30, 35 e 40 oC), salinidade (0.5 % e 7.5%) e déficit hídrico (0, -0.2, -0.4, -0.6, -0.8, -1.0 e -1.2 Mpa) simulado por PEG com 10 repetições e avaliado o crescimento. No segundo ensaio, as plantas de arroz inoculadas com: BRM-32111, BRM-32113 e controle, foram submetidas as LAS de: 100, 70, 50 e 30 % da CC, com três vasos por tratamento e cinco plantas por vaso. Foram avaliados: água evapotranspirada e potencial hídrico (ψam), biomassa e comprimento da planta e raiz, área foliar, teor relativo de clorofila. No terceiro ensaio, foram usadas plantas controle, inoculadas com BRM-32111 e BRM-32113, submetidas a 100% e 50% da CC, e mantido até aos 28 dias, e avaliado crescimento, fisiologia e anatomia. No quarto ensaio, foram utilizados quatro tratamentos constituídos de sementes de arroz inoculados com P. fluorescens BRM32111 em solo com resíduo, com B. pyrrocinia BRM-32113 em solo com resíduo e plantas controle em solo com resíduo (CR) e plantas controle em solo sem resíduo (SR) de raízes de plantas de arroz (resíduos de Aleloquímicos), todos os ensaios foram em DIC, e os resultados submetidos ANOVA, teste de Duncan (p < 0.05). No primeiro ensaio, verificou - se que as PGPR foram tolerantes aos estresses abióticos de: salinidade (80%), temperatura (96%) e seca (96%), no segundo ensaio, as plantas atingiram a LAS crítica de até 63 % da CC, as plantas com BRM-32111 tiveram os efeitos amenizador aos danos causado pelo déficit hídrico, na biomassa 30%, comprimento da raiz 88% em LAS de 30% de CC quando comparados a plantas controle. No terceiro ensaio, as sementes com BRM-32111 diferiram do controle na germinação, e com BRM 32113 diferiram do controle no IVG. As plantas inoculadas com BRM 32111 e BRM-32113 tiveram menor redução no diâmetro radicular, número de poros de protoxilema, espessura do córtex, e aumento na densidade de estômatos, e aumento em taxa de assimilação de carbono (A), eficiência do uso da água (EUA) e eficiência de carboxilação da rubisco (A / Ci), maior acumulo de clorofila a, prolina e redução na concentração de aldeído malônico (MDA), em relação a plantas controle submetidas a mesma LAS. No quarto ensaio, o crescimento de plântulas e plantas de arroz controle (CR) foi afetado negativamente pelos compostos alelopáticos. Em semeio com resíduo, plantas inoculadas com as rizobactérias P. fluorescens BRM-32111 e B. pyrrocinia BRM-32113 induziram aumento em 88% na biomassa, 3% na área foliar, 40% e 67% no comprimento e biomassa radicular, respectivamente, 21% na clorofila a, 50% na A, e 63% no EUA comparado as plantas controle CR. Estes resultados, evidenciam que as rizobactérias são tolerantes aos estresses de temperatura, salinidade e pressão osmótica e auxiliam na amenização do efeito danosos causados pelo déficit hídrico, e aumentam a tolerância de plantas de arroz ao estresse com aleloquímicos no arroz de terras altas

A major factor in the ability of Pseudomonas fluorescens Pf-5 to act as a biological control agent is its production of antibiotics, including pyoluteorin (PLT), 2,4-diacetylphloroglucinol (2,4-DAPG) and pyrrolnitrin (PRN). The data provided in this thesis demonstrate that the presence of any of these antibiotics in the extracellular milieu affects production of that same antibiotic, as well as others, by Pf-5. Amending the growth medium with antibiotics had multiple effects on secondary metabolism in Pf-5. i) PLT positively regulated its own production, ii) 2,4-DAPG positively regulated its own production. iii) PLT suppressed 2,4-DAPG production. iv) 2,4- DAPG inhibited PLT production. v) PLT suppressed transcription of a heterologous ferric-pyoverdine uptake gene. vi) PRN exerted a slight inhibitory effect on PLT gene transcription and production. PLT autoinduction by Pf-5 was extensively characterized, and was shown to require concentrations of exogenous PLT in the nanomolar range. These low concentrations are comparable to those of many molecules proposed to function in signaling roles. PLT served as a signal between distinct populations of cells within the rhizosphere, where it prompted autoinduction by those cells. Aside from effects of Pf- 5 antibiotics on one another, I also described the positive effect of exogenous PLT on expression of a set of transport genes flanking the PLT biosynthetic gene cluster. Sequence data and experimental evidence suggests that these genes encode a transport apparatus for PLT. The deduced amino acid sequences for four adjacent open reading frames together resemble Type I secretion apparatuses, which typically function in transport of proteins rather than secondary metabolites. The intact transporter genes are necessary for optimal PLT production. Taken together, the data from the studies described herein demonstrate that i) the production of PLT by Pf-5 can affect the production of PLT by neighboring cells, and ii) PLT and other exogenous secondary metabolites have both autoregulatory and cross-regulatory effects in culture. Because Pf-5 derivatives engaged in PLT crossfeeding in the rhizosphere, it is likely that cross-feeding occurs for other secondary metabolites as well. Thus, production of an antibiotic by one cell can profoundly affect secondary metabolism in neighboring cells occupying natural habitats. / Graduation date: 2004

Competitive exclusion has been the mechanism hypothesized to account for the biological control of fire blight disease of pear and apple by the bacterium Pseudomonas fluorescens A506 (A506). Recent laboratory assays demonstrated, however, that A506 produces an antibiotic that is toxic to the fire blight pathogen, Erwinia amylovora, when cultured on media amended with iron (Fe����� or Fe�����). This study investigated this iron-dependent antibiosis by A506 by: 1) examining bioavailability of iron to A506 on blossom surfaces, 2) mutagenizing A506 to disrupt genes involved in antibiotic production, and 3) evaluating suppression of fire blight by A506 when co-treated with an iron chelate (FeEDDHA). Bioavailability of iron on blossoms was investigated with an iron biosensor [iron-regulated promoter (pvd) fused to an ice nucleation reporter gene (inaZ)] in A506. A506 (pvd-inaZ) expressed high ice nucleation activity (INA) on blossoms indicating a low-iron environment unlikely to induce antibiosis by A506. Spraying blossoms with FeEDDHA at concentrations ���0.1 mM significantly suppressed INA by A506 (pvd-inaZ). Transposon mutagenesis was used to generate and select mutants of A506 exhibiting altered antibiotic production profiles. One antibiotic-deficient mutant, A506 Ant���, was recovered; this mutant showed reduced epiphytic fitness on blossoms of apple and pear trees compared to the parent stain, A506. Another mutant, A506 Ant���, lost the characteristic fluorescent phenotype and exhibited iron-independent antibiotic production in defined culture media. A506 Ant��� established high populations on blossoms of apple and pear trees, similar to populations attained by A506, and reduced incidence of fire blight between 20 to 40%, levels comparable to A506 in orchard trials. In orchard trials, A506 was co-treated with FeEDDHA and fire blight suppression was evaluated. Bacterial strains established high populations on blossoms when co-treated with 0.1 mM FeEDDHA or in water. Significantly enhanced suppression of fire blight incidence by antibiotic producing strains of A506 amended with 0.1 mM FeEDDHA was observed in 2 of 5 trials, providing some evidence that iron-induced antibiosis can be a contributing mechanism in disease control. Lack of disease control by the antibiotic deficient strain, A506 GacS, and by 0.1 mM FeEDDHA alone added support to this hypothesis. / Graduation date: 2004

Graduation date: 1998