Mass spectrometry based proteomics has emerged as a powerful methodology for investigating protein expression. “Bottom up” techniques in which proteins are first digested, and resulting peptides separated via multi-dimensional chromatography then analyzed via mass spectrometry provide a wide depth of coverage of expressed proteomes. This technique has been successfully and extensively used to survey protein expression (expression proteomics) and also to investigate proteins and their associated interacting partners in order to ascertain function of unknown proteins (functional proteomics). Azospirillum brasilense is a free-living diazotrophic soil bacteria, with world-wide significance as a plant-growth promoting bacteria. Living within the rhizosphere of cereal grasses, its diverse metabolism is important for its survival in the competitive rhizospheric environment. The recently sequenced genome of strain Sp245 provided a basis for the proteome studies accomplished in this work. After initial mass spectrometer parameter optimization studies, the expressed proteomes of two strains of Azospirillum brasilense, Sp7 and Sp245, grown under both nitrogen fixing and optimal growth (non nitrogen fixing) conditions were analyzed using a bottom up proteomics methodology. Further proteome studies were conducted with A. brasilense strain Sp7 in order to ascertain the effect of one chemotaxis operon, termed Che1. In this study, proteomic surveys were conducted on two bacterial derivative strains, created earlier, which lacked either a forward signaling pathway or an adaptation pathway. The proteomic surveys conducted in this work provide a foundation for further biochemical investigations. In order to facilitate further investigation and a movement into functional proteomics, a set of destination vectors was created that contain a variety of tandem affinity tags. The addition of tandem affinity tags to a protein allow for generic purification schemes, and can facilitate future studies to investigate proteins of interest discovered in the first expression proteomic surveys of A. brasilense. Taken together, this dissertation provides a valuable data set for investigation into the physiology of A. brasilense and further provides biochemical tools for analysis of the functional protein interactions of A. brasilense cells.

Previous studies have shown that chemotaxis plays an important role in the colonization of the wheat roots surfaces by Azospirillum brasilense and a chemotaxis operon shown to control motility and chemotaxis in A. brasilense has been isolated. This study looked at the effects of mutations in individual genes coding for chemotaxis proteins from this operon on the ability of the cells to colonize the surface of sterile wheat roots. Using both quantitative and qualitative assays, the study shows differences in the colonization ability of the mutants relative to the wild type: the cheB, cheR, cheBR, and cheOp mutants were significantly impaired in wheat root colonization. Interestingly, the cheA mutant was not affected in its ability to colonize the wheat root surface relative to the wild type. Future studies will look for the factors that compensate for cheA impairment in the rhizosphere.

Previous studies have shown that chemotaxis plays an important role in the colonization of the wheat roots surfaces by Azospirillum brasilense and a chemotaxis operon shown to control motility and chemotaxis in A. brasilense has been isolated. This study looked at the effects of mutations in individual genes coding for chemotaxis proteins from this operon on the ability of the cells to colonize the surface of sterile wheat roots. Using both quantitative and qualitative assays, the study shows differences in the colonization ability of the mutants relative to the wild type: the cheB, cheR, cheBR, and cheOp mutants were significantly impaired in wheat root colonization. Interestingly, the cheA mutant was not affected in its ability to colonize the wheat root surface relative to the wild type. Future studies will look for the factors that compensate for cheA impairment in the rhizosphere.

Thèse (Ph.D.)--Université Laval, 2006. / Bibliogr. Publié aussi en version électronique dans la Collection Mémoires et thèses électroniques.

