Development of the Athabasca Oil Sands Region (AOSR) has increased atmospheric nitrogen emissions, a trend which is expected to increase in the future. The area surrounding development is comprised of Boreal upland forests and peatlands. Improved understanding of the hydrological connectivity between Boreal peatlands and uplands is needed to predict the fate and transport of atmospheric N deposited across the region. Two field sites: Jack Pine High (JPH, located 45 km north of Fort McMurray) and Mariana Lakes (ML, located 100 km south of Fort McMurray) were instrumented with piezometers nests and water table wells for this study (n= 108 sampling locations). The wells were placed along transects that cover target landscape units (bog, fen, upland). Wells were sampled for water isotopes and geochemical parameters during the summers of 2011-2014 to characterize the baseline geochemistry of groundwater in the different landscape units. Inorganic (nitrate, ammonium) and organic forms of nitrogen (dissolved organic nitrogen), major and minor ions and water isotope tracers (18O, 2H and 3H) were measured to identify the various forms of nitrogen in the different landscape units, as well as to assess connectivity and potential for nitrogen transport between the different units. At JPH surface and groundwater flow is from the uplands to the fen. There was little (<0.1-1.5 mg/L) nitrate, ammonium, or dissolved organic nitrate (DON) found throughout JPH. At ML nitrogen concentrations were higher (<0.1-30 mg/l) and concentrations of ammonium and DON increased at depths throughout ML. The distribution of 3H with depth within the peatland reveals limited connectivity between the peat and underlying mineral soils. Tritium sampling at ML indicates that at some locations the wetland residence time is greater than 50 years. Nitrogen movement out of peatlands may take longer due to conversions and storage. At ML nitrogen (NH4 and DON) is produced and stored at depth in the wetlands. At JPH higher nitrogen concentrations are found in the shallow groundwater of the fen. Increases in nitrogen inputs to JPH and ML are likely to be utilized by plants, but dramatic changes to the peatland may cause stored nitrogen to become mobile. / Graduate

Nitrate assimilation is an extremely important part of the nitrogen cycle and is carried out by most bacteria, fungi and plants. A relatively short catalytic pathway reduces nitrate to nitrite (via nitrate reductase activity) and then nitrite to ammonium ions (via nitrite reductase activity) which are converted into organic nitrogen by further metabolic pathways. A considerable amount of information is known about the biochemistry, genetics and recently, the molecular biology of these two enzymes. Much less is known about the transport of nitrate and nitrite into cells as well as the synthesis of the molybdenum cofactor needed for nitrate reductase catalytic activity. Research work reported in this thesis focus on these latter two processes in the eukaryotic model organisms. Aspergillus nidulans and to a lesser extent Arahidopsis thaliana. Genetic characterisation of 47 crn mutants shows that there are three additional genes (i.e. to the original identified crnA gene) likely to be involved in nitrate transport. These additional genes are unlinked to each other or to crnA. Although it was shown that the nitrate uptake into cells of these mutants are lower than the wild-type, their exact involvement in nitrate transport requires their molecular cloning. Certain mutations generated in the crnA gene have been investigated at the molecular level and the disruptions in the protein determined. During the genetic studies of crn mutants, two other genes were postulated. The first is chlA, mutation which results in resistance to chlorate (unlike the wild-type) and caesium (like the wild-type). The second is cesA mutation. These latter mutants lead to caesium sensitivity but are chlorate sensitive like the wild-type. These two genes are unlinked to crnA, crnB, crnC and crnD genes. The bases of these phenotypes is unclear and need further investigation. A study of nitrite uptake