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 cofactor's polypeptide environment appears to be intimately involved in the delicate control of the MoFe protein'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 "fine-tune" 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.

Applications of fertilizers derived from non-renewable resources, along with improved land management practices have contributed greatly increased crop yields in the past 70 years. Biofertilizers and organic amendments, provide alternative sources of nutrients for increased plant yields and resistance against abiotic stress. The objective of this work was to evaluate the effectiveness of various biofertilizers and an organic amendment on improve plant health and/or crop yield. The first study focused on the organic amendment, glucoheptonate and found that applications of 800-1600 kg/ha can increase available water capacity in fine textured soils by up to 3%. The second study evaluated the effectiveness of dual-inoculating biofertilizers Mung beans (Vigna radiata (L.) Wilczek) with two, bradyrhizobium spp. and arbuscular mycorrhizal fungi. Dual inoculation significantly increased grain yield (+33%) compared to a synthetic N fertilizer application but did not significantly increase grain yield compared to the control (+22%). Dual inoculation may increase grain yields of mung beans compared to synthetic fertilizer regime but does not show evidence of improving N fixation. The final study was a greenhouse experiment focused on evaluating some mung bean cultivars to determine their susceptibility to salt stress while also evaluating the effect of inoculation in combating saline soils. Germination was significantly decreased at 6 dS/m in all cultivars by about 36% when compared to the control treatment (0 dS/m). Seed yields, pods per plant and seeds per plant, increased as salt concentration increased. No factors recorded where affected by inoculation. Overall, our research suggests that the use of biofertilizers and organic amendments can improve crop health, but other management and environmental considerations need to be accounted for when reporting effectiveness of such alternative soil amendments / Doctor of Philosophy / Applications of fertilizers derived from non-renewable resources, along with improved land management practices have contributed greatly to increased crop yields in the past 70 years. Biofertilizers and organic amendments, provide alternative sources of nutrients for increased plant yields and resistance against abiotic stress. The objective of this work was to evaluate the effectiveness of an organic amendment and various biofertilizers to improve plant health and/or crop yield. The first study focused on the organic amendment, glucoheptonate and found that applications of 800-1600 kg/ha can increase available water capacity in fine textured soils by up to 3%. The second study evaluated the effectiveness of dual-inoculating Mung beans (Vigna radiata (L.)Wilczek) with two biofertilizers, bradyrhizobium spp. and arbuscular mycorrhizal fungi. Dual inoculation significantly increased grain yield (+33%) compared to a synthetic N fertilizer application but did not significantly increase grain yield compared to the control (+22%). Dual inoculation may increase grain yields of mung bean compared to synthetic fertilizer regime but does not show evidence of improving N fixation. The final study was a greenhouse experiment focused on evaluating mung bean cultivars (4) to determine their susceptibility to salt stress while also evaluating the effect of inoculation in combating the effect of saline soils. Germination was significantly decreased by about 36% at a salinity of 6 dS/m across all cultivars compared to the control at 0 dS/m. Seed yields, pods per plant and seeds per plant, increased as salt concentration increased for some cultivars. No factors recorded were observed to be affected by inoculation. Overall, our research suggests that the use of biofertilizers and organic amendments can improve crop health, but other management and environmental considerations need to be accounted for when reporting effectiveness of such alternative soil amendments.

