Universiẗat, Diss., 2004--München.

Several nitrogen fixers were isolated from the roots and rhizosphere of Quebec crops. Many of these nitrogen fixers were discarded when the production of N(,2)O in the presence of C(,2)H(,2), as a test for denitrifying ability, was included as a selected characteristic. Further characterization of the Nir('+) strains showed that they were Azospirillum lipoferum. / The cytochrome composition of Azospirillum brasilense (a denitrifier) grown under various conditions in a defined medium was investigated. Optical absorbance difference spectra of the particulate fraction of cells grown under aerated conditions indicated the presence of cytochromes of type b, c and a+a(,3). Under low aeration there was a quantitative increase in cytochromes b and c with a concomitant decrease in the a+a(,3)-type cytochrome. At high aeration, a CO spectrum indicated the possible participation of an o-type cytochrome. / At both high and low oxygen concentrations, the supernatant fraction revealed only one c-type cytochrome. Its abundance was increased at low oxygen concentrations. / Cytochrome spectra of anaerobically grown cells using different nitrogen oxides (NO(,3)('-), NO(,2)('-) and N(,2)O) as final electron acceptors revealed the presence of the different cytochromes involved in anaerobic respiration. The reduction of NO(,2)('-) was associated with the cytochrome cd (peak at 620 nm) found only in the supernatant fraction of NO(,2)('-)-grown cells. / Growth on NO(,3)('-) was characterized by a diauxic type of curve in which the first logarithmic phase corresponded to the reduction of NO(,3)('-). The second logarithmic phase corresponded to the reduction of NO(,2)('-). / Growth of Azospirillum brasilense with NO(,2)('-) and N(,2)O as final electron acceptor was possible only when a small amount of NO(,3)('-) was present initially. In contrast with other bacteria, growth of Azospirillum brasilense with tungstate instead of molybdate did not result in NO(,3)('-) reductase-deficient cells. / The NO(,2)('-) accumulation observed with NO(,3)('-)-grown cells possibly resulted from the different NO(,3)('-)- and N(,2)('-)-reductase specific activities. However, the longer lag in the NO(,2)('-) reduction when higher concentrations of NO(,3)('-) were used may be due to a direct effect of NO(,3)('-) on the synthesis or activity of the NO(,2)('-) reductase.

Ureases (ureia amido-hidrolases; EC 3.5.1.5) são enzimas níquel dependentes, amplamente distribuídas em bactérias, fungos e plantas, que catalisam a hidrólise da ureia à amônia e a dióxido de carbono. Em plantas e fungos, as ureases são hexâmeros formados por subunidades idênticas. Em bactérias, as ureases são formadas por duas ou três subunidades distintas, como é o caso em Helicobacter pylori e Azospirillum brasilense, respectivamente. As bactérias do gênero Azospirillum são microrganismos diazotróficos (capazes de fixar nitrogênio atmosférico), encontrados no solo e em associação com raízes de plantas como o arroz, o trigo e o milho. A principal função da urease em plantas parece estar relacionada à reciclagem do nitrogênio, mas diversas atividades biológicas que independem da atividade ureolítica têm sido demonstradas, sugerindo que esta possa estar envolvida na defesa da planta contra insetos e fungos. A fim de estudar esta enzima, a primeira parte do trabalho objetivou obter a urease recombinante de Azospirillum brasilense FP2. Para isso, o inserto foi clonado no vetor pET-23a por recombinação homóloga in vivo. Seis aminoácidos mostraram-se diferentes, em relação a sequência de proteínas predita do A. brasilense sp245, o que parece diferença entre as cepas de A. brasilense sp245 e FP2. A expressão da enzima recombinante foi otimizada, seguida de purificação feita por cromatografia de afinidade a níquel. No segundo capítulo desse trabalho é apresentada a clonagem, a expressão do gene e a purificação de um peptídeo derivado da urease ubíqua de soja. Esse peptídeo, denominado soyuretox, é colinear com o jaburetox, uma peptídeo recombinante derivado da urease de Canavalia ensiformis. Dados prévios do nosso laboratório demonstram diversas propriedades biológicas do jaburetox, tais como atividade inseticida, capacidade de interagir com