Cianobactérias são micro-organismos que realizam fotossíntese oxigênica e têm uma distribuição cosmopolita. Algumas cianobactérias são capazes de realizar fotossíntese e fixação biológica de nitrogênio (FBN), dois dos mais importantes processos na natureza, simultaneamente. As cianobactérias da ordem Nostocales são capazes realizar uma separação espacial destes dois processos por meio da formação de células especializadas, os heterócitos, onde acontece a fixação de nitrogênio, restringindo a fotossíntese às células vegetativas. Embora tenham importância ecológica, econômica e social, as cianobactérias foram muito pouco estudas com enfoque genômico. Este trabalho teve como objetivo a caracterização dos agrupamentos gênicos envolvidos na fixação biológica de nitrogênio e na diferenciação de heterócitos de três isolados brasileiros de cianobactérias da ordem Nostocales. Para este fim, culturas das linhagens Sphaerospermopsis torquesreginae ITEP-024, Nostoc sp. CENA67 e Fischerella sp. CENA161 foram sequenciadas com a plataforma MiSeq e então foi realizada a montagem ab initio do genoma com as leituras obtidas. Além desses três isolados, os genomas de outras 31 linhagens disponíveis no banco de dados GenBank foram recuperados e utilizados para comparação. A anotação dos genes foi realizada por meio do alinhamento de sequências nucleotídicas já conhecidas de outras linhagens contra os genomas dessas linhagens, utilizando a ferramenta BLASTn, e por meio do servidor antiSMASH. Além disso, análises filogenéticas foram realizadas a partir dos genes anotados. O sequenciamento dos genomas das três linhagens apresentou altos valores de qualidade de bases e elevada cobertura genômica. A anotação dos agrupamentos envolvidos na FBN revelou a presença de um total de 22 genes envolvidos na síntese da Mo-nitrogenase, sendo 19 presentes em todas as linhagens. Os isolados brasileiros apresentaram sintenia com outras linhagens próximas filogeneticamente, apresentando variação apenas na presença de regiões de excisão e do gene glbN. A linhagem CENA161 apresentou um agrupamento gênico completo para síntese da V-nitrogenase, assim como apenas outras 5 linhagens em toda a ordem. Foram encontradas nas três linhagens sequenciadas os genes devACB, relacionados à formação da camada polissacarídica do heterócito. Os genes hglEGDCA e hetM, que estão relacionados à formação da camada glicolipídica de heterócitos, foram encontrados completos nas linhagens ITEP-024 e CENA67, mas apenas parcialmente na CENA161. Genes reguladores do processo de diferenciação também foram acessados nas três linhagens brasileiras, entretanto apenas os reguladores globais do processo formam encontrados em CENA161. As análises filogenéticas mostraram que o gene nifH não reflete a filogenia do táxon e não é um bom marcador filogenético. Entretanto, a análise de todo o agrupamento refletiu o padrão filogenético de acordo com a taxonomia. Os resultados contribuem para a melhor compreensão dos aspectos genéticos e evolutivos do processo de FBN em cianobactérias. / Cyanobacteria are oxygenic photosynthetic microorganisms that have a worldwide distribution. Some cyanobacteria are capable of photosynthesis and biological nitrogen fixation (BNF), two of the most important processes in nature, simultaneously. Cyanobacteria from the Nostocales order perform a spatial separation of these processes through the formation of specialized cells, heterocytes, where nitrogen fixation is carried out, while photosynthesis is limited to vegetative cells. Although cyanobacterial have ecological, economic and social importance, they have been understudied with genomic approaches. This study aimed to characterize gene clusters involved in nitrogen fixation and heterocytes differentiation from three Brazilian strains of cyanobacteria from Nostocales order. For this purpose, nonaxenic cultures of the strains Sphaerospermopsis torque-reginae ITEP-024, Nostoc sp. CENA67 and Fischerella sp. CENA161 were sequenced with the Illumina MiSeq platform and ab initio genome assembly was performed. In addition to these strains, genomes from 31 strains available in the GenBank database were retrieved and used for comparison. Gene annotation was performed through alignments between known genes present in other cyanobacteria and the strains genomes, using the BLASTn tool, and through the antiSMASH server. Furthermore, phylogenetic analyses were performed on the annotated genes. The genome sequencing showed high bases quality values and genomic coverage. The annotation of clusters involved in the BNF revealed the presence of a total of 22 genes involved in the synthesis of Mo-nitrogenase, among 19 were present in all strains. Brazilian isolates showed synteny with phylogenetically related strains, with variations only in the presence of excision regions and glbN gene. The CENA161 strain showed a complete gene cluster for synthesis of V-nitrogenase, present in only 5 other strains in this order. devACB genes, related to the heterocyte polysaccharide layer formation, were found in the three strains sequenced. The hglEGDCA and hetM genes, related to the formation of heterocyte glycolipid layer, were complete in ITEP-024 and CENA67 strains, but only partial in CENA161. Gene regulation of the heterocyte differentiation process have also been accessed in the three Brazilian strains, but only the general process regulatory genes were found in CENA161. Phylogenetic analysis showed that the gene nifH does not reflect the phylogeny of this group and should not be considered a good phylogenetic marker. However, the complete genes cluster analysis reflected the patterns of phylogenetic grouping according to taxonomy. The results contribute to a better understanding of the genetic and evolutionary aspects of the BNF process in cyanobacteria.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1958. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Bibliography: leaves 50-54.

