Tesi sul tema "Azotobacter. Nitrogen"

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::lacZ fusion are presented and discussed.
Master of Science

Olive Mill Wastewater (OMW), by-product of oil industry, is a dark liquid with a characteristic fetid smell, bitter taste and bright appearance
having a high pollution potential, creating serious problems in countries producing olive oil. Azotobacter chroococcum as a Nitrogen-fixing bacteria can bioremediate OMW, by degrading its toxic constituents. With the help of this detoxification process OMW can be used as biofertilizer. In this study, the dynamics of growth and nitrogen fixation at different physiological conditions and nutrient requirements of A. chroococcum in chemically defined N-free medium was determined. These parameters were cultivation conditions such as pH, temperature and aeration and some additives such as inorganic salts, boric acid and nitrogen. Consequently, the maximum cell concentration were obtained when A. chroococcum was grown at neutral pH, 35&
#61616
C, 150 rpm and in medium supplemented with manganese salt at 0.01% concentration. The maximum nitrogen fixation products were attained when A. chroococcum was grown under the same conditions except at pH 8. Further, bioremediation of OMW by A. chroococcum was examined. When A. chroococcum was cultivated in OMW containing basal medium at 10% OMW concentration, a cell density 12 times higher than in the OMW free medium was achieved. Also, it was found to have maximum increase in extracellular protein concentration (112 mg/l) at 10% OMW containing medium and maximum increase in ammonia concentration (9.05 mg/l) at 5% OMW containing medium.

A model describing the potential amino acid ligands to the four 4Fe-4S centers (P-clusters) within the Azotobacter vinelandii nitrogenase MoFe protein is presented. Based on interspecies and intersubunit amino acid comparisons of the α- and ß-subunits of the MoFe protein, and the FeMoco biosynthetic proteins, NifE and NifN, four conserved residues (Cys62, His83, Cys88, Cys154 all proposed P-cluster ligands) within the α- subunit were targeted for site-directed mutagencsis studies. In order to define a range of acceptable substitutions, 35 specific site-mutants have been constructed, each with a different amino acid replacement at one of the four targeted positions. Previous studies indicated that these residues were important for MoFe activity, and may act as metallocenter ligands. Unusual redox and spectroscopic properties of the Fe-S centers suggest the involvement of ligands other than the four typical cysteines, though extrusion requirements indicate that some thiol ligands are likely. Surprisingly, mutants with an Asp, Gly, Thr, or Ser substituted for Cys88 are still capable of diazotrophic growth (Nif+), though whole cell and crude extract acetylene reduction activity is lowered. Several substitutions (Cys, Asp, Phe, Asn, Met, Tyr, Leu) are tolerated at the His83 position, these Nif+ mutant strains also have varying acetylene reduction rates and growth rates. All mutants with substitutions at positions 62, 154, resulted in complete loss of diazotrophic growth. The results could be interpreted by the following explanations: 1) Our proposed model for the P-cluster ligation within the MoFe protein is incorrect. 2) Some substitutions permit P-cluster rearrangement to a semi-functional state. 3) Either, P-clusters are not absolutely essential for diazotrophic growth, or the enzyme can function with a reduced number of these metal centers.
Master of Science
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Site-directed mutagenesis and gene replacement procedures were used to isolate mutant strains of Azotobacter vinelandii that produce altered MoFe proteins where the α-subunit residue-195 position, normally occupied by a histidine residue, was individually substituted by a variety of other amino acids. Structural studies have revealed that this histidine residue is associated with the FeMo-cofactor binding domain and probably provides an NH→S hydrogen bond to a central bridging sulfide located within FeMo-cofactor. The present study investigates the role of the α-histidine-195 residue in nitrogenase catalysis by examining the altered MoFe proteins. Comparisons of the catalytic and spectroscopic properties of altered MoFe proteins produced by the Azotobacter vinelandii mutant strains suggest that the α-histidine-195 residue has a structural role which serves to keep the FeMo-cofactor attached to the MoFe protein and to correctly position the FeMo-cofactor within the polypeptide matrix such that N₂ binding is accommodated. Substitution of the α-His-195 residue by a glutamine residue results in an altered MoFe protein that binds but does not reduce N₂, the physiological substrate. Stopped-flow spectroscopic analyses indicate that the α-195^gln MoFe protein is unable to reduce N₂ even though the altered MoFe protein can reach the redox state necessary for N₂ reduction. Although, N₂ is not a substrate for the altered MoFe protein, it is an inhibitor of both acetylene and proton reduction, both of which are otherwise effectively reduced by the altered MoFe protein. This result provides evidence that N₂ inhibits proton and acetylene reduction by simple occupancy of the active site. The α-195^gln MoFe protein catalyzes HD formation in the presence of N₂ and D₂. Moreover, N₂ binding at the active site of the altered MoFe protein is inhibited by the addition of D₂. These observations indicate that binding of nitrogen to the enzyme is necessary but its reduction is not required for the formation of HD. N₂ uncouples MgATP from proton reduction catalyzed by the α-195gln MoFe protein, but does so without lowering the overall rate of MgA TP hydrolysis. Thus, the quasi-unidirectional flow of electrons from the Fe protein to the MoFe protein that occurs during nitrogenase turnover is controlled, in part, by the substrate serving as an effective electron sink. N₂-induced uncoupling of ATP hydrolysis from substrate reduction by the α-195^gln MoFe protein is reversed by the addition of H₂ (D₂) in the assay atmosphere. This observation can successfully be explained if it-is assumed that the altered MoFe protein has a much greater binding affinity for H₂ (D₂) than for N₂. Substitution of the α-histidie-195 residue by glutamine also imparts hypersensitivity of acetylene reduction and N2 binding to inhibition by CO, indicating that the imidazole group of the α-histidine- 195 residue might protect an Fe contained within FeMo-cofactor from attack by CO.
Ph. D.