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-10-27Bitstream added on 2014-06-13T20:24:21Z : No. of bitstreams: 1 nunes_m_dr_jabo.pdf: 789915 bytes, checksum: d33780e723c4af1036139660c9582f18 (MD5) Bitstreams deleted on 2016-10-27T11:27:29Z: 000681810.pdf,. Added 1 bitstream(s) on 2016-10-27T11:28:04Z : No. of bitstreams: 1 000681810.pdf: 562297 bytes, checksum: 2a5bb2ece78e1bfaa1bd6aca60814546 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / A microalga Haematococcus pluvialis é conhecida como o microrganismo que apresenta maior rendimento final de astaxantina em sua biomassa, quando submetida a fatores de estresse celular eficientes. Contudo ainda apresenta uma característica de crescimento desfavorável quando comparada a outras espécies de microalgas cultivadas em escala comercial, visto que possui uma taxa de crescimento intrínseca, resultando em baixa densidade celular ao final dos cultivos. Assim, o rendimento final do pigmento é relativamente baixo. Neste sentido, este estudo teve por objetivo avaliar a resposta de Haematococcus pluvialis ao processo de indução à carotenogênese por meio de estresse luminoso e nutricional, e promover o aumento de biomassa vegetativa da microalga através da adição da rizobactéria promotora de crescimento em plantas, Azospirillum brasilense V6, estirpe produtora do fito hormônio AIA (ácido indol – acético), em cultivos estanques, visando obter maior rendimento final de astaxantina. O trabalho foi dividido em duas fases. Primeiramente, no cultivo referente ao controle (contendo apenas H. pluvialis) foram aplicadas duas combinações de fatores de estresse celular para indução a síntese de astaxantina pela microalga, visando a escolha do método de estresse a ser aplicado na fase seguinte. As células de H. pluvialis foram submetidas ao estresse por alta intensidade luminosa (350 μmol photons m-2 s-1) ou ao mesmo estresse luminoso aliado a suplementação com CO2 (4%) na aeração fornecida na fase exponencial de crescimento. Avaliou-se, entre outros fatores de resposta celular, a concentração final de astaxantina na biomassa após 10 dias de estresse. As concentrações do pigmento foram 4,15 e 17,66 mg g-1, respectivamente, para as culturas submetidas ao estresse luminoso e para... / The microalgae Haematococcus pluvialis is known as the microorganism that has a highest final yield of astaxanthin in its biomass, when induced by efficient stress factors. However, still presents a characteristic of unfavorable growth when compared to other species of microalgae grown on a commercial scale, once it has an intrinsic growth rate that provides low cell density in the culture. Thus, the final yield of the pigment is relatively low. Thus, this study aimed to evaluate the response of Haematococcus pluvialis submitted to carotenogenesis induction process through light and nutrient stress, and promotion of vegetative microalgae biomass increase through the addition of the growth promoter bacteria in plants, Azospirillum brasilense V6, a strain that produce the phyto hormone IAA (indole - acetic acid). The experiments were performed in “batch” systems, aiming to obtain higher final yield of astaxanthin. The work was divided into two phases. First, to the control (containing only H. pluvialis) it was applied two combinations of stress factors to induce cellular synthesis of astaxanthin by the microalgae, to define the method of stress to be applied in the next phase. The cells of H. pluvialis were submitted to stress by high light intensity (seven times the original) or the same light stress combined with CO2 supplementation (4%) in the aeration provided in the exponential growth phase. We evaluated, among other factors of cellular response, the final concentration of astaxanthin in the biomass after 10 days of stress. The pigment concentrations were 4.15 and 17.66 mg g-1, respectively, for cultures submitted to light stress and to those who received the same light stress add with the increase of C / N ratio in the culture medium. So, for the next experimental phase, the combination... (Complete abstract click electronic access below)