bicamadas lipídicas, efeito fungitóxico, dentre outras. Estudos comparativos com o soyuretox mostraram que esse peptídeo apresenta toxicidade para duas leveduras testadas, Candida tropicalis e Saccharomyces cerevisiae. / Urease (urea amidohydrolase, EC 3.5.1.5) are nickel-dependent enzymes widely distributed in bacteria, fungi and plants which catalyze the hydrolysis of urea to ammonia and carbon dioxide. In plants and fungi, ureases are hexamers formed by one type of subunit. In bacteria, ureases are formed by two or three distinct subunits, such as in Helicobacter pylori and in Azospirillum brasilense, respectively. Bacteria of the genus Azospirillum are diazotrophic (capable of fixing atmospheric nitrogen), found in the soil and in association with roots of plants such as rice, wheat and corn. The main function of urease in plants appears to be related to nitrogen recycling, but several biological activities that are independent of ureolytic activity have been demonstrated, suggesting that it may be involved in the defense of plants against insects and fungi. In order to study this enzyme, the first part of the study aimed to obtain the recombinant Azospirillum brasilense FP2 urease. For this end, the insert was cloned into the vector pET-23a by in vivo homologous recombination. Six amino acids were different in relation to the predicted protein sequence of A. brasilense sp245, which seems to difference among strains of A. brasilense sp245 and FP2. Expression of the recombinant protein was optimized, following purification by nickel affinity chromatography. Cloning, expression of the gene and purification of a peptide derived from soybean ubiquitous urease is presented in the second chapter of this work. This peptide, called soyuretox, aligns with jaburetox, a recombinant peptide derived from Canavalia ensiformis urease. Previous data from our laboratory have shown diverse biological properties for jaburetox, such as insecticidal activity, ability to interact with lipid bilayers, fungitoxic effects, among others. Comparative studies with soyuretox, showed that this peptide presents toxicity against yeast, Candida tropicalis and Saccharomyces cerevisiae.

Ureases (ureia amido-hidrolases; EC 3.5.1.5) são enzimas níquel dependentes, amplamente distribuídas em bactérias, fungos e plantas, que catalisam a hidrólise da ureia à amônia e a dióxido de carbono. Em plantas e fungos, as ureases são hexâmeros formados por subunidades idênticas. Em bactérias, as ureases são formadas por duas ou três subunidades distintas, como é o caso em Helicobacter pylori e Azospirillum brasilense, respectivamente. As bactérias do gênero Azospirillum são microrganismos diazotróficos (capazes de fixar nitrogênio atmosférico), encontrados no solo e em associação com raízes de plantas como o arroz, o trigo e o milho. A principal função da urease em plantas parece estar relacionada à reciclagem do nitrogênio, mas diversas atividades biológicas que independem da atividade ureolítica têm sido demonstradas, sugerindo que esta possa estar envolvida na defesa da planta contra insetos e fungos. A fim de estudar esta enzima, a primeira parte do trabalho objetivou obter a urease recombinante de Azospirillum brasilense FP2. Para isso, o inserto foi clonado no vetor pET-23a por recombinação homóloga in vivo. Seis aminoácidos mostraram-se diferentes, em relação a sequência de proteínas predita do A. brasilense sp245, o que parece diferença entre as cepas de A. brasilense sp245 e FP2. A expressão da enzima recombinante foi otimizada, seguida de purificação feita por cromatografia de afinidade a níquel. No segundo capítulo desse trabalho é apresentada a clonagem, a expressão do gene e a purificação de um peptídeo derivado da urease ubíqua de soja. Esse peptídeo, denominado soyuretox, é colinear com o jaburetox, uma peptídeo recombinante derivado da urease de Canavalia ensiformis. Dados prévios do nosso laboratório demonstram diversas propriedades biológicas do jaburetox, tais como atividade inseticida, capacidade de interagir com bicamadas lipídicas, efeito fungitóxico, dentre outras. Estudos comparativos com o soyuretox mostraram que esse peptídeo apresenta toxicidade para duas leveduras testadas, Candida tropicalis e Saccharomyces