The purpose of this study was to compare the stiffness of a Type II external skeletal fixator (ESF) to a 3.5 mm locking compression plate (LCP) in axial compression, mediolateral, and craniocaudal bending in a fracture gap model. The hypothesis was that the Type II ESF would demonstrate comparable stiffness to the LCP. A bone simulant consisting of short fiber reinforced epoxy cylinders and a 40 mm fracture gap was used. The LCP construct consisted of a 12 hole 3.5 mm plate with three 3.5 mm bicortical locking screws per fragment. The Type II ESF construct consisted of 3 proximal full fixation pins (Centerface®) per fragment in the mediolateral plane, and 2 carbon fiber connecting rods. Five constructs of each were tested in non-destructive mediolateral and craniocaudal bending, and axial compression. Stiffness was determined from the slope of the elastic portion of force-displacement curves. A one-way ANOVA and a Tukey-Kramer multiple comparisons test were performed, with significance defined as p < 0.05. In mediolateral bending, the stiffness of the Type II ESF (mean ± standard deviation; 1584.2 N/mm ± 202.8 N/mm) was significantly greater than that of the LCP (110.0 N/mm ± 13.4 N/mm). In axial compression, the stiffness of the Type II ESF (679.1 N/mm ± 20.1 N/mm) was significantly greater than that of the LCP (221.2 N/mm ± 19.1 N/mm). There was no significant difference between the constructs in craniocaudal bending. This information can aid in decision-making for fracture fixation, although ideal stiffness for healing remains unknown. / Master of Science / Optimum fracture stabilization requires a balance between providing a stable mechanical environment and preserving the blood supply to healing tissues. When the complexity of a fracture precludes reconstruction of the bony column, the fixation method chosen for repair must counteract the forces of weight bearing, including compression and bending. Knowledge of the relative construct stiffness is important for a clinician to determine the ability of a fixation technique to withstand all forces acting on a fracture, while supporting bone healing. The purpose of this study was to compare the stiffness of a Type II external skeletal fixator (ESF) and a locking compression plate (LCP) when non-destructive physiologic loads are applied in axial compression, mediolateral bending, and craniocaudal bending. Five constructs of each were tested in non-destructive mediolateral and craniocaudal bending, and axial compression. Stiffness was determined from the slope of the elastic portion of force-displacement curves. There was a significant difference between the stiffnesses of the Type II ESF and the LCP in all modes of loading except craniocaudal bending. The Type II ESF was significantly stiffer in mediolateral bending than the LCP, and the Type II ESF was significantly stiffer in axial compression compared to the LCP. There was no statistically significant difference in stiffness in craniocaudal bending. This information will aid a clinician in selecting an appropriate fixation method for a non-reconstructable fracture, but further studies are required to assess the importance of increased stiffness in a clinical setting.

The herbicide simazine (2-chloro-4,6-bis(ethylamino)- 1,3,5-triazine) was found to have an inhibitory effect on nitrification in pure and mixed cultures of the nitrifying bacteria. Simazine concentrations of 6 and 9ppm inhibited the rate of nitrification in soil perfusion units, and an abnormally high level of nitrite nitrogen was observed in the herbicide treatments. The herbicide had no effect on the growth of Nitrosomonas europaea in shake flask culture, but did inhibit the growth of Nitrobacter agilis. The addition of yeast extract to the culture medium did not reverse the inhibition. Simazine did not inhibit the respiration of N. agilis even at a 100ppm concentration. Five organisms were isolated which could utilize simazine as a sole source of carbon and nitrogen. Pure cultures of these organisms were made and several were identified. / Master of Science

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

Previous studies revealed that α-275-Cys provides an essential ligand to one of the Fe atoms on the FeMo-cofactor, and its substitutions resulted in inactive nitrogenase. In order to study the structural-functional relationship of the protein environment in this region with respect to the FeMo-cofactor, subtle changes were introduced through substitutions using a site-directed mutagenesis and gene-replacement method at α-274-His, α-276-Tyr and α-277-Arg in Azotobacter vinelandii nitrogenase. Characterization of mutants strains resulting from amino acid substitutions at residues, α-274-His, α-276-Tyr or α-277-Arg, using activity assays, resulted in mixed Nif phenotypes. Therefore, none of these residues is absolutely required for nitrogenase activity. However, the changed EPR spectra of the altered MoFe proteins from some strains with substitutions at either α-276-Tyr or α-277-Arg indicated that the FeMo-cofactor environment had been perturbed by these substitutions. Together with its changed EPR spectrum, substituting α-277-Arg with His showed some extraordinary catalytic features, such as its inability to reduce N₂ while retaining respectable C₂H₂- and H⁺-reduction activities. It was also found that this altered protein used a higher percentage of total electron flux for H₂ evolution under an C₂H₂/Ar atmosphere than did wild type. Further characterization of the purified α-277^his MoFe protein in parallel with its wild type counterpart revealed that the alteration in the α-277^his MoFe protein caused a lower affinity for C₂H₂ binding, whereas it did not affect the CO binding. Interestingly, CO-induced cooperativity during C₂H₂ reduction was observed in this altered MoFe protein clearly indicating two sites for C₂H₄ evolution, one of which might be in the vicinity of this residue. Furthermore, the α-277^his MoFe protein does not bind or reduce N₂ leading to the proposal of a nonexistent E₄ redox state in the MoFe protein catalytic cycle which was supported by stopped-flow spectrophotometric evidence. This altered α-277^his MoFe protein showed comparable physical stabilities to that of the wild-type protein, and its ATP hydrolysis rates remained constant under a number of substrates assayed. Therefore, the substitution has not affected the overall protein structure, rather, it has changed the local FeMo-cofactor environment. When we studied the purified α-276^his and α-274^gin/α-276^his MoFe proteins and compared the results with the data from the α-277^his and wild-type MoFe protein, we found that there is no direct correlation between the additional set of EPR signals observed in these altered MoFe proteins and their catalytic activities. The current understanding concerning the functionality of these residues is that they are involved in maintaining a proper environment for FeMoco to bind and in stabilizing the different redox states of the enzyme during catalysis. / Ph. D.