Orientadora : Profª Drª Emanuel M. de Souza / Coorientador : Prof. Dr. Vivian Rotuno Moure / Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Ciências : Bioquímica. Defesa: Curitiba, 25/05/2017 / Inclui referências : f. 86-97 / Resumo: A fixação biológica de nitrogênio é catalisada pelo complexo enzimático nitrogenase. Este complexo contém duas metaloproteínas: as proteínas Fe e MoFe. Em Azospirillum brasilense, objeto deste estudo, a regulação pós-traducional envolve a modificação covalente de uma das subunidades da proteína Fe por ADP-ribosilação. Esta ADPribosilação inativa reversivelmente a proteína Fe e é catalisada pela dinitrogenase redutase ADP-ribosiltransferase (DraT) em resposta ao aumento de íons amônio ou depleção da energia celular. O grupo ADP-ribosil é removido pela dinitrogenase redutase glicohidrolase (DraG), promovendo reativação da proteína Fe e consequentemente, da nitrogenase. Em resposta ao aumento de íons amônio, as proteínas PII, assim como a proteína de membrana AmtB, estão envolvidas no sistema de regulação atuando principalmente por interação direta com proteínas alvo. No caso de A. brasilense, as proteínas PII são denominadas de GlnB e GlnZ e interagem com DraT e DraG, respectivamente, regulando suas atividades. Porém o mecanismo pelo qual estas enzimas são reguladas pelos níveis energéticos celulares não é totalmente conhecido. O objetivo deste trabalho foi contribuir para elucidação do mecanismo da regulação póstraducional da nitrogenase em condições de anaerobiose. Vários aspectos da regulação do metabolismo energético nesta bactéria foram estudados. Para avaliar o envolvimento de AmtB e GlnZ de A. brasilense na regulação da nitrogenase, foi realizado um ensaio de desligamento / religamento da nitrogenase por anaerobiose em mutantes nos respectivos genes. Os resultados mostraram que as proteínas GlnZ e AmtB não participam diretamente da regulação por ADP-ribosilação da nitrogenase. Análises de LC-MS dos níveis de ATP e ADP indicaram redução substancial quando a cultura foi exposta à anaerobiose. Além disso, a adição do desacoplador CCCP, que diminui o ATP intracelular, causou desligamento e ADP-ribosilação da nitrogenase. Portanto, razão ATP/ADP pode estar relacionada ao mecanismo de regulação da atividade da nitrogenase nessas condições. Análise das frações celulares mostrou que, diferentemente da regulação por íons amônio, as proteínas PII não vão para a membrana durante a ADP-ribosilação da nitrogenase. O mesmo ocorre com DraG que permanece no citoplasma. Os resultados sugerem a existência de diferentes vias para o controle da atividade da nitrogenase, que depende do estímulo ao qual as células são submetidas. Palavras-chave: Azospirillum brasilense, regulação pós-traducional, ADP-ribosilação da proteína Fe, sistema DraT/DraG, anaerobiose. / Abstract: The biological nitrogen fixation is catalyzed by a nitrogenase enzymatic complex. This complex contains two metalloproteins: the Fe and MoFe proteins and undergoes to post-translational modification. In Azospirillum brasilense, object of this study, this posttranslational regulation involves the covalent modification in one of the subunits of Fe protein by ADP-ribosylation. This ADP-ribosylation reversibly inactivates the Fe protein and is catalyzed by dinitrogenase reductase ADP-ribosyltransferase (DraT) in response to an increase of ammonium concentration or decrease of cellular energy. The ADP-ribosyl group is removed by dinitrogenase reductase glycohydrolase (DraG), promoting Fe protein and, consequently, nitrogenase re-activation. In response to increased ammonium ions, the PII proteins, as well as the NH3 - channel AmtB, are involved in the regulatory system acting primarily by direct interaction with the target proteins. In A. brasilense, the PII proteins are denominated GlnB and GlnZ and interact with DraT and DraG, respectively, regulating DraT and DraG activities. However, the mechanism by which these enzymes are regulated by cellular energy levels is not fully known. The goal of this study was to contribute to the elucidation of the mechanism of post-translational nitrogenase regulation under anaerobic conditions. Several aspects of energy metabolism regulation in this bacterium were studied. To evaluate the involvement of A. brasilense AmtB and GlnZ in the regulation system, switch-off / on assays of nitrogenase by anaerobiosis were performed. The results confirmed that GlnZ and AmtB proteins