cerevisiae. / Urease (urea amidohydrolase, EC 3.5.1.5) are nickel-dependent enzymes widely distributed in bacteria, fungi and plants which catalyze the hydrolysis of urea to ammonia and carbon dioxide. In plants and fungi, ureases are hexamers formed by one type of subunit. In bacteria, ureases are formed by two or three distinct subunits, such as in Helicobacter pylori and in Azospirillum brasilense, respectively. Bacteria of the genus Azospirillum are diazotrophic (capable of fixing atmospheric nitrogen), found in the soil and in association with roots of plants such as rice, wheat and corn. The main function of urease in plants appears to be related to nitrogen recycling, but several biological activities that are independent of ureolytic activity have been demonstrated, suggesting that it may be involved in the defense of plants against insects and fungi. In order to study this enzyme, the first part of the study aimed to obtain the recombinant Azospirillum brasilense FP2 urease. For this end, the insert was cloned into the vector pET-23a by in vivo homologous recombination. Six amino acids were different in relation to the predicted protein sequence of A. brasilense sp245, which seems to difference among strains of A. brasilense sp245 and FP2. Expression of the recombinant protein was optimized, following purification by nickel affinity chromatography. Cloning, expression of the gene and purification of a peptide derived from soybean ubiquitous urease is presented in the second chapter of this work. This peptide, called soyuretox, aligns with jaburetox, a recombinant peptide derived from Canavalia ensiformis urease. Previous data from our laboratory have shown diverse biological properties for jaburetox, such as insecticidal activity, ability to interact with lipid bilayers, fungitoxic effects, among others. Comparative studies with soyuretox, showed that this peptide presents toxicity against yeast, Candida tropicalis and Saccharomyces cerevisiae.

Ureases (ureia amido-hidrolases; EC 3.5.1.5) são enzimas níquel dependentes, amplamente distribuídas em bactérias, fungos e plantas, que catalisam a hidrólise da ureia à amônia e a dióxido de carbono. Em plantas e fungos, as ureases são hexâmeros formados por subunidades idênticas. Em bactérias, as ureases são formadas por duas ou três subunidades distintas, como é o caso em Helicobacter pylori e Azospirillum brasilense, respectivamente. As bactérias do gênero Azospirillum são microrganismos diazotróficos (capazes de fixar nitrogênio atmosférico), encontrados no solo e em associação com raízes de plantas como o arroz, o trigo e o milho. A principal função da urease em plantas parece estar relacionada à reciclagem do nitrogênio, mas diversas atividades biológicas que independem da atividade ureolítica têm sido demonstradas, sugerindo que esta possa estar envolvida na defesa da planta contra insetos e fungos. A fim de estudar esta enzima, a primeira parte do trabalho objetivou obter a urease recombinante de Azospirillum brasilense FP2. Para isso, o inserto foi clonado no vetor pET-23a por recombinação homóloga in vivo. Seis aminoácidos mostraram-se diferentes, em relação a sequência de proteínas predita do A. brasilense sp245, o que parece diferença entre as cepas de A. brasilense sp245 e FP2. A expressão da enzima recombinante foi otimizada, seguida de purificação feita por cromatografia de afinidade a níquel. No segundo capítulo desse trabalho é apresentada a clonagem, a expressão do gene e a purificação de um peptídeo derivado da urease ubíqua de soja. Esse peptídeo, denominado soyuretox, é colinear com o jaburetox, uma peptídeo recombinante derivado da urease de Canavalia ensiformis. Dados prévios do nosso laboratório demonstram diversas propriedades biológicas do jaburetox, tais como atividade inseticida, capacidade de interagir com bicamadas lipídicas, efeito fungitóxico, dentre outras. Estudos comparativos com o soyuretox mostraram que esse peptídeo apresenta toxicidade para duas leveduras testadas, Candida tropicalis e Saccharomyces cerevisiae. / Urease (urea amidohydrolase, EC 3.5.1.5) are nickel-dependent enzymes widely distributed in bacteria, fungi and plants which catalyze the hydrolysis of urea to ammonia and carbon dioxide. In plants