was undertaken which showed that wild-type A. nidulans has an active nitrite transport system. The activity of this system is repressed by ammonium and is nitrate induced. Mutants which are hypersensitive to chlorate taken up much higher levels of nitrite as compared to wild-type. 2,082 cnx mutants were isolated and 456 of these were classified as cnxA, cnxB, cnxC, cnxE, cnxF, cnxG and cnxH mutants on the basis of phenotypic complementation. No novel cnx genes were found. More importantly a number of temperature -conditional mutants were isolated, 10 mutants were found to be temperature-sensitives and 10 cryo-sensitives. Of the isolated temperature-sensitives 1 located in cnxA, 1 in cnxB, 2 in cnxC, 1 cnxE, 2 in cnxF and 3 in cnxH. Of the crysosensitives 4 in cnxB, 3 in cnxC and 3 in cnxF. These mutants will be particularly useful to relate structure and function when data is forthcoming regarding their protein sequence. Two temperature-sensitive mutants, cnxH255 and cnxH261 showed reduced nitrate reductase thermostability which indicates that the cnxH product could be associated with the NR protein. One of the Aspergillus nidulans genes required for the synthesis of the molybdenum cofactor was isolated using molecular self-cloning transformation approaches. This gene, cnxH, was sequenced at the nucleotide level as well as three mutant alleles (one temperature sensitive and two temperature non-conditional). The results show that the cnxH product is the homologue of Escherichia, coli moaE whose role is in the synthesis of the molybdenum cofactor specifically to convert the large subunit to active converting factor. Sequence analysis of the two non-conditional mutants indicates that such mutants generated stop codons which provides little or no information about the structure / function relationships. The mutation in the temperature-sensitive mutant lead to a glycine insertion at position +443 and it is postulated that this additional amino acid caused the heat liability of the NR enzyme. Studies of cnxH expression show that the cnxH is in very low abundance and not regulated at the transcriptional level at least since similar transcript levels were seen in both nitrate and ammonium grown cells- conditions, which making difference for nitrate reductase activity. Finally attempts at isolating Arabidopsis thaliana cnx genes failed.

Nitrite reduction is the third step of the nitrate assimilation pathway in higher plants and is catalysed by nitrite reductase. The whole-plant barley mutants STA1010, STA2760 and STA4169 accumulate nitrite in the leaf after treatment with nitrate and, like the nir1 mutant STA3999 (Duncanson et al, 1993), lack detectable nitrite reductase cross-reacting material in the leaf and root. STA1010, STA2760 and STA4169 carry a recessive mutation in a single nuclear gene, identified as the Nir1 locus. RFLP analysis of the nir1 mutant STA3999 has allowed the Nir1 locus to be mapped to within 0.3cM of the nitrite reductase apoprotein gene, Nii. Studies to confirm the identity of the Nir1 locus as Nii, by establishing the full-length Nii cDNA sequences from STA3999 and from its wild-type cv Tweed for comparative purposes, were unsuccessful as attempts to isolate a Nii cDNA clone from a barley cv Tweed cDNA library yielded only partial-length Nii clones. These nirl mutants display greatly reduced nitrite reductase activity and increased NADH-nitrate reductase activity in the leaf, as compared to wild-type plants, suggesting a regulatory perturbation in the expression of the Nar1 gene. Northern analysis shows that the nir1 mutants possess nitrite reductase apoprotein (nii) transcript of wild-type size (2.3kb) and at approximately wild-type levels. Since nir1 mutants possess a phenotype that might be anticipated for a Nii mutant, it is likely that the nir1 mutation is present in the nitrite reductase apoprotein gene Nii and affects translation of the nii transcript. Studies of barley wild-type cv Golden Promise have demonstrated that nitrite reductase in leaf tissue is up-regulated by a coaction of nitrate and light which acts, at least partly, at the transcriptional level.