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-07-03T17:23:46Z No. of bitstreams: 1 texto completo.pdf: 529263 bytes, checksum: 519329022e5e2f52db8e5958ffce55b7 (MD5) / Made available in DSpace on 2017-07-03T17:23:46Z (GMT). No. of bitstreams: 1 texto completo.pdf: 529263 bytes, checksum: 519329022e5e2f52db8e5958ffce55b7 (MD5) Previous issue date: 2001-06-18 / Fundação de Amparo à Pesquisa do Estado de Minas Gerais / Este estudo objetivou avaliar, em dois cultivares de trigo irrigado, os efeitos da inoculação, peletização das sementes e aplicação de Mo em cobertura sobre a atividade da nitrato redutase (ANR), compo sição mineral e características agronômicas. Um experimento, em condições de campo, foi conduzido no Departamento de Fitotecnia (DFT) (Estação Experimental Diogo Alves de Melo), de junho a outubro de 1999, sendo outro experimento, em vasos, conduzido em casa de vegetação do DFT e na mesma época. O experimento de campo foi constituído de 16 tratamentos, dispostos num esquema fatorial 2 x 2 x 2 x 2 [dois -1 cultivares (EMBRAPA-22 e BR-26), Mo (0 e 90 g.ha ), peletização (presente e ausente) e inoculação com Azospirillum brasilense (presente e ausente)], no delineamento em blocos casualizados com quatro repetições. O experimento em vasos foi constituído de 18 tratamentos, num esquema fatorial, acrescidos de duas -1 parcelas [(EMBRAPA-22 + 90 g.ha de Mo + peletização + inoculação - P e - S) e -1 (BR-26 + 90 g.ha de Mo + peletização + inoculação - P e - S)], no delineamento em blocos casualizados com quatro repetições. Avaliaram-se a ANR em quatro estádios da cultura, o teor de macro e micronutrientes na parte aérea e nos grãos e -1 características radiculares e agronômicas. A aplicação de 90 g.ha de Mo e a inoculação das sementes aumentaram significativamente a ANR, nas quatro épocas avaliadas (28, 48, 68 e 88 dias após a emergência das plântulas). Ocorreu redução -1 na atividade da enzima com o avanço do ciclo da cultura. A aplicação de 90 g.ha de Mo e a inoculação das sementes com Azospirillum brasilense aumentaram a matéria seca, o comprimento e área radiculares e os teores de nutrientes das plantas, além de diminuírem o diâmetro radicular destas. A matéria seca e o comprimento e área radiculares e os teores de nutrientes foram superiores no -1 cultivar EMBRAPA-22. A aplicação de 90 g.ha de Mo, assim como a inoculação de Azospirillum brasilense, aumentou a altura das pl antas, a produção de grãos, a biomassa seca e o peso de mil grãos, nos dois experimentos realizados. / The objective of this study was to evaluate the effects from seed inoculation, seed pelletization and Mo application on the activity of nitrate reductase (ANR), the mineral composition and agronomic characteristics. An experiment was carried out under field conditions at the Estação Experimental Diogo Alves de Melo from June to October 1999, and at the same time another one was accomplished in pots under greenhouse conditions, both places pertaining to the Departmento de Fitotecnia-UFV. The field experiment consisted of 16 treatments arranged in a factorial scheme of 2 x 2 x 2 x 2 [two cultivars -1 (EMBRAPA-22 and BR-26), Mo (0 and 90 g.ha ), pelletization (present and absent) and inoculation with Azospirillum brasilense (present and absent)] on a randomized block experimental design with four replicates. The experiment in pots consisted of 18 treatments on a factorial scheme with two additional plots -1 [(EMBRAPA-22 + 90 g.ha of Mo + pelletization + inoculation - P and - S) and (BR-26 + 90 g.ha -1 of Mo + pelletization + inoculation - P and - S)] on a randomized block experimental design with four replicates. The ANR was evaluated at four crop stages, as well as the macronutrient and micronutrients in the aerial part and the grains, the agronomic and root characteristics were also evaluated. The -1 application of 90 g.ha Mo and seed inoculation significantly increased the ANR at the four appraised times (28, 48, 68 and 88 days after emergency of the seedlings). There was a reduction in enzyme activity as the crop cycle advanced. Either -1 application of 90 g.ha Mo and the seed inoculation with Azospirillum brasilense increased the root dry matter, root length, root area and the nutrient contents in plants, whereas the root diameter was reduced. The root dry matter, root length, root area and the nutrient contents in plants were superior in cultivar EMBRAPA- -1 22. The application of 90 g.ha Mo as well as the seed inoculation with Azospirillum brasilense increased the plant heights, grain yield, dry biomas s and the weight of a thousand grains in both experiments. / Tese importada do Alexandria

