La réplication de l’ADN est une fonction cellulaire responsable de la duplication du matériel génétique. Elle est assurée par un complexe protéique appelé réplisome. Ce processus est hautement régulé en fonction des conditions de croissance cellulaire. Durant cette thèse je me suis intéressé principalement au contrôle de la réplication par le Métabolisme Central Carboné (MCC) et, dans une moindre mesure, au fonctionnement du réplisome chez la bactérie modèle Bacillus subtilis. J’ai analysé la réplication de l’ADN dans des mutants métaboliques, par deux techniques ; la QPCR et la cytométrie en flux. Mes analyses révèlent que la réplication de l’ADN est dérégulée dans des cellules mutées dans les cinq dernières réactions de la glycolyse et dans celles affectées dans des réactions connectant cette petite région du métabolisme aux autres réactions du MCC (haut de la glycolyse, voie des pentoses phosphate et cycle de Krebs) et au milieu extérieur (voies overflow qui éliminent les métabolites du MCC produits en excès). J’ai constaté que dans ces mutants la réplication commence plutôt et dure plus longtemps que dans une souche sauvage. L’ensemble de ces résultats montre que les réactions situées au cœur du MCC sont importantes pour assurer un bon contrôle temporel de la réplication. J’ai aussi établi que le ppGpp, une petite molécule fonctionnant comme une alarmone de l’état nutritionnelle des cellules, ne joue pas un rôle déterminant dans le contrôle de la réplication par le métabolisme dans des cellules à l’état d’équilibre. L’ensemble de nos connaissances actuelles sur les réplisomes repose essentiellement sur les données accumulées à partir de la dissection du réplisome de la bactérie modèle Escherichia coli et des phages T4 et T7. Chez Bacillus subtilis, deuxième modèle bactérien le mieux connu et représentant des Gram+ à faible GC%, il existe deux ADN polymérases essentielles à la réplication : PolC et DnaE. Nous avons montré que DnaE, comme PolC, fait partie du réplisome. Nos études fournissent une explication moléculaire à la spécialisation de DnaE dans la synthèse du brin d’ADN discontinu. En conclusion, nos résultats montrent que les réplisomes bactériens ont beaucoup plus évolué qu’attendu tant dans leur composition protéique que dans leur organisation et leur fonctionnement. Ils montrent également, et pour la première fois, que le contrôle temporel de la réplication dépend de réactions situées au cœur du MCC chez B. subtilis. Ces données et d’autres de la littérature suggèrent que cette propriété pourrait être universelle et pourrait jouer un rôle important dans la carcinogenèse. / DNA replication is a central cellular function for the duplication of the genetic material. A protein complex that is called replisome carries out this function. The process of replication is highly regulated with respect to cell growth conditions. During my thesis I was primarily interested in the control of replication by the central carbon metabolism (CCM) and to a lesser extent, to the functioning of the replisome in the bacterium Bacillus subtilis. The thesis studied the DNA replication in metabolic mutants by employing two techniques; QPCR and flow cytometry. The analyses showed that DNA replication is deregulated in cells that carry the following mutations: First, cells with mutations in the last 5 reactions of glycolysis. Second, cells with mutations in the reactions that connect the last part of glycolysis to the other parts of CCM (upper part of glycolysis pathway, pentose phosphate and Krebs cycle). Third, cells mutated in the overflow genes (channels that eliminate overflow metabolites produced in excess in CCM). The results demonstrate that in these mutants the replication begins and lasts longer than in the wild strain. All of these results show that the reactions that are centrally located to the CCM are important to ensure a correct control of replication timing. I also found that the ppGpp, a small molecule that functions as an alarmone of nutritional state in the cells, does not play a decisive role in the control of replication by metabolism in cells in steady state. The current knowledge of replisomes is mainly based on accumulated data from the dissection of the replisome of the model bacterium Escherichia coli and the phages T4 and T7. Bacillus subtilis is the second well studied bacterial model, a representative of Gram+ low GC%, it carries –unlike E. coli- two essential DNA polymerases for replication: PolC and DnaE. The thesis showed that DnaE as PolC form a part of the replisome in B. subtilis and provide a molecular explanation to the specialization of DnaE in the synthesis of the DNA lagging strand. In conclusion, the results show that there is much more diversity in the protein composition, organization and functioning of replisomes in bacteria than it is expected. In addition, the thesis concluded for the first time that the temporal control of replication depends on reactions located in the heart of CCM in B. subtilis. This property, in combination with other data from the literature, suggests that it could be universal and play an important role in carcinogenesis.