and fungi, ureases are hexamers formed by one type of subunit. In bacteria, ureases are formed by two or three distinct subunits, such as in Helicobacter pylori and in Azospirillum brasilense, respectively. Bacteria of the genus Azospirillum are diazotrophic (capable of fixing atmospheric nitrogen), found in the soil and in association with roots of plants such as rice, wheat and corn. The main function of urease in plants appears to be related to nitrogen recycling, but several biological activities that are independent of ureolytic activity have been demonstrated, suggesting that it may be involved in the defense of plants against insects and fungi. In order to study this enzyme, the first part of the study aimed to obtain the recombinant Azospirillum brasilense FP2 urease. For this end, the insert was cloned into the vector pET-23a by in vivo homologous recombination. Six amino acids were different in relation to the predicted protein sequence of A. brasilense sp245, which seems to difference among strains of A. brasilense sp245 and FP2. Expression of the recombinant protein was optimized, following purification by nickel affinity chromatography. Cloning, expression of the gene and purification of a peptide derived from soybean ubiquitous urease is presented in the second chapter of this work. This peptide, called soyuretox, aligns with jaburetox, a recombinant peptide derived from Canavalia ensiformis urease. Previous data from our laboratory have shown diverse biological properties for jaburetox, such as insecticidal activity, ability to interact with lipid bilayers, fungitoxic effects, among others. Comparative studies with soyuretox, showed that this peptide presents toxicity against yeast, Candida tropicalis and Saccharomyces cerevisiae.

No description available.

The present study was aimed at revealing the early signalling events during the interaction of the diazotrophic soil bacterium Azospirillum brasilense with its host plant Arabidopsis thaliana. Furthermore, taking advantage of the micro array technique, a comprehensive overview of Arabidopsis genes has been undertaken which are affected upon association with A. brasilense The characterization of the early responses of Arabidopsis plants upon inoculation with Azospirillum brasilense strain Sp7 clearly indicated parallels with the initial events in plant pathogen interaction. For instance, not only bacterial preprations (lysates) form Azospirillum elicited an apoplastic alkalinization of the culture medium, but also the live bacteria, which were even more effective. Besides, in a luminol based assay, the bacterial lysates triggered production of the reactive oxygen species (ROS) in the Arabidopsis leaf discs. Interestingly, the elongation factor receptor mutants (efr) were completely insensitive to Azospirillum, suggesting elongation factor Tu (EF-TU) recognition as elicitor by Arabidopsis. This hypothesis was further validated with a bioinformatic approach. The N terminus initial 26 amino acids from Azospirillum EF-TU gene (elf26) showed more similarity to the elf26 sequences of bacteria like Agrobacterium tumefaciens which elicit responses in the plants through EF-TU rather than Pseudomonas syringae where the potent elicitor is flagellin 22. Universal transcriptome profiling of Arabidopsis thaliana seedlings upon inoculation with Azospirillum brasilense over a time course of six, twenty four and ninty six hours revealed very little genetic responses in the early time points. However, a bulk of genes was differentially regulated in 96 hours post inoculation (96hpi). The nature of these genes indicated that the bacterial treatment, among others, greatly affect the processes like cell wall modification, hormone metabolism, stress and secondary metabolism. Additionally expression levels of a numer of transcription factors (TFs) related to basic helix loop helix (BHLH) and MYB domain containing TF families were altered with Azospirillum inoculation. Particularly the BHLH TFs were among the most highly regulated genes. The array results from Azospirillum treated plants were further compared with the already available data emnating from treatment with flagellin 22 (flg22), oligogalacturonides (OGs) and Agrobacterium tumefaciens. Noteworthy, very