Made available in DSpace on 2014-12-02T11:16:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-02-21Bitstream added on 2014-12-02T11:20:55Z : No. of bitstreams: 1 000798405.pdf: 931361 bytes, checksum: 83395259e8516b722468ec16b1d9101f (MD5) / A produtividade média de feijão no Brasil é baixa. Alternativas que possam aumentar a produtividade de forma sustentável devem ser preconizadas, tais como a redução do uso de fertilizantes nitrogenados pela adoção da fixação biológica de nitrogênio. Neste cenário, uma técnica alternativa de co-inoculação ou também denominada de inoculação mista com bactérias simbióticas e assimbióticas tem sido estudada em leguminosas, no entanto existem poucos estudos em campo com feijoeiro e sobre a influência da quantidade de água irrigada. Dessa forma, o objetivo deste trabalho foi avaliar a eficiência da inoculação e co-inoculação de feijão com Rhizobium tropici e Azospirillum brasilense sob duas lâminas de irrigação na produção e qualidade fisiológica de sementes. O experimento foi desenvolvido no período de outono/inverno dos anos de 2012 e 2013, no município de Selvíria-MS. O delineamento experimental foi de blocos casualizados em esquema fatorial 5x2, com quatro repetições. Os tratamentos foram constituídos pela combinação de cinco formas de fornecimento de nitrogênio (testemunha sem inoculação com 40 kg ha-1 de nitrogênio em cobertura; testemunha sem inoculação com 80 kg ha-1 de N em cobertura; inoculação de A. brasilense com 40 kg ha-1 de N em cobertura; inoculação de R. tropici com 40 kg ha-1 de N em cobertura; e co-inoculação de A. brasilense e R. tropici com 40 kg ha-1 de N em cobertura) e duas lâminas irrigação (recomendada e 75% da recomendada). A aplicação de nitrogênio em cobertura foi realizada utilizando a ureia. Concluiu-se que a co-inoculação, a inoculação de R. tropici, a inoculação de A. brasilense associadas a 40 kg ha-1 de nitrogênio em cobertura e a testemunha sem inoculação com aplicação de 80 kg ha-1 de N em cobertura não proporcionam acréscimos de produtividade em relação a testemunha não inoculada com 40 kg ha-1 ... / The average yield of beans in Brazil is low. Alternatives that can increase yield in a sustainable manner should be preconized, such as reducing the use of nitrogen fertilizers through the adoption of biological nitrogen fixation. In this scenario, an alternative technique of co-inoculation or also called the mixed inoculation with symbiotic and non-symbiotic bacteria has been studied in leguminous, however there are few studies in field with common bean under the influence of the amount of water irrigated. Thus the objetive of this study was to evaluate the efficiency of inoculation and co-inoculation of common bean with Rhizobium tropici and Azospirillum brasilense under two irrigation depths on production and physiological seed quality. The experiment was performed in the fall/winter of the years 2012 and 2013, in Selviria-MS. A randomized block design in a 5x2 factorial scheme with four repetitions was used. The treatments were constituted by the combination of five forms of nitrogen supply (control non-inoculated with 40 kg ha- 1 of nitrogen in topdressing; control non-inoculated with 80 kg ha- 1 of N in topdressing; A. brasilense inoculation with 40 kg ha-1 of N in topdressing; R. tropici inoculation with 40 kg ha-1 of N in topdressing; and co-inoculation of A. brasilense and R. tropici with 40 kg ha- 1 of N in topdressing) and two irrigation depths (recommended and 75% of the recommended). Was concluded that the co-inoculation, the inoculation of R. tropici, the inoculation of A. brasilense associated with 40 kg ha-1 of nitrogen in topdressing and the control non-inoculation with application of 80 kg ha-1 of N topdressing not increase of grain yield compared to control non-inoculation with 40 kg ha-1 of nitrogen in topdressing. Considering the development conditions of the experiment, the use of 75% of the recommended irrigation depth provides similar or greater grain yield than the ...

Orientador: Leandro Borges Lemos / Resumo: A utilização de manejos sustentáveis para maximizar a produtividade das culturas sem aumentar os custos de produção é um dos grandes desafios da agricultura atual. Assim, objetivou-se avaliar o efeito de manejos de fornecimento de nitrogênio, associando formas de inoculação com Rhizobium tropici, co-inoculação com Azospirillum brasilense e adubação mineral de cobertura, no desempenho agronômico de cultivares de feijão comum do grupo comercial carioca de ciclo precoce. O experimento foi instalado em delineamento em blocos casualizados (DBC), no esquema fatorial 2x12 (2 cultivares e 12 manejos de fornecimento de N), com quatro repetições. Para os manejos de fornecimento de N foram associados adubação mineral de cobertura com N (45 e 90 kg ha-1 de N), inoculação de sementes com Rhizobium tropici e coinoculação via semente e foliar com Azospirillum brasilense. Foram avaliados o número de trifólios por planta, área foliar, índice de clorofila, teor de nitrogênio foliar e nos grãos, massa seca de plantas, número de vagens por planta, número de grãos por planta e produtividade, número de nódulos por planta, massa seca de nódulos por planta, massa seca de raízes e eficiência de nodulação. Os dados foram submetidos à análise de variância (teste F) e, quando necessário, as médias foram agrupadas pelo teste de Scott-Knott a 5% de probabilidade. Com exceção da massa seca de plantas, todas as variáveis analisadas apresentaram máxima resposta no tratamento com inoculação de sementes com Rh... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The use of sustainable managements techniques to maximize crop productivity without increasing production costs is one of the great challenges of modern agriculture. The objective of this study was to assess the effect of different nitrogen management by testing the inoculation with Rhizobium tropici, co-inoculation with Azospirillum brasilense, and topdressing N fertilization on the agronomic performance of common bean cultivars of early cycle. The experiment was carried out in a randomized block design (RBD), in a 2x12 factorial scheme (2 cultivars and 12 N supply management), with four replications. For N supply management, it was used topdressing N fertilization (45 and 90 kg ha-1 N), seed inoculation with Rhizobium tropici, and seed and leaf co-inoculation with Azospirillum brasilense. It was evaluated: the number of trifolium per plant, leaf area, chlorophyll index, leaf and grain nitrogen content, dry mass of plants, number of pods per plant, number of grains per plant and yield, number of nodules per plant, dry matter of nodules per plant, dry matter of root, and nodulation efficiency. Data were subjected to analysis of variance (F test) and, when necessary, means were grouped by Scott-Knott test at 5% probability. Except for the dry mass of plants, all variables analyzed showed maximum response in the treatment with seed inoculation with Rhizobium tropici, foliar application of Azospirillum brasilense, and topdressing fertilization with 90 kg ha-1 of N. In general, t... (Complete abstract click electronic access below) / Mestre

La croissance et la capacité d'absorption d'azote de la microalgue d'eau douce Chlorella vulgaris co-immobilisée dans des billes d'alginate, en présence de la bactérie promotrice de croissance des microalgues Azospirillum brasilense Cd, ont été étudiés. La co-immobilisation des microalgues avec A. brasilense conduit à deux phénomènes indépendants directement affectés par des facteurs de culture, tels que la forme azotée, le pH et la présence d'une source de carbone. Premièrement, la croissance de la population de microalgues augmentait sans une augmentation de la capacité d'absorption d'azote par les cellules seules, ou deuxièmement, la capacité des cellules à absorber l'azote augmentait sans une augmentation de la population entière de microalgues. Ces phénomènes étaient dépendants de la densité de la population des microalgues, qui était à son tour affectée par les facteurs de culture. Cela renforce la conclusion selon laquelle la taille de la population de microalgues contrôle l'absorption d'azote chez les cellules de C. vulgaris -plus la population est élevée (peu importe les paramètres expérimentaux), moins les cellules absorbent d'azote. Les résultats ont montré que YAzospirillum affectait la croissance de la Chlorella vulgaris. L'un des processus proposés par lequel XAzospirillum promeut la croissance des plantes supérieures, est la production de phytohormones, principalement 1' acide 3-indole acétique (auxine ou AI A). Par conséquent, l'effet de cette phytohormone, produite par plusieurs souches de VAzospirillum et favorisant la croissance de microalgue, a été étudié. Cinq souches de type sauvage â!Azospirillum {A. brasilense souches Cd, Sp6, 8-1 et Sp245 et A. lipoferum JA4) et leurs mutants déficients en production d'AIA : A. brasilense SpM 7918 (modifié de A. brasilense Sp6), A. brasilense FAJ0009 (modifié de A. brasilense Sp245), A. brasilense S-l-gfp (modifié de A. brasilense 8-1) et A. lipoferum 3A4::ngfp\5 (modifié de A. lipoferum JA4) ont été co-immobilisés avec C. vulgaris en billes d'alginate. Aucune production d'AIA n'est possible sans l'apport de tryptophane, nos résultats corroborant des observations antérieures selon lesquelles la production d'AIA chez Azospirillum est induite par la présence de tryptophane. Les taux élevés de tryptophane étaient délétères sur la croissance des pousses de C. vulgaris utilisées. Cette recherche montre que que l'AIA produit par 1' Azospirillum spp. est impliqué dans la croissance du C vulgaris immobilisé dans des billes d'alginate, et que la co-immobilisation avec différentes souches