Bacillus subtilis est une bactérie à Gram positif ubiquitaire vivant dans différents environnements terrestres et aquatiques. Différents polymères extracellulaires entrant dans la composition de la matrice des biofilms à B. subtilis ont été décrits. Des polysaccharides sont à la base de ces propriétés mécaniques, la viscoélasticité étant modulée par la teneur du biofilm en différents polymères extracellulaires comme les protéines amyloïdes et l’ADN extracellulaire.L’objectif de ce travail était d’étudier le rôle des exopolymères dans les biofilms à B. subtilis en utilisant la souche type de l’espèce CIP52.65T et différentes autres souches sauvages, cliniques et mutantes. La composition de la matrice varie en fonction de la présence de saccharose dans le milieu de culture, ainsi les effets d’une supplémentation du milieu Trypticase Soja (TS) en saccharose (20% p/v) ont été étudiés sur la croissance planctonique, la production de polymères de matrice et la formation de biofilm pour toutes ces souches de B. subtilis. Enfin, parmi les protéines de la matrice, B. subtilis produit une protéine formant des fibres amyloïdes appelée TasA. Son rôle exact dans le biofilm reste encore mal connu. Le but de cette seconde étude était de mieux comprendre le mécanisme d'auto-assemblage de TasA et de comprendre son rôle dans la matrice. En regroupant toutes les caractérisations effectuées sur les biofilms et sur les peptides amyloïdes, la conception de matrice biomimétique a permis d’effectuer de première approche sur les propriétés mécaniques de celle-ci, en reproduisant des matrices artificielles à base d’exopolysaccharides (lévane), de peptides amyloïdes et d’ADN. / Bacillus subtilis is a ubiquitous gram-positive bacterium that lives in different terrestrial and aquatic environments. Various extracellular polymers involved in the composition of the B. subtilis biofilm matrix have been described. Polysaccharides are the basis of these mechanical properties, the viscoelasticity being modulated by the content of the biofilm in different extracellular polymers such as amyloid proteins and extracellular DNA.The aim of this work was to study the role of exopolymers in B. subtilis biofilms using the type CIP52.65T strain and various other wild, clinical and mutant strains. The composition of the matrix varies according to the presence of sucrose in the culture medium, so the effects of supplementation of the medium Trypticase Soy (TS) sucrose (20% w/v) were studied on the planktonic growth, matrix polymer production and biofilm formation for all these B. subtilis strains. Finally, among the proteins in the matrix, B. subtilis produces an amyloid-forming protein called TasA. Its exact role in the biofilm remains poorly understood. The purpose of this second study was to better understand the self-assembly mechanism of TasA and to understand its role in the matrix. By grouping all the characterizations carried out on the biofilms and the amyloid peptides, the biomimetic matrix design made it possible to carry out a first approach on the mechanical properties of this one, by reproducing artificial matrices based on exopolysaccharides (levan), amyloid peptides and DNA.

No description available.

No description available.