different set of genes were affected upon inoculation with Azospirillum in relation to other treatments. Secondly a cluster of proteins involved in the biosynthesis of aliphatic glucosinolates (GSL) were uniquely induced upon Sp7 exposure. Genes operating in flavonoid biosynthesis also showed a distinct regulation trend in the comparative analysis. Taken together, the study in question provides insights into the early signalling events in the context of Azospirillum-Arabidopsis association and the bacterial signals recognized by the plants. The array data, at the same time, elucidates the genetic factors of Arabidopsis triggered upon association with Azospirillum brasilense. / Die vorliegende Arbeit befasst sich mit den physiologischen und genetischen Reaktionen im Zuge der Interaktion von Arabidopsis thaliana mit dem freilebenden, Stickstoff-fixierenden Bodenbakterium Azospirillum brasilense. Qualitativ konnten gemeinsame Mechanismen der frühen physiologischen Antworten von Arabidopsis auf Lysate von mutualistischen (Azospirillum brasilense) oder pathogenen (Pseudomonas syringae und Agrobacterium tumefaciens) Mikroorganismen festgestellt werden. So reagierten Arabidopsis (Col-0 ) Pflanzen auf Lysate dieser Bakterien mit einem Anstieg der cytosolischen Calcium-Konzentration sowie des extrazellulären pH Werts, mit der Bildung reaktiver Sauerstoffspezies und einer Depolarisierung des Membranpotentials. Diese Antworten untschieden sich jedoch zum Teil erheblich in ihrer Amplitude. Weitere Untersuchungen konnten zeigen, dass Flagellenproteine von Azospirillum nicht durch Arabidopsis erkannt werden. Somit unterscheidet sich der Erkennungsmechanismus der Azospirillen von dem der Pseudomonaden, welche aufgrund ihrer Flagellenproteine durch den FLAGELLINSENSING-2 (FLS2) Rezeptor in Arabidopsis perzipiert werden. Die Arabidopsis Mutante ELONGATIONFACTOR RECEPTOR (efr) war insensitiv gegenüber Azospirillumlysaten. Dies legte nahe, dass die Erkennung von Azosprillum über eine Erkennung des bakteriellen Elongationsfaktors (EF-Tu) durch den EFR Rezeptor verläuft. Die anschließende Klonierung des Azospirillum EF-Tu Gens zeigte positionspezifische Unterschiede in der abgeleiteten Aminosäuresequenz gegenüber Referenzsequenzen aus Escherichia coli oder Agrobacterium tumefaciens und erklärt somit die „imperfekte“ Erkennung durch den EFR Rezeptor. Der zeitliche Verlauf der genetischen Antwort von Arabidopsis im Zuge der Interaktion mit Azospirillum wurde mit Hilfe „Micro-Array“ basierter Transkriptionansanalysen 6, 24 und 96 Stunden nach Inokulation (hpi) der Pflanzen untersucht. Dabei wurden nach 6 und 24 hpi lediglich 30 bzw. 60 differenziell regulierte Transkripte gefunden. Diese Beobachtung steht im Gegensatz zu Studien pathogener Elizitoren wie Flagellinen, in welchen bereits nach wenigen Stunden mehr als eintausend differenziell regulierte Transkripte in Arabidopsis gefunden wurden. Dieser Effekt konnte in den Interaktionsstudien mit Azospirillum erst nach 96 hpi beobachtet werden. Die Analyse der genetischen Antwort ergab, dass 96 hpi insbesondere Gene in ihrer Expression verändert waren, deren Produkte im Zusammenhang mit Zellwandmodifikationen, dem Hormonmetabolismus, der Stressanpassung sowie der sekundären Metabolismus stehen. Darüber hinaus konnten Gene aus der Familie der sog. „basic-helix-loop-helix“ und „MYB“ Transkriptionsfaktoren identifiziert werden, die einer spezifischen Regulation durch Azospirillum unterlagen. Die vergleichende Analyse der Araydaten mit Datensätzen, die im Zuge von Pathogen-Arabidopsis Interaktionen gewonnen wurden zeigte, dass insbesondere die Biosynthese von aliphatischen Glykosiden und Flavonolen eine typische Antwort der Pflanze auf die mutualistischen Azospirillum Bakterien darstellt. Die vorgestellte Arbeit liefert somit erste Erkenntnisse zur physiologischen und genetischen Antwort von Arabidopsis auf Azospirillum und ermöglicht die vergleichende Betrachtung dieser Antworten im Kontext der Interaktion von Pflanzen mit pathogenen Mikroorganismen. Die im Rahmen dieser Arbeit identifizierten, differenziell regulierten Gene bieten neue Ansatzpunkte zum vertieften Studium der Wechselwirkung von mutualistischen, wachstumsfördernden Bakterien mit höheren Pflanzen.

