No description available.

No description available.

Thesis (M.S.)--Rutgers University, 2008. / "Graduate Program in Oceanography." Includes bibliographical references (p. 29-32).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1966. / Typescript. Vita. Description based on print version record. Includes bibliographical references.

Thesis--Wisconsin. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1969. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

No description available.

The enzyme nitrogenase, which catalyzes nitrogen fixation in Azotobacter vinelandii, consists of two components, the Fe protein (NifH) and the MoFe protein (NifDK). NifK contains several highly conserved residues implicated in its functions throughout the protein. However, the carboxyl terminus of NifK is not implicated in any of the known functions of this protein. Therefore, the present study explored the role of carboxyl terminal region of NifK. The results of growth analysis showed that when the media was adjusted to be slightly acidic, the strain that expresses the mutated NifK yielded a lower growth compared to the wild type. These observations implied that the carboxyl terminus of the NifK contributes to the formation of a stable nitrogenase complex when A. vinelandii is grown in acidic environment. The proper interaction between NifH and NifK is essential for the nitrogenase conformation. To determine how the interaction is influenced by the characteristics of the amino acids available at position 112 of NifH, we introduced residue mutations to the codon encoding for Glu112. Growth analyses indicated that mutant strains are capable of propagation under nitrogen-deficit conditions although the growth rate is lower than that of wild type strain. Therefore the charge carried by the amino acid at position 112 of NifH plays a minor role in the interaction whereas; a more important factor is the length of the side chains. The research on hydrogenase expressed by bacteria shed light on the possibilities of utilizing this novel energy source. We endeavored to take advantage of the nature of A. vinelandii and construct an A. vinelandii mutant strain expressing Fe-hydrogenase. This ongoing research involves molecular manipulation of the enzyme-encoding gene hydA. The synthetic hydA was incorporated and expressed in A. vinelandii strain DJ54. At the same time, we screened several biomass materials for their capabilities in sustaining diazotrophic growth of A. vinelandii. The result indicated that the HydA protein can be expressed in A. vinelandii under certain conditions and a number of biomass substances can be supportive ingredients for putative biohydrogen media.

The site of substrate binding and reduction for the nitrogenase complex is located on the iron molybdenum cofactor (FeMo-co) which is contained within the a-subunit of the molybdenum iron protein. FeMo co consists of a metal sulfur core composed of an FeS cluster bridged by three inorganic sulfides to a MoFeS cluster. An organic acid, homocitrate, is coordinated to the Mo atom through its 2-carboxy and 2-hydroxy groups. Homocitrate is formed by the condensation of acetyl-CoA and a-ketoglutarate, which is catalyzed by a homocitrate synthase encoded by nifV. By deleting the nifV gene from Azotobacter vinelandii we were able to study the role of homocitrate in nitrogenase catalysis. A poly-histidine tail was incorporated into the C-termini of the a-subunit permitting isolation of the homocitrateless MoFe protein by using metal affinity chromatography. We have found that the addition of a poly-histidine tag does not alter the catalytic behavior of the native enzyme. In NifV- strains of Klebsiella pneumoniae, citrate has been found to replace homocitrate as the organic constituent of FeMo-co. We have found no evidence this is so in A. vinelandii. Gas chromatography mass spectrophotometry studies indicate little or no organic acids are associated with FeMo-co. We examined the catalytic properties of the NifV- MoFe protein In the mutant, H2 evolution is inhibited by the addition of CO, unlike in the wild type. We have found that the NifV- MoFe protein from A. vinelandii is able to catalyze the reduction of acetylene to both ethylene and ethane. / Master of Science

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.