Mo-nitrogenase consists of two component proteins, the Fe protein and the MoFe protein. The site of substrate binding and reduction within the Mo-nitrogenase is provided by a metallocluster, the FeMo cofactor, located in the a-subunit of the MoFe protein. The FeMo cofactorâ s polypeptide environment appears to be intimately involved in the delicate control of the MoFe proteinâ s interactions with its substrates and inhibitors (Fisher K et al., 2000c). In this work, the a-subunit 278-serine residue of the MoFe protein was targeted because (i) a serine residue at this position is conserved both in the Mo-nitrogenase from all organisms examined and in the alternative nitrogenases (Dean, DR and Jacobson MR, 1992); (ii) its hydroxyl group hydrogen bonds to the Sg of the a-subunit 275-cysteine residue that directly ligates the FeMo cofactor; and (iii) its proximity to the a-subunit 277-arginine residue, which may be involved in providing the entry/exit route for substrates and products (Shen J et al., 1997). Altered MoFe proteins of A. vinelandii nitrogenase, with the a278Thr, a278Cys, a278Ala and a278Leu substitutions, were used to study the interactions of H+, C2H2, N2 and CO with the enzyme. All strains, except the a278Leu mutant strain, were Nif+. From measurement of the Km for C2H2 (C2H4 formation) for the altered MoFe proteins, the a278Ala and a278Cys MoFe proteins apparently bind C2H2 similarly to the wild type, whereas the a278Thr and the a278Leu MoFe proteins both have a Km ten-times higher than that of the wild type. Unlike wild type, these last two altered MoFe proteins both produce C2H6. These results suggest that C2H2 binding is affected by substitution at the a-278 position. Moreover, when reducing C2H2, the a278Ala and a278Cys MoFe proteins respond to the inhibitor CO similarly to the wild type, whereas C2H2 reduction catalyzed by the a278Thr MoFe protein is much more sensitive to CO. Under nonsaturating concentrations of CO, the a278Leu MoFe protein catalyzes the reduction of C2H2 with sigmoidal kinetics, which is consistent with inhibitor-induced cooperativity between at least two C2H4-evolving sites. This phenomenon was previously observed with the a277His MoFe protein, in which the a-subunit 277-arginine residue had been substituted (Shen J et al., 1997). Together, these data suggest that the MoFe protein has at least two C2H2-binding sites, one of which may be located near the a277-278 residues and, therefore, most likely on the Fe4S3 sub-cluster of the FeMo cofactor. Like the wild type, N2 is a competitive inhibitor of the reduction of C2H2 by the a278Thr, a278Cys and a278Ala MoFe proteins. Apparently, the binding of N2 in these altered MoFe proteins is similar to that with the wild type MoFe protein, suggesting that the aSer278 residue is not directly involved in N2 binding and reduction. Previous work suggested that both a high-affinity and low-affinity C2H2-binding site were present on the MoFe protein (Davis LC et al., 1979; Christiansen J et al., 2000). Our results are generally consistent with this suggestion. Currently, there is not much information about the proton donors and how the protons necessary to complete all substrate-to-product transformations are transferred. The dependence of activity on pH (activity-pH profiles) has provided useful information about the nature of the groups involved in proton transfer to the FeMo cofactor and the bound substrate. Approximately bell-shaped activity-pH profiles were seen for all products from catalysis by all the MoFe proteins tested whether under Ar, in the presence of C2H2 as a substrate, or with CO as an inhibitor. The profiles suggested that at least two acid-base groups were required for catalytic activity. The pKa values of the deprotonated group and protonated group were determined from the pH that gave 50% maximum specific activity. These pKa values for the altered a278-substituted MoFe proteins and the a195Gln MoFe protein under various assay atmospheres were compared to those determined for the wild type. It was found that the pKa value of the deprotonated group was not affected by either substitution or changing the assay atmosphere. The wild type MoFe protein has a pKa (about 8.3) for the protonated group under 100% argon that was not affected very much by the substitution by Cys, Ala and Leu, whereas the Thr substitution shifted the pKa to about 8, which was the same as that of the wild type MoFe protein in the presence 10% CO. The pKa values for the protonated group for all the altered MoFe proteins were not changed with the addition of 10% CO. These results suggest that the aSer278 residue, through hydrogen bonding to a direct ligand of the FeMo cofactor, is not one of the acid-base groups required for activity. However, this residue may â fine-tuneâ the pKa of the responsible acid-base group(s) through interaction with the aHis195 residue, which has been suggested (Dilworth MJ et al., 1998; Fisher K et al., 2000b) to be involved in proton transfer to substrates, especially for N2 reduction. The activity-pH profiles under different atmospheres also support the idea that more than one proton pathway appears to be involved in catalysis, and specific pathway(s) may be used by individual substrates.
Ph. D.