do not directly participate in ADP- ribosylation of nitrogenase. LC-MS analysis of ATP and ADP levels showed a marked reduction when the culture was exposed to anaerobic conditions. Furthermore, addition of CCCP, which decreases intracellular ATP, caused switch-off and ADP-ribosylation of nitrogenase. Therefore, the ATP / ADP ratio, lead nitrogenase switch-off. Analysis of the cellular fractions, showed that, unlike the regulation by ammonium ions, the PII proteins did not migrate to the membrane during nitrogenase switch-off, and DraG also remained in the cytoplasm. The results suggest distinct pathway for the control of nitrogenase activity depending on which stimulus the cells are submitted to. Key words: Azospirillum brasilense, post-translational regulation, ADP-ribosylation, DraT / DraG system, anaerobiosis.

Nitrogen (N) is a chemical constituent for almost all biological molecules including proteins, DNA, RNA, lipids and is therefore vital for life. The ultimate source of nitrogen is the atmospheric dinitrogen (N2) but that only becomes bioavailable through a process of nitrogen fixation, the process that converts N2 to ammonia (NH3). The industrial Haber-Bosch process and biological nitrogen fixation account for the majority of nitrogen fixed every year. However, due to its high temperature, pressure and fossil fuel requirements, Haber-Bosch is an expensive process. Every year, approximately 3% of the global energy demand is used to manufacture ammonia through Haber-Bosch process. On the other hand, biological systems produce ammonia by reducing dinitrogen at ambient temperature and pressure using an anaerobic enzyme called nitrogenase. Research in understanding the mechanism of nitrogenase could eventually allow researchers to mimic the enzyme and fix nitrogen efficiently at standard temperature and pressure. In this research nitrogenase of Azotobacter vinelandii was studied to understand the mechanism of delivery of electrons/protons to the active site and how these accumulated reducing equivalents are used for substrates reduction. Through a series of studies, it has been demonstrated that the electrons and protons are added to the active site in a concerted manner which are then stored as bridging hydrides. The accumulated hydrides are used in four different mechanisms, namely reductive elimination, hydride protonolysis, migratory insertion and proton coupled electron transfer, to catalyze the reduction of varieties of unsaturated molecules. This fundamental understanding of molecular detail of nitrogenase catalysis could eventually help in development of more efficient, robust and selective catalysts.

The enzyme nitrogenase catalyzes the energy-dependent reduction of dinitrogen to ammonia via biological nitrogen fixation. Nitrogenase is composed of two metalloproteins known as the molybdenum-iron (MoFe) protein and the iron (Fe) protein. The Fe protein, a 60-kDa dimer of the product of the nifH gene, contains a single 4Fe-4S cluster and two Mg-ATP-binding sites, one at each subunit. The Fe protein is the obligate electron donor to the MoFe protein. To date, no other mutual protein has shown to substitute Fe protein in biological fixation, and the NifH is functional only in the presence of the nifessory protein NifM. Interestingly, the protochlorophyllide reductase (ChlL) encoded by the chlL gene of Chlamydomonas reinhardtii shows significant homology and structural similarity with NifH. Previously, our laboratory has shown that the ChlL can substitute the Fe protein in the functioning nitrogenase only in the absence of NifM. We have also shown that the NifM is a PPIase and the Pro-258 located in the C-terminus of NifH is one of the substrates for NifM. Since the least structural homology exists between NifH and ChlL at the C-terminal region, we hypothesized that we can generate a NifM-independent NifH-ChlL chimeric protein by replacing the C-terminus of NifH (that spans the substrate of PPIase) with that of ChlL. To test this idea we created a chimeric construct by replacing the NifH C-terminal region (residues 248-291) with the ChlL C-terminal region (residues 240-294). The chimeric gene was then transformed into the nifM- Azotobacter vinelandii strain AV98. While the wild type nifH could not render a Nif+ phenotype to the nifM- AV98, the chimera could impart Nif+ phenotype to this nifM- strain. This result demonstrated that the NifH-ChlL chimeric protein is NifM-independent.