O nitrogênio é um elemento essencial à vida na Terra. Em geral, a disponibilização desse elemento para os seres vivos se dá por meio da fixação biológica do nitrogênio (FBN). Os micro-organismos capazes de realizar a FBN são denominados de diazotróficos e contêm o complexo enzimático da nitrogenase. Por ser um processo extremamente dispendioso, a FBN é regulada, principalmente, em nível transcricional, em resposta à quantidade de nitrogênio fixado e aos níveis de oxigênio. Os mecanismos de regulação do processo em bactérias Gram-negativas estão bem caracterizados, porém, em bactérias Gram-positivas, os estudos ainda são escassos. Paenibacillus riograndensis é uma bactéria Gram-positiva diazotrófica aeróbia facultativa e formadora de esporos, cujo sequenciamento completo do genoma a capacita como um interessante modelo para o estudo da regulação da FBN. No genoma de P. riograndensis foram identificados três agrupamentos contendo genes relacionados à FBN. Um deles, com uma organização estrutural menos conservada, foi considerado inativo a partir de análises de PCR em tempo real e de atividade de promotor. Os outros dois tiveram seus transcritos identificados e induzidos sob condições de fixação de nitrogênio, sendo um deles responsável pela codificação de um sistema alternativo da nitrogenase, independente de molibdênio. Esse sistema alternativo foi identificado como sendo aquele composto apenas por ferro e validado tanto pela análise das sequências dos genes estruturais, como pela atividade enzimática em meio sem molibdênio. Sequências localizadas a aproximadamente 250 pares de bases (pb) a montante do início da tradução dos primeiros genes dos dois agrupamentos funcionais também tiveram suas atividades como regiões reguladoras validadas pelo reconhecimento em Escherichia coli, com um provável padrão de iniciação da transcrição constitutivo. Uma menor atividade de transcrição foi observada no fragmento de 500 pb localizado a montante do agrupamento dos genes da nitrogenase alternativa, indicando a presença de regiões contendo motivos de regulação negativa do processo. Investigações mais detalhadas dessas sequências podem revelar padrões inéditos para a regulação da FBN em bactérias Gram-positivas, em geral, e em P. riograndensis, em particular. / Nitrogen is an essential element for life. In general, it becomes available to biosphere mainly through biological nitrogen fixation (BNF). Microorganisms named diazotrophs perform BNF and they have the nitrogenase enzyme. As BNF is a very energetic expensive process, it is tightly regulated mainly at transcriptional level in response to available nitrogen and oxygen levels. Regulatory networks comprising BNF systems in Gramnegative bacteria are well characterized, while studies related to Gram-positive bacteria are scarce. Paenibacillus riograndensis is a Gram-positive endospore-forming facultative anaerobic diazotroph, whose complete genome sequence presents it as an interesting model for the study of BNF regulation. In P. riograndensis genome three cluster comprising BNF related genes were identified. One of them, displaying a less conserved structural organization, was stated as inactive from real time PCR and promoter activity analysis. The other ones had their transcripts identified and responded to nitrogen fixation conditions. One of the active clusters comprises genes coding for an alternative nitrogenase system independent of molybdenum, the iron-only system. This alternative system was validated by enzymatic activity under Mo-depleted conditions. Sequences 250 base pairs (bp) upstream from the first open reading frame (ORF) of each active cluster had their promoter activities validated by recognizing in Escherichia coli, showing a probable constitutive expression pattern. A weaker promoter activity was identified in a fragment 500 bp upstream of the first ORF from the alternative cluster, suggesting the presence of a negative regulation motif. Future investigations may provide us with new patterns of BNF regulation in Gram-positive bacteria, in general, and in P. riograndensis in particular.

Benzene and its derivatives are ubiquitous in chemical research, with applications ranging from material science to biomedical research. 1,2-Dihydro-1,2-azaborine is a benzene mimic which replaces a CC bond with a BN bond. The basic science and applications of 1,2-azaborines is relatively underdeveloped. This thesis expands the fundamental understanding of 1,2-azaborines. Chapter I describes the air and moisture stability of 1,2-azaborines. Chapter II introduces nucleophilic aromatic substitution reactions that the parent 1,2-dihydro-1,2-azaborine will undergo. Chapter III discusses a trimerization reaction that 1,2-dihydro-1,2-azaborine can perform, which is unique from benzene. Chapter IV examines a novel protection free synthesis of 1,2-azaborines, which provides a more direct route to functionalized 1,2-azaborines. Chapter V discusses the novel deprotection of the N-silicon using an amide, giving one of the first 1,2-azaborine pharmaceutical mimics. Finally, chapter VI summarized miscellaneous contributions I have made to the basic science of 1,2-azaborines. This dissertation includes previously published and unpublished co-authored material. / 10000-01-01

