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 / incomplete_metadata

Young mine soils constructed following surface-mining for coal contain low levels of organic matter and nitrogen. It was hypothesized that nitrogen-fixing plants could be incorporated into a reforestation system in a manner that would meet the short term and long-term nitrogen needs of crop trees while rebuilding the soil and litter nitrogen pools and restoring a stable nitrogen cycle. The nitrogen status of two interplanting studies containing legume ground covers was examined. The first study site was mined prior to 1940 and subsequently abandoned. In 1988, the land was reclaimed, and an interplanting study was established in 1990. Pitch x loblolly pines (Pinus x rigitaeda) and eastern white pines (Pinus strobus L.) were interplanted with four nitrogen-fixing species: black alder (Alnus glutinosa L. IGaertn.l), black locust (Robinia pseudoacacia L.}, bicolor lespedeza (Lespedeza bicolor) and autumn olive (Elaeagnus umbel/ata Thwnb.). Pitch x loblolly pine survival was good; however, eastern white pine survival was only fair, averaging 54%. All of the nurse tree species with the exception of black alder had fair survival rates. Black alder survival was extremely low at 19%. After the third growing season, a ground cover dominated by legumes averaged 79% cover. The black mine spoil on the site was extremely rich in nitrogen, averaging 5,115 mg·kg-1. When the black mine spoil was mixed with the native topsoil, a mine soil that was created had an extremely variable total nitrogen content. However, low mineralizeable nitrogen levels suggested that the nitrogen in the mine spoil was not generally available. Pine responses to the four nitrogen-fixing species were compared. Nurse trees had no effect on pine growth after three years. Pine foliar nitrogen levels were adequate across the study site even in the control treatments and in the microsites where few legumes were present. Microsites surrounding 50 pines of each species were studied. The pitch x loblolly pines grew larger at lower pH levels and higher coarse fragment content reflecting the pines' preference for moderately acid soils, coarse-textured soils. Some competition was present between the crop trees and herbaceous cover, but it was not a controlling factor in pine tree growth. / Master of Science

Master of Science

Nitrogen fixation is no longer considered to be a minor factor of the nitrogen cycle in oceanic ecosystems. Recent geochemical and biological efforts have led to a significant increase in the estimated input of nitrogen to marine ecosystems by biological fixation, while molecular studies have increased our knowledge of the number and diversity of nitrogen fixers known to be active in the ocean. Although Trichodesmium spp. have long been viewed as the primary marine nitrogen fixers, recent efforts have shown that various members of the picoplankton community are also actively involved in nitrogen fixation. The relative abundance of different nitrogen fixers is an important ecosystem parameter since nitrogen fixers may differ significantly in their physiology, life history and ecology. Here we combine rate measurements and stable isotope natural abundance measurements to constrain the impact of N2 fixation in the waters north of Australia. Samples were collected in the Coral, Arafura, and East Timor Seas, thus spanning three distinct hydrographic regions. Our data show that Trichodesmium has a significant influence on the stable nitrogen isotope ratios of particulate and zooplankton biomass and suggest that Trichodesmium is a significant source of nitrogen for the pelagic ecosystem. Based on stable carbon isotope ratios, it is also likely that the pathways are indirect and nitrogen fixed by Trichodesmium enters the higher trophic levels via decomposition as dissolved organic and inorganic nitrogen. Picocyanobacteria showed high diazotrophic activity at some stations, but unlike Trichodesmium, their N2 fixation rate was not reflected in the stable N isotope ratios of particulate and zooplankton biomass. Our results suggest an important N contribution to biomass by diazotrophs in the Coral Sea, Arafura Sea and East Timor Sea.