O ácido ƴ-poliglutâmico (ƴ-PGA) é uma homopoliamida aniônica, biodegradável, comestível e atóxica, sintetizada por bactérias do gênero Bacillus, podendo ser utilizado nas indústrias alimentícia e de cosméticos, na medicina e no tratamento de águas residuais. Este trabalho teve como objetivo caracterizar e identificar potenciais aplicações para o ƴ-PGA obtido através do cultivo submerso de Bacillus subtilis BL53, conduzido sob condições otimizadas em trabalhos anteriores. Além disso, foi avaliado o efeito de diferentes inóculos e da adição de precursores da rota metabólica na produção do biopolímero. A melhor condição obtida foi testada em biorreatores com adição de polidimetilsiloxano (PDMS) como carreador de oxigênio, com o objetivo de aumentar a produtividade do biopolímrero. A massa molar média (Mw), obtida através de espalhamento de luz estático, na ordem de 106 g mol-1 não apresentou diferenças significativas para o biopolímero obtido após 48 e 96 h de cultivo. As análises reológicas conduzidas em viscosímetro rotacional indicaram que os polímeros obtidos após 48 e 96 horas apresentaram comportamento Newtoniano, sendo que após 96 horas a viscosidade absoluta foi maior. As análises térmicas (calorimetria diferencial exploratória e análise termogravimétrica) indicaram a temperatura de fusão (Tm) de 134 ºC e 128 ºC e o intervalo de degradação (Td) entre 120 ºC - 190 ºC e 120 ºC - 215 ºC, para os biopolímeros obtidos após 48 e 96 horas de cultivo respectivamente. O caldo LB apresentou-se como o melhor inóculo para a produção de ƴ-PGA. A adição dos precursores L-glutamina e ácido -cetoglutárico aumentou em 20 % a produção do biopolímero. A adição de 10 % de PDMS nos cultivos em biorreatores aumentou o coeficiente volumétrico de transferência de massa (KLa) e a produção e produtividade do ƴ-PGA, sendo produzidos 23.5 g L-1 do biopolímero em 24 horas de cultivo, uma produtividade aproximadamente 40 % superior às obtidas por outros autores utilizando o mesmo microrganismo. / Poly-ƴ-glutamicacid (ƴ-PGA) is an anionic, biodegradable, non-toxic and edible homopolyamide, synthesized by bacteria of the genus Bacillus, being used in food, cosmetics, medicine and waste water treatment. The aim of this study is to characterize and indentify potencial applicatiions for the ƴ-PGA obtained by submerged cultivation of Bacillus subtilis BL53, conducted under optimized conditions in previous studies. We also evaluated the effect of different inoculants and addition of precursors in the metabolic pathway of production of the biopolymer. The best condition obtained yet been tested in bioreactors with addition of polydimethylsiloxane (PDMS) as a carrier of oxygen in order to further increase the productivity of biopolymer. The average molecular weight (Mw) obtained by static light scattering, on the order of 106 g mol-1, showed no significant differences for biopolymer obtained after 48 and 96 h of cultivation. Analyses conducted in rotational viscometer indicated that biopolymers after 48 and 96 h have a Newtonian behavior, and the 96 hours had higher absolute viscosity. The thermal analysis (differential scanning calorimetry and thermo gravimetric analysis) indicated the melting temperature (Tm) as 134 ºC and 128 ºC and degradation temperature range (Td) of 120 ºC - 190 ºC and 120 ºC - 215 ºC, after 48 and 96 hours respectively. It was found that the best inoculum medium for biopolymer production was the LB broth. The addition of the precursors L-glutamine and -ketoglutaric acid increased in 20% the ƴ-PGA production. The addition of 10% of PDMS in bioreactors cultures increased the mass transfer volumetric coefficient (KLa) and the production and productivity of ƴ-PGA, being produced 23.5 g l-1 of the biopolymer in 24 hours of cultivation, a productivity about 40 % higher than those obtained by other authors using the same microorganism.