d'Azospirillum spp. atténue les effets délétères du tryptophane sur le C. vulgaris. Co-immobilisation de Chlorella vulgaris avec Azospirillum brasilense affecte l'absorption d'ammonium par les microalgues par le biais de métabolismes indéfinis. Les résultats présentés dans ce travail montrent clairement qu'il y a un effet & Azospirillum sur les activités spécifiques de la Glutamine synthétase (GS) et de la Glutamate déshydrogénase (GDH) de Chlorella. Lorsque Chlorella coimmobilisée absorbe plus d'ammonium que Chlorella seule, les deux enzymes fonctionnent en même temps, et les activités spécifiques par cellule de la GS et de la GDH sont plus élevées. Toutefois, dans les cas où seule l'activité GS augmente, mais pas celle de la GDH, l'activité GS plus élevée n'est pas suffisante pour atteindre une absorption d'ammonium plus élevée au niveau de la culture. En résumé, il y a un effet positif d'A. brasilense sur l'activité de la glutamine synthétase et de la glutamate déshydrogénase chez Chlorella. La régulation et la relation de l'activité enzymatique avec la concentration initiale en ammonium demeurent floues .Les données présentées dans ce chapitre dans leur ensemble montrent que les activités plus élevées de GS et de GDH augmentent l'absorption d'ammonium par Chlorella. / Growth and capacity to absorb nitrogen in the freshwater microalgae Chlorella vulgaris when co-immobilized in alginate beads with the microalgae growth-promoting bacterium Azospirillum brasilense Cd and the mechanisms involved were studied in this work. Co-immobilization of the microalgae with A. brasilense leads to two independent phenomena directly affected by culturing factors, such as nitrogen species, pH, and presence of a carbon source. First, growth of the microalgae population increased without an increase in capacity of the single cells to absorb nitrogen; second, the capacity of cells to absorb nitrogen increased without increasing the microalgae population. These phenomena were dependent on population density of the microalgae, which was, in turn, affected by culturing factors. This supports the conclusion that the size of the microalgae population controls absorption of nitrogen in C. vulgaris cells—the higher the population (whatever the experimental parameters), the less nitrogen each cell takes up. The results show that Azospirillum affects the growth of Chlorella vulgaris. One of the mechanisms proposed by which Azospirillum promotes growth of higher plants is the production of phytohormones, mainly the auxin indole-3-acetic acid (IAA); therefore the effect of this phytohormone produced by several strains of Azospirillum in promoting growth of the microalgae was studied. Five wild type strains of Azospirillum {A. brasilense strains Cd, Sp6, 8-1, and Sp245 and A. lipoferum JA4) and their indole-3-acetic-acid déficient mutants, A. brasilense SpM 7918 (modifïed from A. brasilense Sp6), A. brasilense FAJ0009 (modified from A. brasilense Sp245), A. brasilense %-\-gfp (modified from A, brasilense 8-1), and A. lipoferum lA4::ngfp\5 (modified from A. lipoferum JA4) were coimmobilized with C. vulgaris in alginate beads. No IAA production occurred without tryptophan. Our results corroborate previous observations that IAA production in Azospirillum is induced by the presence of tryptophan. It was found that high levels of tryptophan are deleterious to the growth of the C. vulgaris strain we used. This study demonstrated that IAA produced by Azospirillum spp. is involved in growth promotion of C. vulgaris immobilized in alginate beads, and that co-immobilization with several strains of Azospirillum spp. alleviated the deleterious effects of tryptophan in C. vulgaris. Co-immobilization of Chlorella vulgaris with Azospirillum brasilense affects ammonium absorption by the microalgae via undetermined metabolism. The results presented in this work show clearly that there is an effect of Azospirillum on the specific activity of the enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) in Chlorella. When coimmobilized Chlorella absorbs more ammonium than Chlorella alone, both enzymes are operating at the same time, and the specific activity per cell of GS and GDH are higher. However, in cases when only the activity of GS increases, but not of GDH, this GS higher activity was not sufficient to yield a higher absorption of ammonium at the culture level. There is a positive effect of A. brasilense on the activity of glutamine synthetase and glutamate dehydrogenase in Chlorella. However, the regulation and the relation of the enzymatic activity with initial ammonium concentration are still unclear.