Biological nitrogen fixation is accomplished in the bacterium Azotobacter vinelandii by means of three metalloenzymes: The molybdenum, vanadium, and iron-only nitrogenase. The knowledge regarding biological nitrogen fixation has come from studies on the Mo-dependent reaction. However, the V- and Fe-only-dependent reduction of nitrogen remains largely unknown. By using homology modeling techniques, the protein folds that contain the metal cluster active sites for the V- and Fe-only nitrogenases were constructed. The models uncovered similarities and differences existing among the nitrogenases regarding the identity of the amino acid residues lining pivotal structural features for the correct functioning of the proteins. These differences, could account for the differences in catalytic properties depicted by these enzymes. The quaternary structure of the dinitrogenases also differs. Such component in the Mo-nitrogenase is an α2β2 tetramer while for the V- an Fe-only nitrogenase is an α2β2δ2 hexamer. The latter enzymes are unable to reduce N2 in the absence of a functional δ subunit, yet they reduce H+ and the non-physiological substrate C2H2. Therefore, the δ subunit is essential for V- and Fe-only dependent nitrogen fixation by a mechanism that still remains unknown. In attempt to understand why the δ subunit is essential for V-dependent N2 reduction from a structural stand point, this work presents the strategy followed to clone the vnfG gene and purify its expression product, the δ subunit. The purified protein was subjected to crystallization trials and used to stabilize a histidine-tagged VFe protein that would otherwise purify with low Fe2+ content and poor H+ and C2H2 reduction activities. The VFe preparation was used to conduct substrate reduction assays to assess: i) The electron allocation patterns to each of the reduction products of the substrates C2H2, N2, N2H4, and N3−; and ii) Inhibition patterns among substrate and inhibitor of the nitrogenase reaction. This work also reports on the effect N2H4 and N3− has on the electron flux to the products of the C2H2 reduction. The work presented herein provides information with which to compare and contrast biological nitrogen fixation as catalyzed by the Mo- and V-nitrogenases from Azotobacter vinelandii.

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.

碩士
國立中興大學
土壤環境科學系所
105
Rice is one of the most important food crops in the world, but climate change may affect its yield. The purpose of this study was to study the effects of CO₂ concentrations, soil types, Azotobacter inoculations, and nitrogen fertilization rates on rice growth. A factorial design experiment was carried out including two CO₂ concentrations (500 and 1000 ppm), two soil types (Dali and Houlong soils), four N application rates (0, 60, 120, and 180 kg ha-¹), and four Azotobacter inoculation treatments with a non-inoculated control. Each treatment included three replicates and was arranged in controlled greenhouses using the randomized complete block design. Inoculation of rice plants with A.chroococcum strain CHB869 significantly increased total dry weight under 1000 ppm CO₂, suggesting that the strain considerably promoted rice growth under stresses. Under 1000 ppm CO₂, the harvest index for rice plants grown in Dali soil decreased significantly because excessive N fertilization might result in stresses. In contrast, the harvest index for rice plants grown in Houlong soil under 1000 ppm CO₂ increased significantly with increasing N fertilization. The panicle weight, thousand grain weight, and harvest index of rice plants grown under 1000 ppm CO₂ in Dali soil were significantly increased by CHB869 inoculation. However, only under 1000 ppm CO₂ rice plants inoculated with A.beijerinckii strain CHB461 and grown in Houlong soil showed a significant increase in the thousand weight. Under 1000 ppm CO₂, nutrients uptake of rice plants grown in Dali soil increased significantly with increasing N fertilization. However, the N application rate of 180 kg ha-¹ significantly reduced N, P and K uptake of rice plants grown in Houlong soil. In addition, nutrient uptake of vegetative organs of rice plants grown in Dali soil with the tree individual Azotobacter strains and in Houlong soil with CHB 461 was significantly reduced probably because the nutrients translocated into grains. Under 1000 ppm CO₂, Dali soil inoculated with CHB869 and Houlong soil inoculated with CHB461 significantly reduced soil C/N ratio, providing more nitrogen for root uptake and consequently increasing yields Taken together, the use of Azotobacter spp. along with an appropriate soil fertility management program based on soil properties may ensure yields for rice production under climate change impacts, contributing to food security.