O ácido y-poliglutâmico (y-PGA) é um biopolímero solúvel em água, aniônico, atóxico, biodegradável e biocompatível, produzido por Bacillus e que possui aplicações nas áreas química, médica, ambiental e de alimentos. Este trabalho tem como objetivo estudar o processo de produção do y-PGA a partir do cultivo de uma nova linhagem de Bacillus isolada de ambiente amazônico, o Bacillus subtilis BL53. Este estudo inclui a seleção de culturas, a otimização de condições de cultivo e a prospecção de substratos de produção alternativos. A habilidade de produção de y-PGA por linhagens de Bacillus isoladas na região amazônica foi investigada. A linhagem BL53 foi a linhagem selecionada, sendo identificada como Bacillus subtilis, através da análise da seqüência do gene 16S DNAr e de suas características bioquímicas. A avaliação das condições de cultivo submerso foi conduzida em agitador orbital mediante delineamento composto central rotacional (DCCR) que apontou como ponto ótimo de produção de y-PGA, a temperatura de 37 oC, o pH inicial de 6,9 e a concentração de 1,22 mM de Zn2+ suplementada ao Caldo E. Nas condições otimizadas, a produção de y-PGA foi igual a 10,4 g/L, cerca de três vezes maior que a obtida em condições convencionais de cultivo em Caldo E, utilizando a mesma linhagem. A influência da disponibilidade de oxigênio sobre a produção de y-PGA por B. subtilis BL53 foi avaliada em biorreator agitado de 5 L, com o emprego das velocidades de agitação de 500, 750 e 1000 rpm, sendo mantida fixa a taxa de aeração de 2 vvm. A produção de y-PGA mostrou-se altamente dependente da transferência de oxigênio, sendo que o teor de oxigênio dissolvido decaiu rapidamente nas primeiras 15 horas de cultivo, como resultado da elevada demanda de oxigênio pelas bactérias. O aumento na velocidade de agitação no biorreator possibilitou uma maior transferência de oxigênio e induziu o aumento na taxa de consumo de oxigênio pelas bactérias, conduzindo a maior produção e maior produtividade de y-PGA. A intensificação da agitação também influenciou os parâmetros cinéticos de crescimento do Bacillus subtilis BL53, provocando um aumento na velocidade específica de crescimento na fase logarítmica (max) sem provocar a perda da viabilidade celular. Com a utilização da velocidade de agitação de 1000 rpm em biorreator, o tempo de cultivo pode ser reduzido para menos de 48 h, cerca de 50% do tempo necessário para operação a 500 rpm. A investigação de substratos alternativos para produção de y-PGA mostrou o glicerol residual de biodiesel e o resíduo fibroso de soja como substratos promissores, apontando para a possibilidade de investigação em trabalhos futuros. / The poly-gamma-glutamic acid (y-PGA) is a water-soluble biopolymer, anionic, non toxic, biodegradable and biocompatible, it is produced by Bacillus and it has applications in chemical, medical, environmental and food industries. This work aims to study the process of production of y-PGA through cultivation of a new strain of Bacillus isolated from the Amazonian environment, Bacillus subtilis BL53. This study includes the screening of strains, the optimization of culture conditions and the investigation of alternative substrates. The ability of y-PGA production by Bacillus strains isolated from Amazonian environment was investigated. The BL53 strain was selected and identified as Bacillus subtilis, through analysis of 16S rDNA gene sequence and its biochemical characteristics. Evaluation of culture conditions in submerged cultivation was conducted in shaker using central composite design (CCD), which showed the temperature of 37 oC, the initial pH 6.9 and concentration of 1.22 mM Zn2+ in Medium E as the optimal conditions to y-PGA production. Under optimized conditions, the production of y-PGA was 10.4 g/L, about threefold what was obtained using this strain under conventional cultivation in Medium E. The influence of oxygen availability on the production of y-PGA by B. subitlis BL53 was evaluated in a 5 l stirred bioreactor with the use of stirring rates of 500, 750 and 1000 rpm, using a fixed aeration rate of 2 vvm. Production of y-PGA was highly dependent on oxygen transfer, and the dissolved oxygen content decreased rapidly in the first 15 hours of culture as a result of high oxygen demand by bacteria. The increase in stirring rate in the bioreactor allowed a better oxygen transfer and induced a rise in the oxygen uptake rate by bacteria, leading to higher production and higher productivity of y-PGA. The intensification of the stirring also influenced the kinetics growth parameters of Bacillus subtilis BL53, producing an increase in specific growth rate in the logarithmic phase (max) without causing reduction of cell viability. Using the stirring rate of 1000 rpm in a bioreactor, the cultivation time can be reduced to less than 48 h, about 50% of the time required for operation at 500 rpm. The investigation of alternative substrates for the production of y-PGA showed crude glycerol from biodiesel and soybean industrial fibrous residue as promising ones, pointing to research possibilities in future work.