O nitrogênio é um elemento essencial à vida na Terra. Em geral, a disponibilização desse elemento para os seres vivos se dá por meio da fixação biológica do nitrogênio (FBN). Os micro-organismos capazes de realizar a FBN são denominados de diazotróficos e contêm o complexo enzimático da nitrogenase. Por ser um processo extremamente dispendioso, a FBN é regulada, principalmente em nível transcricional, em resposta à quantidade de nitrogênio fixado e aos níveis de oxigênio. Os mecanismos de regulação do processo em bactérias Gram-negativas estão bem caracterizados, porém, em bactérias Gram-positivas, os estudos ainda são escassos. Paenibacillus riograndensis SBR5T é uma bactéria Gram-positiva diazotrófica aeróbia facultativa e formadora de esporos, interessante modelo para o estudo da regulação da FBN com o genoma completo disponível. O fator de transcrição GlnR foi proposto como regulador dos genes nif em Paenibacillus spp. a partir de análises in silico. O presente trabalho identificou sítios de ligação de GlnR em promotores de genes envolvido com a FBN e validou o papel de GlnR como regulador negativo dos genes nif em P. riograndensis. Também foi demonstrado que a enzima glutamina sintetase interage com GlnR quando está inibida por feedback e estabiliza a ligação de GlnR às regiões reguladoras dos genes nif. Um modelo de repressão baseado em operadores múltiplos foi proposto integrando a regulação da FBN à regulação global do nitrogênio exercida por GlnR. Na tentativa de identificar elementos específicos relacionados à regulação do molibdênio (componente do cofator enzimático) e da nitrogenase alternativa (com cofator independente de molibdênio), foi acessado o perfil transcricional de P. riograndensis sob condições de limitação de nitrogênio e molibdênio. Alguns fatores de transcrição especificamente induzidos sob tais condições e provavelmente relacionados à homeostase de metais foram identificados. Com relação à glutamina sintetase, além da demonstração da interação entre a enzima e o fator de transcrição GlnR, duas proteínas adicionais homólogas de glutamina sintetase e que não participam dessa regulação foram identificadas codificadas no genoma. Uma delas foi caracterizada como glutamina sintetase funcional, enquanto a outra não apresentou atividade bioquímica. Além disso, um protocolo para transformação da linhagem em estudo foi estabelecido e otimizado, o que permitirá aprofundar os estudos de genética molecular tanto da FBN como de outros processos de interesse em P. riograndensis. / Nitrogen is an essential element for life. In general, it becomes available to biosphere mainly through biological nitrogen fixation (BNF). Microorganisms named diazotrophs perform BNF and they have the nitrogenase enzyme. As BNF is a very energetic expensive process, it is tightly regulated mainly at transcriptional level in response to available nitrogen and oxygen levels. Regulatory networks comprising BNF systems in Gram-negative bacteria are well characterized, while studies related to Gram-positive bacteria are scarce. Paenibacillus riograndensis SBR5T is a Grampositive endospore-forming facultative anaerobic diazotroph, whose complete genome sequence presents it as an interesting model for the study of BNF regulation. The transcription factor GlnR has been proposed as the nif regulator in Paenibacillus spp. based on in silico analysis. The present work identified GlnR-binding sites at BNFrelated promoters and validated GlnR role as nif negative regulator in P. riograndensis. It was also demonstrated that the feedback-inhibited glutamine synthetase enzyme interacts with GlnR and stabilizes its binding activity in the nif genes promoters. A multiple operator model was proposed to integrate BNF regulation and the global nitrogen regulation coordinated by GlnR. In an effort to identify specific regulatory elements related to molybdenum (enzymatic cofactor component) and the alternative nitrogenase (which presents a molybdenum-independent cofactor), the transcriptional profile of P. riograndensis was accessed under nitrogen and molybdenum limiting conditions. Transcription factors specifically induced under such conditions and probably related to metal homeostasis were identified. Regarding the glutamine synthetase, two additional glutamine synthetase homologs that do not take part in GlnR regulation were identified. One of them was characterized as a functional glutamine synthetase, while the other did not display biochemical activity. Also, a protocol to transform the model in study was established and optimized. This protocol allows the development of further molecular research to unveil BNF and other interesting processes in P. riograndensis.

The aim of this study was to assess amount of available nitrogen and phosphorous in forest soils using the ion exchange resin (IER) bag method in watersheds of Plešné and Čertovo lake from 2003 to 2012. Watersheds were exposed to acidic deposition for long time. The results indicate higher amount of total available nitrogen and phosphorous in soils from the watershed of Plešné lake because the forest there has been infected with bark beetle (Ips typographus) and it influences transformations through the whole ecosystem.

