Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Thesis (M.S.)--University of North Carolina Wilmington, 2007. / Includes bibliographical references (leaves: 120-121)

Thesis (M.S.)--University of Montana, 2007. / Title from title screen. Description based on contents viewed Aug. 8, 2007. Includes bibliographical references.

No description available.

Sequence analysis of the entire 3.5 kb <i>Hind</i>III genomic DNA fragment previously isolated from <i>Nostoc commune</i> UTEX 584 (Defrancesco and Potts 1988), determined the exact locations of the <i>nifU, nifH</i>, and <i>nifD</i> genes and identified two potential stem loop structures, a direct repeat, and an ORF that codes for a protein with a predicted amino acid sequence similar to that of myoglobin from <i>Paramecium caudatum</i>. The <i>N. commune</i> UTEX 584 myoglobin-like protein has a predicted length of 118 amino acids and molecular mass of 12,906 Da. A PCR copy of the gene (<i>glbN</i>) was cloned for overexpression of the protein. The recombinant protein was purified and used for spectral analysis and for the production of polyclonal antisera. Treatment of the recombinant protein with dithionite and CO resulted in spectral shifts characteristic of hemoproteins that bind oxygen. While some of the spectral characteristics are unique to the protein, in general the spectra were more like those of globins than cytochromes. Based on these characteristics and the sequence similarity to the P. caudatum mnyoglobin, we proposed the name cyanoglobin, with the gene designation glbN and the protein designation GlbN. Western analysis of GlbN expression was performed on N. commune UTEX 584 and two species of Anabaena (Anabaena sp. PCC 7120 and Anabaena variabilis). In N. commune UTEX 584 a protein with a molecular mass similar to that predicted for GlbN was detected. This protein was produced in increased amounts under the same growth conditions that resulted in increased production of nitrogenase reductase (the nifH gene product). No proteins of similar size to GlbN were detected in Anabaena sp. PCC 7120 or A. variabilis. A possible function of GlbN may be for oxygen storage, transport, or protection of the nitrogenase system. These functions as well as those of the direct repeat and the potential stem loop structures and their relationship to nitrogen fixation or other physiological processes in N. commune UTEX 584 require further analysis. / Ph. D.

The products of nifE and nifN from Azotobacter vinelandii, which are involved in the biosynthesis of the iron-molybdenum cofactor (FeMo-co) co) from nitrogenase, have been analyzed using a variety of mutagenic techniques. NifE was the object of several site-specific, amino acid substitutions that were designed to elicit information regarding metal cluster ligands, subunit-subunit interactions, and the proposed transfer of FeMo-co.from a nifEN-products complex to the apo-MoFe protein. A model of metal cluster binding; regions within the nifEN-products is discussed insofar as it relates to the rationale for the targeting of particular amino acids for-substitution. A translational fusion between nifN and lacZ was constructed and used to study the regulation of nifEN. This gene fusion was regulated in the same manner as wild type nifN and produced a fusion protein which was enzymatically active with respect to substrates of β-galactosidase. Results from mutant strains which carry lesions in nifH or nifA in addition to the nifN / Master of Science

The present study investigates the structural and functional roles of the metalloclusters present within the MoFe protein of nitrogenase from Azotobacter vinelandii. A gene replacement strategy was developed for oligonucleotide-directed mutagenesis of these proteins and the resulting biological and biochemical effects of these changes were examined. Identification of structurally important regions in the MoFe protein subunits and assignment of specific amino acid residues as potential metal cluster ligands were based upon several criteria: i. metallocluster extrusion requirements; spectroscopic properties of the MoFe protein; interspecies and intersubunit comparisons; iv. comparison of the MoFe protein subunit sequences to iron-molybdenum cofactor biosynthetic gene products. This mutagenesis strategy has permitted the construction of thirty-three mutant strains having specific amino acid substitutions within the MoFe protein subunits. Based on the diazotrophic growth characteristics and substrate reduction capabilities of these mutant strains, a model is presented in which potential metallocluster binding sites within the MoFe protein subunits are defined. In addition to analysis of the MoFe protein subunits, this site-directed mutagenesis and gene replacement strategy can be used to place specific mutations into any gene product encoded within the A. vinelandii nif gene cluster. Finally, nucleotide sequence analysis of the regions flanking the nifEN genes revealed the presence of three nif genes (nifT, nifY, and nifX) and four open reading frames (ORF1, ORF2, ORF3, and ORF4). Two of these genes, nifX and ORF3, were shown to be under nif control and synthesis of their products elevated in response to a demand for fixed nitrogen. Mutant strains with deletions in ORF3 appeared to accumulate an excess amount of MoFe protein when compared to wild type. The ORF3 gene product has been overproduced in E. coli. This provides an important step toward characterizing the protein and elucidating the molecular basis for its control of nifDK gene expression. / Ph. D.

Thesis (M.S.)--University of Hawaii at Manoa, 2006. / Includes bibliographical references (leaves 48-53). / viii, 53 leaves, bound ill. (some col.) 29 cm

Thesis (M.S.)--Mississippi State University. Department of Biological Sciences. / Title from title screen. Includes bibliographical references.

Nitrogenase fixation occurred in a number of habitats in and nearby several antarctic lakes. The observed acetylene reduction occurred in bluegreen algal mats in littoral areas that received maximal sunlight. The benthic bluegreen algal communities in reduced light under 5-6 m of permanent ice showed no detectable nitrogenase activity. The observed nitrogen fixation potential correlated with the presence of heterocystous bluegreen algae considered to be the major nitrogen fixing organisms in these habitats. The relatively low acetylene reduction rates suggest that a small but significant contribution of ammonia to these environments deficient in nitrogen may occur through nitrogen fixation. / Master of Science