With the increase of agricultural production, there is an improvement in the use of mineral fertilizers, which may cause different environmental problems, besides the soil salinization. A possible alternative for reducing the application of these products is the use of plant growth-promoting bacteria (PGPB), that can be used alone or in co-inoculation, resulting in an alternative environmentally and economically feasible. Better results can be obtained if the interaction among bacteria-bacteria and bacteria-plant be elucidated, and strategy developed to optimize these interactions. Thus, the plant growth-promoting Bacillus sp. RZ2MS9, previous described as a potential PGPB in maize and soybean, was GFP-tagged and monitored alone and co-inoculated with Azospirillum brasilense (Ab-v5::pWM1013) during maize colonization. The interaction of tagged strains in maize were monitored by fluorescent microscopy (FM) and quantitative PCR (qPCR), demonstrating an endophytic behavior of Bacillus sp. RZ2MS9. Although the non-detection of Ab-v5::pWM1013, the co-inoculation resulted in the best increase in root and shoot dried weight, root volume and in root diameter, showing that inoculation with more than one strain can be a good choice to development of bio-fertilizers. One important system to bacterial interaction is the quorum sensing (QS). The QS is an important cell-cell communication system that allows bacterial cells to recognize their own population and modulate their gene expression. This, system is also involved in the interspecific communication, including other bacterial species and plants. In the other hand, enzymes able to detect and degrade these molecules evolved, the called quorum quenching (QQ) system, that has been evolved in some bacteria as competitive advantage for niches colonization. The aiiA gene, was one of the first gene related with the QQ in Bacillus. The aiiA was found in Bacillus sp. RZ2MS9 genome. Through construction of a new QQ biosensor, Agrobacterium tumefaciens At11006, and validated by A. tumefaciens NTL4, the ability of RZ2MS9 to degrade QS molecules was confirmed. The knockout of aiiA gene was performed using the CRISPR-Cas9 system, confirming this gene function. By these results, the influence of QQ system of Bacillus sp. RZ2MS9 during maize colonization and RZ2MS9 - A. brasilense - maize can be better investigated, opens the possibility to better understand the role of QQ system in the interaction among PGPB and plants. / Concomitantemente ao aumento da produção agrícola, há o aumento do uso de fertilizantes minerais, que pode acarretar no desenvolvimento de diferentes problemas ambientais, além de causar a salinização dos solos. Uma possível alternativa para tentar reduzir a aplicação desses produtos é o uso de bactérias promotoras de crescimento de plantas (BPCPs), que podem ser usadas isoladamente ou em co-inoculação com outras bactérias, tornando-as uma alternativa ambientalmente e economicamente viável. Melhores resultados podem ser obtidos se a interação bactéria-bactéria e bactéria-planta for elucidada, permitindo que estratégias sejam desenvolvidas para otimizar essas interações. Em vista disso, a bactéria Bacillus sp. RZ2MS9, previamente descrita como uma potencial BPCP em milho e soja, foi marcada com GFP e monitorada durante a colonização de milho inoculada sozinha, bem como em co-inoculação com Azospirillum brasilense (Ab-v5::pWM1013). A interação dessas linhagens marcadas em milho, foi monitorada por microscopia de fluorescência (FM) e PCR quantitativo (qPCR), revelando um comportamento endofítico de Bacillus sp. RZ2MS9. Em plantas co-inoculadas, apesar da linhagem Ab-v5::pWM1013 não ter sido detectada por qPCR, a co-inoculação resultou no aumento do peso seco das raízes e da parte aérea, no volume e no diâmetro do sistema radicular, demonstrando que a inoculação com mais de uma linhagem bacteriana pode ser uma boa alternativa para o desenvolvimento de bio-fertilizantes. O quorum sensing (QS) é um importante sistema de comunicação célula-célula que permite que as bactérias reconheçam sua própria população e modulem sua expressão gênica. Este sistema também está envolvido na comunicação interespecífica, incluindo outras espécies bacterianas e plantas. Co-evolutivamente, enzimas capazes de detectar e degradar essas moléculas evoluíram, dando origem ao chamado quorum quenching (QQ), sistema que evoluiu em algumas bactérias como uma vantagem competitiva para a colonização de nichos. O gene aiiA, foi um dos primeiros genes relacionados ao sistema QQ descrito no gênero Bacillus, gene este que foi anotado no genoma de RZ2MS9. Através da construção de uma nova linhagem biossensora de QQ, Agrobacterium tumefaciens At11006, e validada através da linhagem A. tumefaciens NTL4, a capacidade de RZ2MS9 de degradar moléculas de QS foi confirmada. O knockout do gene aiiA foi realizado utilizando o sistema CRISPR-Cas9, confirmando a função desse gene. Através dos resultados obtidos neste trabalho, a influência do sistema QQ de Bacillus sp. RZ2MS9 durante a colonização do milho, bem como a interação RZ2MS9 - A. brasilense - milho pode ser melhor investigada, abrindo a possibilidade de uma melhor compreensão do papel do sistema QQ na interação entre bactérias promotoras de crescimento e plantas.