Submitted by Alexia Morello da Silva Cascaldi null (alexia.morello@yahoo.com.br) on 2017-09-19T05:33:05Z No. of bitstreams: 1 Dissertação_Alexia_Morello_da_Silva_Cascaldi.pdf: 844332 bytes, checksum: 98e363c052f3354ad72b45c70bf2f3bd (MD5) / Approved for entry into archive by Monique Sasaki (sayumi_sasaki@hotmail.com) on 2017-09-19T20:48:59Z (GMT) No. of bitstreams: 1 cascaldi_ams_me_jabo.pdf: 844332 bytes, checksum: 98e363c052f3354ad72b45c70bf2f3bd (MD5) / Made available in DSpace on 2017-09-19T20:48:59Z (GMT). No. of bitstreams: 1 cascaldi_ams_me_jabo.pdf: 844332 bytes, checksum: 98e363c052f3354ad72b45c70bf2f3bd (MD5) Previous issue date: 2017-07-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O nitrogênio é um nutriente essencial para as culturas, entretanto seu aproveitamento está sujeito a muitas perdas no sistema solo-planta-atmosfera. Alguns compostos têm sido utilizados com o propósito de reduzir essas perdas, aumentando a eficiência dos fertilizantes nitrogenados. O objetivo deste trabalho foi avaliar o efeito da utilização do estabilizante de ureia NZone Max, em relação às perdas por volatilização de amônia (NH3) e lixiviação de nitrato (NO3-), em cultivo de Brachiaria brizantha cv. Marandu, adubada com ureia, sob quatro lâminas de irrigação. Os tratamentos consistiram das lâminas de irrigação correspondentes a 0,20; 0,40; 0,60 e 0,80 da evapotranspiração de referência, com adição ou não de estabilizante de ureia, com aplicação de 100 kg ha-1 de N. O delineamento experimental foi em faixa com parcela subdividida, com lâminas na parcela e estabilizante na subparcela, com 4 repetições. A coleta da amônia volatilizada foi feita com câmaras coletoras instaladas em cada parcela, para posterior quantificação em laboratório. Foram também coletadas amostras da solução do solo para análise química. Os dados foram submetidos à análise de variância pelo teste F e a comparação de médias foi feita pelo teste Tukey (5%). A quantidade de amônia volatilizada foi crescente até o terceiro dia após a adubação, quando atingiu os maiores valores, diminuindo a partir de então, tanto na presença quanto na ausência do estabilizante de ureia, para as 4 lâminas de irrigação. A menor lâmina apresentou a maior quantidade de amônia volatilizada, enquanto que a maior lâmina foi a de menor volatilização. Não houve diferença entre os tratamentos com e sem o uso do estabilizante, tanto para a quantidade de amônia volatilizada, quanto para os valores de nitrato, amônio, N-total, pH e condutividade elétrica da solução do solo. Portanto, a adição do estabilizante NZone Max à ureia não promoveu redução das perdas por volatilização de amônia e lixiviação de nitrato. / Nitrogen is an essential nutrient for crops, however its use is subject to many losses in the soil-plant-atmosphere system. Some compounds have been used for the purpose of reducing these losses, increasing the efficiency of nitrogen fertilizers. The objective of this work was to evaluate the effect of NZone Max urea stabilizer in relation to losses by ammonia (NH3) volatilization and nitrate (NO3-) leaching in a Brachiaria brizantha cv. Marandu crop, fertilized with urea, under four irrigation depths. The treatments consisted of irrigation depths corresponding to 0.20, 0.40, 0.60 and 0.80 of the reference evapotranspiration, with and without urea stabilizer, with application of 100 kg ha-1 of N. The treatments were arranged in a split-plot strip design, with irrigation depths in the plot and stabilizer in the subplot, with 4 replications. The volatilized ammonia was collected with chambers installed in each plot, for subsequent quantification in the laboratory. Soil solution samples were also collected for chemical analysis. The data were submitted to analysis of variance by the F test and the means were compared by the Tukey test (5%). The amount of volatilized ammonia increased until the third day after fertilization, when it reached the highest values, decreasing thereafter, both in the presence and absence of the urea stabilizer, for the 4 irrigation depths. The lower irrigation depth presented a higher amount of volatilized ammonia, while the higher depth was lower volatilization. There was no difference between the treatments with or without stabilizer, for the amount of ammonia volatilized, as well as for the nitrate, ammonium, N-total, pH and electrical conductivity of the soil solution. Thus, the addition of the NZone Max stabilizer to the urea did not promote reduction of the losses by ammonia volatilization and nitrate leaching.