No description available.

No description available.

Made available in DSpace on 2017-07-10T17:37:23Z (GMT). No. of bitstreams: 1 Jesica_F_S_Genero.pdf: 1260489 bytes, checksum: da1efddef812f7eb843220dbd5d18a24 (MD5) Previous issue date: 2016-08-02 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The bacteria Azospirillum brasilense and Herbaspirillum seropedicae are nitrogen fixers and promoting plant growth, able to promote partnerships with the trigo. O objective of this study was to evaluate the crop wheat agronomic performance in response to inoculation with Azospirillum brasilense diazotrophs and Herbaspirillum seropedicae with or without nitrogen fertilization and quantify the number of viable cells of bacteria of the wheat plant roots. The experiment was conducted in the crop year 2014/2014 and 2015/2015. The experimental design was a randomized block in factorial 2 x 4, with four repetitions. The first factor relates to the nitrogen topdressing applications (with and without nitrogen). The second factor relates to inoculating seeds with diazotrophs [without inoculation treatment (control), strain A. brasilense, H. seropedicae strain and combination of the two strains. The combined inoculation of strains A. brasilense + H. seropedicae with nitrogen fertilization promoted an increase of 48% and 13% in grain yield, in the 2014 season and 2015 season, respectively, for the plants that were not inoculated. You can check the inoculation Herbaspirillum, showed better total amount of viable cells with the application of nitrogen. The reduction in the number of CFU of Azospirillum under nitrogen fertilization condition can point to a lower competitiveness of these individuals by exudates and nutrients / As bactérias Azospirillum brasilense e Herbaspirillum seropedicae são fixadoras de nitrogênio e promotoras de crescimento vegetal, capazes de promover associações com a cultura do trigo. O objetivo deste trabalho foi avaliar o desempenho agronômico do trigo em resposta a inoculação de bactérias diazotróficas com Azospirillum brasilense e Herbaspirillum seropedicae associada ou não à adubação nitrogenada e quantificar o número de células viáveis de bactérias das raízes de plantas de trigo. O experimento foi realizado nas safras agrícolas 2014/2014 e 2015/2015. O delineamento experimental foi de blocos casualizados em esquema fatorial 2 x 4, com 4 repetições. O primeiro fator refere-se às aplicações de nitrogênio em cobertura (com e sem nitrogênio). O segundo fator refere-se à inoculação de sementes com bactérias diazotróficas [tratamento sem inoculação (testemunha), estirpe de A. brasilense, estirpe de H. seropedicae e combinação das duas estirpes. Na ausência da inoculação, a adubação nitrogenada em cobertura promoveu um acréscimo de 18% no diâmetro do colmo. Na ausência da adubação nitrogenada em cobertura, a inoculação com H. seropedicae promoveu um acréscimo de 16% no diâmetro de colmo, e na presença da adubação nitrogenada esta bactéria promoveu um acréscimo de 24% no número de espiguetas por espiga em relação às plantas que não foram inoculadas. A inoculação combinada das estirpes A. brasilense + H. seropedicae com adubação nitrogenada em cobertura promoveu um acréscimo de 26% no número de espiguetas por espiga e o dobro do número de grãos obtidos, em relação à combinação sem adubação. A inoculação combinada das estirpes A. brasilense + H. seropedicae com adubação nitrogenada em cobertura promoveu um acréscimo de 48% e 13% na produtividade de grãos, na safra de 2014 e na safra de 2015, respectivamente, em relação as plantas que não foram inoculadas. A inoculação de H. seropedicae apresentou maior quantidade de células viáveis totais com a aplicação de nitrogênio. A redução do número de UFC de A. brasilense sob condição de adubação nitrogenada pode apontar para uma menor competitividade destes indivíduos por exsudatos e nutrientes