Okra (Abelmuschus esculentus (L) Moench.) is one of the most popular crops within the Malvaceae family of plants. It is a common vegetable eminently cultivated in regions experiencing constraints to manage climate change. In South Africa climate change coupled with aluminium-enriched soils are responsible to drawbacks crop performance. Therefore, it is worthwhile to whether okra will thrive as an alternative crop in the country. Many studies have identified potential of okra to improve yields of resource poor farmers in Africa. The physiological responses of okra seed to variations in aluminium ions and temperature were not determined. Therefore, a study with okra, cv. Clemson Spineless, seed coated and uncoated with B. subtilis, was initiated to assess germination on moist filter paper in 90mm diameter Petri plates. Germination medium consisted of various concentrations of aluminium chloride (AlCl3), 0M, 0.001M, 0.01M, 0.05M and 0.1M. Each aluminium treatment was allocated into incubators adjusted to 22°C, 25°C and 37°C temperatures. This resulted into a 5 x 3 x 2 factorial experiment with five replicates and was conducted in three cycles. Daily scores of germinated seeds were assessed from the second to the fifth day after initiation of germination. During termination, five days after the initiation of the experiment 10 seeds with the longest coleoptiles had their coleoptiles measured using a digital caliper. At the fifth day after initiation of the experiment, coleoptile lengths from 10 seeds per treatment were measured using digital caliper. A total of 50 plates (10 from 37°C in Cycle 1; 30 from 22°C, 25°C and 37°C from Cycle 2; 10 from 37°C in Cycle 3), were selected and germinated were ground and stored at - 20°C before 1H NMR analysis. Metabolites were extracted from 50mg ground seed material with 750 μL methanol-D4 and 750 μL buffer (deuterium oxide + potassium dihydrogen phosphate). The mixture was vortexed for three minutes, sonicated for 20 minutes, centrifuged at 18000 rpms for 20 minutes and the supernatant filtered through cotton wool. Then the supernatant was dispensed into NMR tubes for further 1H NMR spectroscopic processing using a 600 MHz NMR xiii Varian spectrometer to generate magnetic spectra of the fifty samples. Results of this study demonstrated that in all the experimental cycles, regardless of aluminium concentration and bacterial seed coating, 37°C inhibited germination percentages and coleoptile lengths in okra seed germination. Germination percentages and coleoptile lengths of bacteria-coated seeds growing in 25°C were most stimulated at all aluminium concentrations, but not at 0.1M. In this temperature germination percentages and coleoptile lengths were highly influenced by the interaction of aluminium concentrations and bacterial coating, respectively. 1H NMR metabolomic association showed no distinct grouping, but clusters across treatments showed to be linked through a subset of metabolites amongst aluminium concentrations, bacterial seed coating and temperatures, respectively. This infers that treatment variations in both seed and bacterial physiological responses were associated through shared metabolic pathways. In conclusion, the study proved that 25°C provide temperature environment within which B. subtilis can be able to stimulate growth and remediate physiological constraints from aluminium ions during okra seed germination. / Agriculture, Animal Health and Human Ecology / M. Sc. (Agriculture)

O ácido ƴ-poliglutâmico (ƴ-PGA) é uma homopoliamida aniônica, biodegradável, comestível e atóxica, sintetizada por bactérias do gênero Bacillus, podendo ser utilizado nas indústrias alimentícia e de cosméticos, na medicina e no tratamento de águas residuais. Este trabalho teve como objetivo caracterizar e identificar potenciais aplicações para o ƴ-PGA obtido através do cultivo submerso de Bacillus subtilis BL53, conduzido sob condições otimizadas em trabalhos anteriores. Além disso, foi avaliado o efeito de diferentes inóculos e da adição de precursores da rota metabólica na produção do biopolímero. A melhor condição obtida foi testada em biorreatores com adição de polidimetilsiloxano (PDMS) como carreador de oxigênio, com o objetivo de aumentar a produtividade do biopolímrero. A massa molar média (Mw), obtida através de espalhamento de luz estático, na ordem de 106 g mol-1 não apresentou