O nitrogênio é um elemento essencial à vida na Terra. Em geral, a disponibilização desse elemento para os seres vivos se dá por meio da fixação biológica do nitrogênio (FBN). Os micro-organismos capazes de realizar a FBN são denominados de diazotróficos e contêm o complexo enzimático da nitrogenase. Por ser um processo extremamente dispendioso, a FBN é regulada, principalmente em nível transcricional, em resposta à quantidade de nitrogênio fixado e aos níveis de oxigênio. Os mecanismos de regulação do processo em bactérias Gram-negativas estão bem caracterizados, porém, em bactérias Gram-positivas, os estudos ainda são escassos. Paenibacillus riograndensis SBR5T é uma bactéria Gram-positiva diazotrófica aeróbia facultativa e formadora de esporos, interessante modelo para o estudo da regulação da FBN com o genoma completo disponível. O fator de transcrição GlnR foi proposto como regulador dos genes nif em Paenibacillus spp. a partir de análises in silico. O presente trabalho identificou sítios de ligação de GlnR em promotores de genes envolvido com a FBN e validou o papel de GlnR como regulador negativo dos genes nif em P. riograndensis. Também foi demonstrado que a enzima glutamina sintetase interage com GlnR quando está inibida por feedback e estabiliza a ligação de GlnR às regiões reguladoras dos genes nif. Um modelo de repressão baseado em operadores múltiplos foi proposto integrando a regulação da FBN à regulação global do nitrogênio exercida por GlnR. Na tentativa de identificar elementos específicos relacionados à regulação do molibdênio (componente do cofator enzimático) e da nitrogenase alternativa (com cofator independente de molibdênio), foi acessado o perfil transcricional de P. riograndensis sob condições de limitação de nitrogênio e molibdênio. Alguns fatores de transcrição especificamente induzidos sob tais condições e provavelmente relacionados à homeostase de metais foram identificados. Com relação à glutamina sintetase, além da demonstração da interação entre a enzima e o fator de transcrição GlnR, duas proteínas adicionais homólogas de glutamina sintetase e que não participam dessa regulação foram identificadas codificadas no genoma. Uma delas foi caracterizada como glutamina sintetase funcional, enquanto a outra não apresentou atividade bioquímica. Além disso, um protocolo para transformação da linhagem em estudo foi estabelecido e otimizado, o que permitirá aprofundar os estudos de genética molecular tanto da FBN como de outros processos de interesse em P. riograndensis. / Nitrogen is an essential element for life. In general, it becomes available to biosphere mainly through biological nitrogen fixation (BNF). Microorganisms named diazotrophs perform BNF and they have the nitrogenase enzyme. As BNF is a very energetic expensive process, it is tightly regulated mainly at transcriptional level in response to available nitrogen and oxygen levels. Regulatory networks comprising BNF systems in Gram-negative bacteria are well characterized, while studies related to Gram-positive bacteria are scarce. Paenibacillus riograndensis SBR5T is a Grampositive endospore-forming facultative anaerobic diazotroph, whose complete genome sequence presents it as an interesting model for the study of BNF regulation. The transcription factor GlnR has been proposed as the nif regulator in Paenibacillus spp. based on in silico analysis. The present work identified GlnR-binding sites at BNFrelated promoters and validated GlnR role as nif negative regulator in P. riograndensis. It was also demonstrated that the feedback-inhibited glutamine synthetase enzyme interacts with GlnR and stabilizes its binding activity in the nif genes promoters. A multiple operator model was proposed to integrate BNF regulation and the global nitrogen regulation coordinated by GlnR. In an effort to identify specific regulatory elements related to molybdenum (enzymatic cofactor component) and the alternative nitrogenase (which presents a molybdenum-independent cofactor), the transcriptional profile of P. riograndensis was accessed under nitrogen and molybdenum limiting conditions. Transcription factors specifically induced under such conditions and probably related to metal homeostasis were identified. Regarding the glutamine synthetase, two additional glutamine synthetase homologs that do not take part in GlnR regulation were identified. One of them was characterized as a functional glutamine synthetase, while the other did not display biochemical activity. Also, a protocol to transform the model in study was established and optimized. This protocol allows the development of further molecular research to unveil BNF and other interesting processes in P. riograndensis.