As a key constituent of proteins, nucleic acids, and other biomolecules, nitrogen is essential to all living organisms including human beings. Dinitrogen represents the largest pool of nitrogen, about 79% of the Earth’s atmosphere, yet it is unusable by most living organisms due to its inertness. There are two ways to fix this inert dinitrogen to usable ammonia. One is the industrial Haber-Bosch process, which needs to be conducted at high temperature and pressure. This process uses a lot of the non-renewable fossil fuel as the energy source. The other major pathway is the biological nitrogen fixation carried out by some microorganisms called diazotrophs. The usable nitrogen output from this biological pathway ultimately supports an estimated 60% of the human population’s demand for nitrogen.The catalyst responsible for the biological nitrogen fixation is called nitrogenase, the most studied form of which contains molybdenum and iron in its active center, so called molybdenum nitrogenase. The work in this dissertation attempts to understand howthis biological catalyst breaks down dinitrogen to ammonia by application of different modern techniques. Firstly, an approach was developed to understand the stepwise reduction mechanism of dinitrogen to ammonia by molybdenum nitrogenase.The second goal of my research is to understand the roles of iron and molybdenum centers in nitrogenase function. My results using carbon monoxide as a probe for genetically modified molybdenum nitrogenase indicate that iron should be the metal sites functioning for nitrogen fixation. This is further supported by another study aimed at understanding the role of molybdenum during nitrogenase functioning.Moreover, an approach was developed to understand the mechanism for the obligatory production of hydrogen gas when nitrogenase activates dinitrogen for reduction. The same study also suggests possible pathways for the addition of hydrogenous species to nitrogen to produce ammonia.As part of this work, we also found that remodeled nitrogenases can use poisonous carbon monoxide and greenhouse-gas carbon dioxide to produce useful hydrocarbons by coupling one or more small molecules, which is hard to be achieved by other catalysts. Further study of these new reactions might give us deep insights on nitrogenase mechanism and inspire scientists to design better catalysts for relevant industrial processes.

This research investigated the existence of <10 £gm nitrogen-fixing unicellular cyanobacteria in the South China Sea. The surveys covered the period from February 2004 to January 2005 and a total of seven cruises. The unicellular cyanobacteria that express orange-yellow from cellular phycoerythrin were observed under a fluorescence microscope. Their expressions in nitrogen-fixation were confirmed by the results from reverse transcription polymerase chain reaction (RT-PCR) and whole cell fluorescence immunolocalization of nitrogenase. The nifH gene sequences of the unicellular cyanobacteria collected from the South China Sea was with >90% identities of their nucleotides similar to the nifH gene sequences of unicellular diazotrophs from ALOHA (Hawaii) as well as Synechocystis sp. WH 8501, Cyanothece sp. ATCC 51142, Cyanothece sp. WH 8902, Cyanothece sp. WH 8904 and Synechococcus sp. RF-1. Positive reactions of fluorescence immunolocalization of nitrogenase were only observed in some, not all, of <10 £gm unicellular cyanobacteria, suggesting that cell counting alone can not be used to estimate nitrogen fixation rate. There was great seasonal and spatial variation in the unicellular cyanobacteria cell density. There was, however, no significant relationship between cell density and the investigated environmental factors. Cell density was high when temperature was high or where stratification index of water column was high, such as in summer or in basin in contrast to other seasons or the shelf-slope regions.

The cyanide-degrading bacterium Klebsiella oxytoca SYSU-011 was isolated from the waste water of a metal-plating plant. In this study, we found out that K. oxytoca was capable of utilizing tetracyanonickelate {K2[Ni(CN)4]}(TCN) as its sole nitrogen source. This organism could degrade TCN both aerobically (D.O.¡×100¢H) and anaerobically (D.O.¡×0¢H).The addition of ammonia (5 mM) in the growth medium would inhibit TCN-degrading. The TCN-degrading by-product, a greenish precipitate, was found in the spent medium and was identified as nickel cyanide [Ni(CN)2] by FT-IR spectroscopic studies. Ammonia was demonstrated as a product of the TCN-degrading process by K. oxytoca resting cells. The addition of glucose could greatly enhance the TCN-degradation. Nitrogenase was found to be the cyanide degrading enzyme in this organism. The activity of nitrogenase was inhibited by ammonia but could be induced by the addition of TCN or KCN.