diferenças significativas para o biopolímero obtido após 48 e 96 h de cultivo. As análises reológicas conduzidas em viscosímetro rotacional indicaram que os polímeros obtidos após 48 e 96 horas apresentaram comportamento Newtoniano, sendo que após 96 horas a viscosidade absoluta foi maior. As análises térmicas (calorimetria diferencial exploratória e análise termogravimétrica) indicaram a temperatura de fusão (Tm) de 134 ºC e 128 ºC e o intervalo de degradação (Td) entre 120 ºC - 190 ºC e 120 ºC - 215 ºC, para os biopolímeros obtidos após 48 e 96 horas de cultivo respectivamente. O caldo LB apresentou-se como o melhor inóculo para a produção de ƴ-PGA. A adição dos precursores L-glutamina e ácido -cetoglutárico aumentou em 20 % a produção do biopolímero. A adição de 10 % de PDMS nos cultivos em biorreatores aumentou o coeficiente volumétrico de transferência de massa (KLa) e a produção e produtividade do ƴ-PGA, sendo produzidos 23.5 g L-1 do biopolímero em 24 horas de cultivo, uma produtividade aproximadamente 40 % superior às obtidas por outros autores utilizando o mesmo microrganismo. / Poly-ƴ-glutamicacid (ƴ-PGA) is an anionic, biodegradable, non-toxic and edible homopolyamide, synthesized by bacteria of the genus Bacillus, being used in food, cosmetics, medicine and waste water treatment. The aim of this study is to characterize and indentify potencial applicatiions for the ƴ-PGA obtained by submerged cultivation of Bacillus subtilis BL53, conducted under optimized conditions in previous studies. We also evaluated the effect of different inoculants and addition of precursors in the metabolic pathway of production of the biopolymer. The best condition obtained yet been tested in bioreactors with addition of polydimethylsiloxane (PDMS) as a carrier of oxygen in order to further increase the productivity of biopolymer. The average molecular weight (Mw) obtained by static light scattering, on the order of 106 g mol-1, showed no significant differences for biopolymer obtained after 48 and 96 h of cultivation. Analyses conducted in rotational viscometer indicated that biopolymers after 48 and 96 h have a Newtonian behavior, and the 96 hours had higher absolute viscosity. The thermal analysis (differential scanning calorimetry and thermo gravimetric analysis) indicated the melting temperature (Tm) as 134 ºC and 128 ºC and degradation temperature range (Td) of 120 ºC - 190 ºC and 120 ºC - 215 ºC, after 48 and 96 hours respectively. It was found that the best inoculum medium for biopolymer production was the LB broth. The addition of the precursors L-glutamine and -ketoglutaric acid increased in 20% the ƴ-PGA production. The addition of 10% of PDMS in bioreactors cultures increased the mass transfer volumetric coefficient (KLa) and the production and productivity of ƴ-PGA, being produced 23.5 g l-1 of the biopolymer in 24 hours of cultivation, a productivity about 40 % higher than those obtained by other authors using the same microorganism.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Parapiptadenia rigida is a forest species that appears in many regions of Brazil, frequently produced in forest nurseries, being the seeds the multiplication form. Usually presents a good germination, however, aspects involving the physiological and health qualities of the lots, still results in lost in the nurseries. In this sense, microorganisms with antagonist characteristics and/or promoters of growth related to many products and coated with polymers, arise as options to improve the growth of the seedlings in the nurseries. This work aimed to evaluate the treatment of Parapiptadenia rigida seeds, for the physiological quality maintenance in the storage and promotion of the seedlings growth in the nursery. The work was split in studies of storage and nursery. In the storage study, the treatments were arranged in completely randomized lineation in trifactorial scheme (4x4x2), represented by storage timing (0, 4, 8, 12 months), products (biological, chemical and witness) and polymers (polymer and witness), with four repetitions. In each time were made evaluations of the germination percentage, vigor tests, seedling length, fresh and dry mass, besides of regular strong and weak seedlings. The nursery studies was subdivided in two experiments: the first using combinations involving Trichoderma sp., polymers and vegetal growth