Throughout the African highlands forage legumes are relied on to add soil N, support increasing livestock populations, and reduce soil erosion. This research addresses a constraint designated by the International Livestock Center for Africa (ILCA) to identify African clover (Trifolium) germplasm and Rhizobium trifolii strain combinations with high productivity potential for the African highlands. A Vertisol and an Eutric Nitosol (Paleudalf), and seed from Trifolium decorum, T. quartinianum, T. rueppellianum, T. steudneri, and T. tembense were obtained from ILCA in Addis Ababa, Ethiopia. A commercial collection and R. trifolii strains isolated from the Ethiopian soils were evaluated for symbiotic effectiveness with these clovers. Effective combinations were evaluated for growth and biological nitrogen fixation (BNF) in a greenhouse on both soils with limited, adequate, and excessive soil moisture. Rhizobia were also evaluated for survival in desiccated soil (12.5 and 17.5 g H₂O g⁻¹ soil) and for competitive nodule forming ability. Effective strains were found among soil isolates but not in the commercial collection. Highest dry matter yields and total BNF accumulation were obtained from T. tembense on all soil and moisture treatments followed by T. decorum and T. quartinianum. Trifolium rueppellianum and T. steudneri had low yields and BNF capacities. Plants receiving adequate and excessive moisture had higher yields than moisture stressed plants. The highest levels of cumulative BNF were obtained on the nitosol soil with either adequate or limited moisture. The moisture limited vertisol supported the lowest BNF levels. Rhizobial strains survived desiccation only in the vertisol at 17.5 g H₂O g⁻¹ soil. All strains could compete with background rhizobia populations to nodulate host plants. Nodule occupancy rates of 20 to 30% were required for high yields. Trifolium tembense, T. decorum, and T. quartinianum are adapted to soils with adequate or excessive moisture, T. rueppellianum and T. steudneri are suited to moisture limited conditions or short growing seasons. Effective rhizobia inoculants and selected clovers have the potential for increasing forage productivity in highland areas. / Ph. D.

Yes / PURPOSE. To compare microperimetric sensitivity around the monocular preferred retinal locus (mPRL) in age-related macular degeneration (AMD) to normative data, and to describe the characteristics of visual field defects around the mPRL in AMD. METHODS. Participants with AMD (total n ¼ 185) were either prospectively recruited (n ¼ 135) or retrospectively reviewed from an existing database (n ¼ 50). Participants underwent microperimetry using a test pattern (37 point, 58 radius) centered on their mPRL. Sensitivities were compared to normative data by spatial interpolation, and conventional perimetric indices were calculated. The location of the mPRL relative to the fovea and to visual field defects was also investigated. RESULTS. Location of mPRL varied approximately 158 horizontally and vertically. Visual field loss within 58 of the mPRL was considerable in the majority of participants (median mean deviation 14.7 dB, interquartile range [IQR] 19.6 to 9.6 dB, median pattern standard deviation 7.1 dB [IQR 4.8–9.0 dB]). Over 95% of participants had mean total deviation worse than 2 dB across all tested locations and similarly within 18 of their mPRL. A common pattern of placing the mPRL just foveal to a region of normal pattern deviation was found in 78% of participants. Total deviation was outside normal limits in this region in 68%. CONCLUSIONS. Despite altering fixation to improve vision, people with AMD exhibit considerable visual field loss at and around their mPRL. The location of the mPRL was typically just foveal to, but not within, a region of relatively normal sensitivity for the individual, suggesting that a combination of factors drives mPRL selection. / This report presents independent research funded by the NIHR