Thesis (Ph. D.)--University of Wisconsin, 1967. / "Nitrogen fixation by growing cells and cell-free extracts of the of the bacillaceae [by] D.F. Witz, R.W. Detroy and P.W. Wilson, [reprinted from] Archiv für mikrobiologie 55, 369-381, 1967" inserted between leaves [9]-[23]. Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

The iron-molybdenum cofactor (FeMo-cofactor) of nitrogenase is the subject of one the most intensive biochemical/genetic detective cases of modern science. At the active site of nitrogenase, the FeMo-cofactor not only represents the heart of biological nitrogen fixation, but its synthesis also serves as a model for complex metallocluster biosynthesis. Research in the Dean Lab is focused on furthering the understanding of Fe-S cluster biosynthesis in the nitrogenase enzyme system. Throughout the years, scientists from a broad range of disciplines have focused their intellectual might on deciphering not only the chemistry of the FeMo-cofactor, but also the biosynthesis of this unique metallocluster. Recent advances in the study of FeMo-cofactor biosynthesis have produced considerable insight regarding the complex series of biological reactions necessary for the synthesis of this metallocluster. The work contained within this dissertation represents my efforts to further the understanding of FeMo-cofactor biosynthesis. The concept of a molecular scaffold in FeMo-cofactor biosynthesis is generally accepted in the field of nitrogenase. Previous work has implicated the products of nifE and nifN as providing the assembly site for FeMo-cofactor synthesis. Researchers were able to purify this molecular scaffold, commonly referred to as the NifEN complex, however, detailed characterization was precluded by the inability to obtain sufficient quantities of NifEN. In an effort to fully characterize the NifEN complex, we initiated a gene fusion approach for the high level production NifEN. In addition to gene fusion, a poly-histidine tag was incorporated into NifEN, allowing purification through the application of immobilized metal-affinity chromatography (IMAC). NifEN obtained in this way was characterized using a variety of biophysical techniques and found to contain two [4Fe-4S] clusters in each NifEN tetramer. These clusters were also shown to be completely ligated by cysteine residues. With the information obtained from this study, it is concluded that the [4Fe-4S] clusters of the NifEN complex are likely to play either a structural or a redox role rather than being transferred and becoming incorporated into the FeMo-cofactor. In addition to the biophysical characterization of the NifEN complex, a separate study was started to characterize the apo-MoFe protein. In this study we used IMAC to purify a poly-histidine-tagged apo-MoFe protein produced by a nifB-deletion mutant of A. vinelandii. Using the poly-histidine fusion approach, apo-MoFe protein was obtained in sufficient quantities for detailed catalytic, kinetic and spectroscopic analyses. This multidisciplinary approach confirmed that apo-MoFe protein contained intact P clusters and P cluster environments, as well as the ability to interact with the Fe protein. It was also shown for the first time that this tetrameric form of purified apo-MoFe protein could be activated by the addition of preformed FeMo-cofactor. The NifEN complex was further characterized to investigate the presence of bound FeMo-cofactor intermediates. NifEN purified by IMAC is produced in the absence of the nitrogenase structural genes (nifHDK). In this genetic background, it is believed that the FeMo-cofactor biosynthetic machinery will become obstructed with unprocessed FeMo-cofactor intermediates, such as the Fe-S precursors of FeMo-cofactor, NifB-cofactor. Previous work indicated that NifEN can exist in either a charged or discharged form, based on the presence or absence of the FeMo-cofactor precursor, NifB-cofactor. EPR and VTMCD spectroscopies showed the presence of a new paramagnetic signal associated with NifEN that is believed to be in the charged or precursor bound state. This represents the first spectroscopic evidence for a precursor to the FeMo-cofactor. Furthermore, an interaction of NifEN and NifX was examined by size exclusion chromatography. From this study, NifX exhibited the capacity to bind a chromophore, presumably an FeMo-cofactor precursor, from the NifEN complex. NifX was also capable of binding to isolated FeMo-cofactor and the FeMo-cofactor precursor, NifB-cofactor. Finally, preliminary investigations involving interaction between the Fe protein and NifEN were initiated. Recent findings indicate that NifEN and the Fe protein have the capacity to interact specifically with one another. The interaction of NifEN and Fe protein appears to be dependent on the association of FeMo-cofactor precursor with NifEN. The NifEN complex also has the capacity to accept electrons from the Fe protein in a MgATP dependent manner. The ability of NifEN to accept electrons from the Fe protein may be involved in the role of Fe protein in FeMo-cofactor biosynthesis. / Ph. D.