stimulators, and the second involved the use of Bacilus subtilis, polymers and vegetal stimulators. For both of them, the treatments was arranged in completely randomized lineation in trifactorial scheme (3x2x4), represented by fungicide product (biological, chemical e witness), polymers (polymers and witness), micronutrients and biostimulating (micronutrients, biostimulating, micronutrients + biostimulating, witness) with four repetitions. In about 60 to 120 days after the emergency, the height of the plant, the stem diameter, and the number of leaves was evaluated. At the experiment ending was made weighing of the dry mass of the aerial part, root and total, besides the relation dry mass of the root/aerial part. The work results showed that: the treatment of the Parapiptadenia rigida seeds, with Trichoderma sp. is efficient to the physiological quality maintenance during the storage; Parapiptadenia rigida Seedlings have higher growth in the nursery when derived from seeds treated with Trichoderma sp. and polymer; Polymerization of Parapiptadenia rigida seeds is a technique that can be used in the nursery; Treatment of Parapiptadenia rigida seeds with Bacillus subtilis is not effective in maintaining the physiological quality during storage, as well as to promote the growth of seedlings in nursery; The application of micronutrients and biestimulant in Parapiptadenia rigida seeds, does not promote the growth of seedlings in nursery. / Parapiptadenia rigida é uma espécie florestal de ocorrência em várias regiões do Brasil, produzida com freqüência em viveiros florestais, sendo a semente sua forma de multiplicação. Geralmente apresenta boa germinação, porém, aspectos envolvendo a qualidade fisiológica e sanitária dos lotes ainda ocasiona perdas em viveiro. Nesse contexto, microrganismos com características antagônicas e/ou promotoras de crescimento associados a diferentes produtos e revestidos com polímeros surgem como alternativa para melhorar o crescimento de mudas em viveiro. Este trabalho teve como objetivo avaliar o tratamento de sementes de Parapiptadenia rigida visando a manutenção da qualidade fisiológica no armazenamento e promoção de crescimento de mudas no viveiro. O trabalho foi dividido em estudos de armazenamento e viveiro. No estudo de armazenamento, os tratamentos foram arranjados em delineamento inteiramente casualizado em esquema trifatorial (4x4x2), representados por tempo de armazenamento (0, 4, 8, 12 meses), produtos (biológico, químico e testemunha) e polímero (polímero e testemunha), com quatro repetições. Em cada tempo foram efetuadas avaliações do percentual de germinação, teste de vigor, comprimento de plântula, massa fresca e seca, além de plântulas normais fortes e fracas. O estudo em viveiro foi subdividido em dois experimentos: o primeiro utilizou combinações envolvendo Trichoderma sp., polímero e estimulantes de crescimento vegetal, e o segundo envolveu o uso de Bacillus subtilis, polímero e estimulantes vegetais. Para ambos, os tratamentos foram arranjados em delineamento inteiramente casualizado em esquema trifatorial (3x2x4), representados por produto biopromotor/fungicida (biológico, químico e testemunha), polímero (polímero e testemunha), micronutrientes e bioestimulante (micronutrientes, bioestimulante, micronutrientes + bioestimulante, testemunha) com quatro repetições. Aos 60 e 120 dias após a emergência avaliou-se a altura de planta, o diâmetro do coleto e o número de folhas. Ao término do experimento foram efetuadas pesagens de massa seca da parte aérea, raiz e total, além da relação massa seca da raiz/parte aérea. Os resultados do trabalho mostraram que: O tratamento das sementes de Parapiptadenia rigida com Trichoderma sp. é eficiente para a manutenção da qualidade fisiológica durante o armazenamento; Mudas de Parapiptadenia rigida apresentam maior crescimento em viveiro quando provenientes de sementes tratadas com Trichoderma sp. e polímero; A polimerização de sementes de Parapiptadenia rigida é uma técnica que pode ser utilizada em viveiro; O tratamento de sementes de Parapiptadenia rigida com Bacillus subtilis não é eficaz na manutenção da qualidade fisiológica durante o armazenamento, bem como para promover o crescimento de mudas em viveiro; A aplicação de micronutrientes e biestimulante vegetal em sementes de Parapiptadenia rigida, não promove o crescimento das mudas em viveiro.