The process of symbiotic nitrogen fixation (SNF) in legume root nodules requires the channeling and exchange of nutrients within and between the host plant cells and between the plant cells and their resident rhizobia. Using a forward genetics approach in the Medicago truncatula Tnt1 mutant population followed by whole genome sequencing, two putative sulfate transporter genes, MtSULTR3;5 and MtSULTR3;4b, were identified. To support the hypothesis that the defective putative sulfate transporter genes were the causative mutation for the mutants' phenotypes, the M. truncatula Tnt1 population was successfully reverse screened to find other mutant alleles of the genes. The F2 progeny of mutants backcrossed with wildtype R108 demonstrated co-segregation of mutant phenotypes with the mutant alleles confirming that the mutated mtsultr3;5 and mtsultr3;4b genes were the cause of defective SNF in the mutant lines mutated in the respective genes. This finding was further established for mtsultr3;4b by successful functional complementation of a mutant line defective in the gene with the wildtype copy of MtSULTR3;4b. A MtSULTR3;4b promoter-GUS expression experiment indicated MtSULTR3;4b expression in the vasculature and infected and uninfected plant cells of root nodules. MtSULTR3;4b was found to localize to the autophagosome membrane when expressed in Nicotiana benthamiana. A transcriptomics study on the mutant nodules revealed the probable impact of mutated mtsultr3;5 and mtsultr3;4b on expression of genes involved in N fixation and on other biological processes, including possible effects of the mutated genes on the transcriptional regulation of sulfate assimilation pathway in the respective mutants' nodules. The RNAseq study also demonstrated the mis-regulation of nodule zone-specific genes in mtsultr3;5 and mtsultr3;4b mutants. A PCR-based approach was used to study the transcription of MtSULTR3;5 and MtSULTR3;4b in the respective mutant lines. The study demonstrated formation of readthrough chimeric gene-Tnt1 transcripts in mtsultr3;5 mutant alleles and truncated chimeric gene-Tnt1 transcripts and aberrantly spliced transcripts or no transcripts in mtsultr3;4b mutant alleles. Gene expression analysis of all MtSULTR genes using qRT-PCR was carried out in wildtype M. truncatula R108 nodules at a time course to evaluate the MtSULTR genes for their potential involvement in the SNF process.

Plants have many nitrate transporters; in the model legume Medicago truncatula, MtNPF1.7 is among them. MtNPF1.7 is important for M. truncatula growth and it has been established that MtNPF1.7 is a high affinity nitrate transporter. M. truncatula plants with mutations in MtNPF1.7 gene show defects during plants growth, with striking abnormalities in nodule development and root architecture. Nitrogen fixation is an energy expensive process; when legumes have sufficient bioavailable nitrogen like nitrate available, it suppresses nodulation and nitrogen fixation. Previous preliminary results in our lab showed that plants constitutively expressing MtNPF1.7 have a growth phenotype in the absence of nitrate, but no data was available on how M. truncatula plants constitutively expressing MtNPF1.7 are affected by the presence of nitrate. For my research, I confirmed the preliminary results on the growth of M. truncatula plants overexpressing NPF1.7 and examined these plants' phenotypes when nitrate was not provided in the growth media and when it was provided at two different concentrations. Compared with wild type A17, plants constitutively expressing MtNPF1.7 gene grow larger, have more lateral roots and more nodules when grown in the absence of nitrate and when 0.2 mM KNO3 was provided. At 1 mM KNO3, there are fewer differences between wild type A17 and plants constitutively expressing the MtNPF1.7 gene. Compared with wild type A17, plants constitutively expressing the MtNPF1.7 gene flower earlier, which indicates MtNPF1.7 gene may have a function in plant flowering.