Nitrogen fixation is an ecologically important microbial process that can contribute bioavailable combined N to habitats low in N. Shipworms, or wood-boring bivalves, host N2-fixing and cellulolytic symbiotic bacteria in gill bacteriocytes, which have been implicated as a necessary adaptation to an N-poor C-rich (wooden) diet. Shipworm symbionts are known to fix N within the gill habitat and newly fixed N is subsequently incorporated into non-symbiont containing host tissue. The presence of N2-fixation in gill bacteriocytes presents a conundrum because N2-fixation is tightly regulated by oxygen in most other diazotrophic microbes. Also, the direct evidence of new N being incorporated into the host tissue indicates that there are potentially complex nutrient cycles in this symbiosis, which have not been investigated. We used the cultivated symbiont Teredinibacter turnerae, which has been isolated from many shipworm species, as a model organism to elucidate controls on N2-fixation and N release in the shipworm symbiosis. Our results indicate that headspace oxygen concentration does not control biomass specific N2-fixation and respiration activity in T. turnerae, but it does influence the magnitude of the growth rate and timing of culture growth. Also, we examined the controls of oxygen on inorganic nutrient uptake rates, and documented a small amount of dissolved inorganic nitrogen release. While the N budget is only partially balanced, we provide indirect evidence for the allocation of fixed N to the excretion of exopolymeric substances and dissolved organic nitrogen; future studies that measure these additional N sinks are necessary to close the N budget. Although there are limitations of using pure cultures to investigate a complex symbiotic system, this study provides direct experimental evidence that T. turnerae has adaptations that are conducive to N2-fixation in gill bacteriocytes.

Legumes play an important role in agriculture as major food sources for humans and as feed for animals. Bioavailable nitrogen is a limiting nutrient for crop growth. Legumes are important because they can form a symbiotic relationship with soil bacteria called rhizobia that results in nitrogen-fixing root nodules. In this symbiosis, rhizobia provide nitrogen to the legumes and the legumes provide carbon sources to the rhizobia. The Medicago truncatula NPF1.7/NIP/LATD gene is essential for root nodule development and also for proper development of root architecture. Work in our lab on the MtNPF1.7/MtNIP/LATD gene has established that it encodes a nitrate transporter and strongly suggests it has another function. Mtnip-1/latd mutants have pleiotropic defects, which are only partially explained by defects in nitrate transport. MtNPF1.7/NIP/LATD is a member of the large and diverse NPF/NRT1(PTR) transporter family. NPF/NRT1(PTR) members have been shown to transport other compounds in addition to nitrate: nitrite, amino acids, di- and tri-peptides, dicarboxylates, auxin, abscisic acid and glucosinolates. In Arabidopsis thaliana, the AtNPF6.3/NRT1.1( CHL1) transporter was shown to transport auxin as well as nitrate. Atchl1 mutants have defects in root architecture, which may be explained by defects in auxin transport and/or nitrate sensing. Considering the pleiotropic phenotypes observed in Mtnip-1/latd mutant plants, it is possible that MtNPF1.7/NIP/LATD could have similar activity as AtNPF6.3/NRT1.1(CHL1). Experimental evidence shows that the MtNPF1.7/NIP/LATD gene is able to restore nitrate-absent responsiveness defects of the Atchl1-5 mutant. The constitutive expression of MtNPF1.7/NIP/LATD gene was able to partially, but not fully restore the wild-type phenotype in the Atchl1-5 mutant line in response to auxin and cytokinin. The constitutive expression of MtNPF1.7/NIP/LATD gene affects the lateral root density of wild-type Col-0 plants differently in response to IAA in the presence of high (1mM) or low (0.1 mM) nitrate. MtNPF1.7/NIP/LATD gene expression is not regulated by nitrate at the concentrations tested and MtNPF1.7/NIP/LATD does not regulate the nitrate-responsive MtNRT2.1 gene. Mtnip-1 plants have an abnormal gravitropic root response implicating an auxin defect. Together with these results, MtNPF1.7/NIP/LATD is associated with nitrate and auxin; however, it does not act in a homologous fashion as AtNPF6.3/NRT1.1(CHL1) does in A. thaliana.