The biosynthetic pathway for the production of the dipeptide antibiotic bacilysin has been the subject of intense research over the past three decades. These studies revealed the role of multiple enzymes in the biosynthesis of this antibiotic. The identification of different enzymes was initially guided by genetic studies on different strains of Bacillus. The functional role of some these enzymes have been validated in vitro in the recent past. Despite this, the in vitro synthesis of bacilysin still remains elusive. The focus of this study was on two oxidoreductases - BacC and BacG. In the course of these studies, several variations to conventional oxidoreductase mechanisms were observed. These studies also provided us an opportunity to examine an oxidoreductase, BacC, at atomic resolution. This thesis describes these structural studies alongside efforts to achieve the biosynthesis of bacilysin in vitro. Chapter 1 provides an introduction to the broad goals of this thesis. First, the diversity of naturally occurring antibiotics is described. This is followed by a description of nonribosomal peptides and their preferred route for antibiotic synthesis. A summary of previous work in this area is provided to place this study in perspective. Earlier studies performed in this laboratory and others provided a framework for understanding the role of BacC and BacG. These studies have been described with an emphasis on the pivotal role of oxidoreductases in this process. In this context, known features of oxidoreductases, classification of the enzyme family, known reaction mechanisms, preferred substrates and cofactors of the enzyme have been summarized in this chapter. Chapter 2 describes the structural and biochemical characterization of B.subtilis BacG. The crystal structures of BacG determined in the apo form and ligand bound states could capture different conformational states of this enzyme. These structures revealed a basis to understand the ping-pong reaction mechanism. The catalytic residues Tyr-Ser-Lys-Asn involved in the proton relay were examined by mutational analysis. These biochemical studies could corroborate our observations derived from structural analysis. Put together, these studies suggest synchronized conformational changes in BacG that can rationalize cofactor specificity and catalytic action on di hydroxyphenyl pyruvate to form tetra hydroxyphenyl pyruvate en route to anticapsin biosynthesis. Bacillus subtilis BacC could be structurally characterized at 1.19Å resolution. The atomic resolution structure formed the basis for the analysis reported in this chapter. The structure revealed aspects of non-covalent interactions that could be unambiguously determined due to the high resolution diffraction data. The atomic resolution structure also enabled us to conduct charge density analysis on this protein. Atomic displacement parameters were used as a tool to explore paths of non-covalent interactions. A commercially available substrate, 3-Quinuclidinone, was used to characterize enzymatic activity. We note that this enzyme follows a rapid equilibrium random mechanism. Furthermore, the kinetic profiles were conclusive to draw inferences on allosteric interactions. A comparison between the NADH-complex and the apo enzyme structure suggests aspects of nuanced atomic displacement that governs the intra structural signal transduction. Taken together, this study provided a template to analyze the role of non-covalent interactions in regulating enzymatic activity. Chapter 4 is based on a survey of oxidoreductases that have been previously described in literature. During this study, we collated the extensive structural and biochemical data in this family of enzymes. However, we noted that the data remains disperse thereby limiting efforts to understand the reaction mechanism from a structural perspective. Here we collate information of known sequences, structures, cofactors, ligand preferences, reaction mechanisms and their influence on higher order association and catalytic activity in this class of enzymes. Chapter 5 summarizes the findings on two oxidoreductases (BacC, BacG). These studies on two closely related oxidoreductases BacC and BacG performing different roles in the same biosynthetic pathway revealed aspects biosynthesis that are often poorly recognized in protein engineering. The role of the reaction mechanism and their influence on the cofactor specificity could be inferred from the studies on these two enzymes. These studies also suggest the feasibility of evaluating aspects of enzyme activity and regulation provided the wealth of a priori information that is currently available. Put together, these studies provide a data-set for protein engineering efforts on oxidoreductases with general inferences for other enzymes in the short-chain dehydrogenases/ reductases (SDR) family. Appendix 1 provides a schematic representation of our efforts to biosynthetically obtain bacilysin in vitro. The identification, mass spectrometry of the products and substrates en route to bacilysin biosynthesis are compiled in this section. Appendix 2 describes the preliminary characterization of B.subtilis BacF. This part of the work describes the cloning, expression and purification of BacF and attempts to obtain suitable diffracting crystals for structural analysis.