O nitrogênio é um elemento essencial à vida na Terra. Em geral, a disponibilização desse elemento para os seres vivos se dá por meio da fixação biológica do nitrogênio (FBN). Os micro-organismos capazes de realizar a FBN são denominados de diazotróficos e contêm o complexo enzimático da nitrogenase. Por ser um processo extremamente dispendioso, a FBN é regulada, principalmente, em nível transcricional, em resposta à quantidade de nitrogênio fixado e aos níveis de oxigênio. Os mecanismos de regulação do processo em bactérias Gram-negativas estão bem caracterizados, porém, em bactérias Gram-positivas, os estudos ainda são escassos. Paenibacillus riograndensis é uma bactéria Gram-positiva diazotrófica aeróbia facultativa e formadora de esporos, cujo sequenciamento completo do genoma a capacita como um interessante modelo para o estudo da regulação da FBN. No genoma de P. riograndensis foram identificados três agrupamentos contendo genes relacionados à FBN. Um deles, com uma organização estrutural menos conservada, foi considerado inativo a partir de análises de PCR em tempo real e de atividade de promotor. Os outros dois tiveram seus transcritos identificados e induzidos sob condições de fixação de nitrogênio, sendo um deles responsável pela codificação de um sistema alternativo da nitrogenase, independente de molibdênio. Esse sistema alternativo foi identificado como sendo aquele composto apenas por ferro e validado tanto pela análise das sequências dos genes estruturais, como pela atividade enzimática em meio sem molibdênio. Sequências localizadas a aproximadamente 250 pares de bases (pb) a montante do início da tradução dos primeiros genes dos dois agrupamentos funcionais também tiveram suas atividades como regiões reguladoras validadas pelo reconhecimento em Escherichia coli, com um provável padrão de iniciação da transcrição constitutivo. Uma menor atividade de transcrição foi observada no fragmento de 500 pb localizado a montante do agrupamento dos genes da nitrogenase alternativa, indicando a presença de regiões contendo motivos de regulação negativa do processo. Investigações mais detalhadas dessas sequências podem revelar padrões inéditos para a regulação da FBN em bactérias Gram-positivas, em geral, e em P. riograndensis, em particular. / Nitrogen is an essential element for life. In general, it becomes available to biosphere mainly through biological nitrogen fixation (BNF). Microorganisms named diazotrophs perform BNF and they have the nitrogenase enzyme. As BNF is a very energetic expensive process, it is tightly regulated mainly at transcriptional level in response to available nitrogen and oxygen levels. Regulatory networks comprising BNF systems in Gramnegative bacteria are well characterized, while studies related to Gram-positive bacteria are scarce. Paenibacillus riograndensis is a Gram-positive endospore-forming facultative anaerobic diazotroph, whose complete genome sequence presents it as an interesting model for the study of BNF regulation. In P. riograndensis genome three cluster comprising BNF related genes were identified. One of them, displaying a less conserved structural organization, was stated as inactive from real time PCR and promoter activity analysis. The other ones had their transcripts identified and responded to nitrogen fixation conditions. One of the active clusters comprises genes coding for an alternative nitrogenase system independent of molybdenum, the iron-only system. This alternative system was validated by enzymatic activity under Mo-depleted conditions. Sequences 250 base pairs (bp) upstream from the first open reading frame (ORF) of each active cluster had their promoter activities validated by recognizing in Escherichia coli, showing a probable constitutive expression pattern. A weaker promoter activity was identified in a fragment 500 bp upstream of the first ORF from the alternative cluster, suggesting the presence of a negative regulation motif. Future investigations may provide us with new patterns of BNF regulation in Gram-positive bacteria, in general, and in P. riograndensis in particular.