South Africa has an array of mining commodities which all play an integral role in our everyday surroundings, income, and most importantly, in the economy of the country. These mining activities also produce vast amounts of discard material, better known as tailings material, which is stored in different ways after extraction has taken place. Usually, storage entails the construction of tailings storage facilities, normal discard or tailings dumps. The upper surfaces of these anthropogenic structures are usually unstable and are, in most cases, characterised by different forms of erosion. This can be due to the chemical and physical properties of the materials of which they are constructed, but mainly due to unstable construction geomorphology, steep slopes, which leads to poor water run-off management and subsequent instability. Therefore, these structures need to be actively managed in order to increase and maintain their stability. Grass establishment, as a stabilisation technique, is the most effective out of all of the techniques, but there are certain constraints regarding this method (Titshall et al. 2013). The most costly constraint is nutrient supplementation during aftercare phases. In order to minimize this cost, new and innovative technologies need to be explored, and trialled. The contribution of soil biological processes in this regard was assessed, in order to minimise anthropogenic inputs. These biological processes refer to the fixation of atmospheric nitrogen by nodular root bacteria that grow on a group of plants referred to as legumes. These bacteria, also known as rhizobia, live in a symbiotic relationship with the host plant where they receive energy in the form of nutrients by trading nitrogen, which is an essential plant nutrient. Nine different tailings materials from different commodities available from South African Mines were selected. For a control medium, a well-drained soil type with an apedel structure and a clay content of approximately 6% was selected in order to promote optimal natural growth. These materials were chemically and physically analysed in order to develop a more holistic understanding on a micro scale level, as well as to ascertain possible constraints in this regard. Pot trials were selected as the experimental method in order to apply more specific control over root growth, plant development and growing conditions. The experimental data were collected over one growing season for both live forms. For this study, seven legume species were selected for establishment in the tailings materials in order to investigate their establishment potential in the growth mediums and their ability to fixate nitrogen. Based on the data, specific species were identified as viable options to include in future tailings amelioration projects; it can be assumed that the nitrogen produced by these species will be available in the growth medium for uptake by neighbouring plants that lack this biological function. These plants will also play a vital role in the long-term sustainable development of vegetation in the anthropogenic growth mediums. Sericea lespedeza had the highest enrichment ability during this study. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

South Africa has an array of mining commodities which all play an integral role in our everyday surroundings, income, and most importantly, in the economy of the country. These mining activities also produce vast amounts of discard material, better known as tailings material, which is stored in different ways after extraction has taken place. Usually, storage entails the construction of tailings storage facilities, normal discard or tailings dumps. The upper surfaces of these anthropogenic structures are usually unstable and are, in most cases, characterised by different forms of erosion. This can be due to the chemical and physical properties of the materials of which they are constructed, but mainly due to unstable construction geomorphology, steep slopes, which leads to poor water run-off management and subsequent instability. Therefore, these structures need to be actively managed in order to increase and maintain their stability. Grass establishment, as a stabilisation technique, is the most effective out of all of the techniques, but there are certain constraints regarding this method (Titshall et al. 2013). The most costly constraint is nutrient supplementation during aftercare phases. In order to minimize this cost, new and innovative technologies need to be explored, and trialled. The contribution of soil biological processes in this regard was assessed, in order to minimise anthropogenic inputs. These biological processes refer to the fixation of atmospheric nitrogen by nodular root bacteria that grow on a group of plants referred to as legumes. These bacteria, also known as rhizobia, live in a symbiotic relationship with the host plant where they receive energy in the form of nutrients by trading nitrogen, which is an essential plant nutrient. Nine different tailings materials from different commodities available from South African Mines were selected. For a control medium, a well-drained soil type with an apedel structure and a clay content of approximately 6% was selected in order to promote optimal natural growth. These materials were chemically and physically analysed in order to develop a more holistic understanding on a micro scale level, as well as to ascertain possible constraints in this regard. Pot trials were selected as the experimental method in order to apply more specific control over root growth, plant development and growing conditions. The experimental data were collected over one growing season for both live forms. For this study, seven legume species were selected for establishment in the tailings materials in order to investigate their establishment potential in the growth mediums and their ability to fixate nitrogen. Based on the data, specific species were identified as viable options to include in future tailings amelioration projects; it can be assumed that the nitrogen produced by these species will be available in the growth medium for uptake by neighbouring plants that lack this biological function. These plants will also play a vital role in the long-term sustainable development of vegetation in the anthropogenic growth mediums. Sericea lespedeza had the highest enrichment ability during this study. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

The growth and Nitrogen fixation by one low salt tolerant alfalfa (Medicago sativa L.) and two germination salt tolerant selections inoculated with were investigated at two salt levels (0, -0.6 Mpa) and two N rates (1, 5ppm) using a system which automatically recirculates a nutrient solution. The high level of salinity (-0.6 Mpa osmotic potential of culture solution) resulted in substantial reduction in the N fixation percentage and total fixed N. The effect of salinity was more pronounced for later cuttings than for the earlier cutting. The N fixation percentages were substantially decreased by increasing N level and the reduction was enhanced by time. The N treatment levels did not exhibit a significant effect on total fixed N. Cultivars did not differ in either growth or N fixation. However, the interaction of N and salinity significantly decreased the percentage and amount of N fixation.

Sanders, Donald W. The University of Manitoba, March, 2017. The effect of preceding crop on soybean (Glycine max) grain yield, mycorrhizal colonization, and biological nitrogen fixation. Major Professor:Yvonne Lawley. Manitoba has seen a twenty-fold increase in soybean acres seeded since 2000, with over 1.6 million acres seeded in 2016. This change presents unique opportunities and challenges to improve crop rotations in Manitoba. This experiment studied the effect of four crop sequences on soybean yield, mycorrhizal colonization, and biological nitrogen fixation. In the first year of this experiment, spring wheat, canola, corn and soybeans were grown at three sites in Manitoba (Carman, Portage la Prairie, and Kelburn). In the second year, soybeans were grown on these same plots as a test crop. This two-year sequence of crops was done twice at each site, in 2012-13 and 2013-14. To determine mycorrhizal colonization, root samples were collected at the V3 stage and then analyzed microscopically for mycorrhizal infection. Nitrogen fixation was estimated using the natural abundance method using soybeans collected at the R5 and R6 stage and canola as a reference crop. Soybean following soybean had significantly higher grain yield than all other crop sequences at one site year, and significantly lower grain yield than all other crop sequences at another site year. There were no other differences in soybean test crop yield between crop sequences. Crop sequence significantly affected mycorrhizal colonization. Soybean following canola had significantly lower mycorrhizal colonization than soybean following soybean or corn. Soybean following spring wheat also had significantly lower mycorrhizal colonization than soybean following soybean or corn. Soil test phosphorus levels also significantly affected mycorrhizal colonization, with increasing soil phosphorus resulting in decreased mycorrhizal colonization. Crop sequence significantly affected biological nitrogen fixation. Soybean following soybean or corn often had significantly greater biological nitrogen fixation than soybean following spring wheat or canola. Soil test nitrate levels affected biological nitrogen fixation, with increasing soil nitrate resulting in decreased biological nitrogen fixation. Soil test nitrate levels were affected by the carbon to nitrogen ratio of the preceding crop, with a higher carbon to nitrogen ratio associated with decreased soil nitrate. These results indicate that although there is often not a yield penalty associated with specific rotations, crop sequence has a strong impact on mycorrhizal colonization and biological nitrogen fixation. The soil organisms associated with those processes affect soil phosphorus uptake and nitrogen fixation. Producers should consider the importance of crop rotation when seeking to maximize productivity through symbiotic processes with mycorrhizae and nodule forming bacteria. / May 2017