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Investigations of the nitrogen fixing (nif) genes in the cyanobacterium Nostoc commune UTEX 584 revealed a gene encoding a hemoprotein, named cyanoglobin. The cyanoglobin gene was isolated and subcloned into Escherichia coli previously. Cyanoglobin possesses a high oxygen affinity. The study presented here investigated the functional role of cyanoglobin, and encompassed the determination of the kinetic basis for the high oxygen affinity of cyanoglobin through kinetic studies utilizing stopped-flow spectrophotometry and flash photolysis. In addition, studies of cyanoglobin, in the presence of a variety of ligands, employed as structural probes of the distal pocket architecture, are presented. These data are interpreted in terms of structural models of cyanoglobin produced by homology modelling and hemoglobins with known crystal structures. Cyanoglobin coordinated oxygen and a variety of ligands with high rates of association, which explained the high oxygen affinity of cyanoglobin. Cyanoglobin possessed high rates of autoxidation and hemin loss. The ligand binding behavior of cyanoglobin was more similar to leghemoglobin than to sperm whale myoglobin. The ligand binding behavior of cyanoglobin is explained in terms of a highly reactive, and solvent exposed, heme-iron. The 5' region of glbN interacted with NtcA, the global regulator of nitrogen metabolism in cyanobacteria, which may provide an indication of the nitrogen deprivation signal required for cyanoglobin expression in vivo. Finally, the isolation and N-terminal sequencing of a potential cyanoglobin homolog in Anabaena sp. strain PCC 7120 is presented. Collectively, the data obtained in this study may support the model of cyanoglobin function described by Hill, et al., that cyanoglobin sequesters oxygen, and presents it to, or is a part of, a terminal cytochrome oxidase complex in Nostoc commune UTEX 584 under microaerobic conditions, when nitrogen fixation, and thus ATP demand, is maximal. / Ph. D.

Nitrification, the chemoautotrophic process via which ammonium-nitrogen (NH₄-N) is converted to nitrate-nitrogen (NO₃-N), is an important nitrogen (N) transformation in stream ecosystems. Experimental addition of dissolved organic carbon (DOC) has been shown to inhibit rates of nitrification, and rates have been stimulated by NH4-N addition. Insights regarding the role of particulate organic matter (POM) in this scenario could further enhance our understanding of linkages between ecosystem carbon (C) and N cycles. Hugh White Creek, a headwater stream located in the southern Appalachian mountains of North Carolina, USA, receives large amounts of allochthonous POM inputs each fall. To address the effects of these inputs on nitrification, I conducted a seasonal survey of organic matter standing stocks and nitrification rates along with experimental manipulation of dissolved C and N supplies in stream sediment microcosms to determine: 1) how rates of nitrification compare across seasons, and 2) to what extent nitrification rates are influenced by seasonal changes in standing stocks and relative abundances of both sedimentary and dissolved forms of C and N. Rates of nitrification were most closely and positively related to rates of ammonification, which, in turn were negatively related to C:N of fine benthic organic matter (FBOM). Uniform additions of C and N throughout the year had different effects on rates of nitrification and ammonification due to their changing relative importance as sediment organic matter stocks were depleted and underwent changes in quality. Slow rates of nitrification for much of the year could be attributed to large quantities of C relative to N in stream sediments. To the extent that changes in OM stocks dictate change in C and N availability, seasonal patterns in OM dynamics represent changes in ecosystem structure relevant to rates of nitrification, emphasizing the importance of terrestrial/aquatic linkages for predicting rates of N transformation in aquatic ecosystems. / Master of Science

Nitrate contamination of ground and surface waters is of serious concern in the United States. Since nitrate is considered one of the most widespread groundwater contaminants, research continues in many disciplines to identify and mediate possible sources (Petrovic, 1990). An opportunity to evaluate a possible source of nitrate contamination exists during bermudagrass sprig establishment. Since most "improved" bermudagrass varieties are established vegetatively, due to lack of viable seed, turf managers apply large quantities of soluble urea weekly to push growth. During this grow-in, turf managers typically apply 48.8 soluble kg N ha-1 wk-1 for an eight to ten week period. We hypothesize that the quantity of nitrogen applied is in excess and that similar 8 to 10 week grow-in can be achieved with less nitrogen especially during the first 4 weeks when there are few plants per unit area. Three interrelated studies were conducted. A "field trial", consists of 1.8 m2 plots in 4 replications of 27 different nitrogen rates ranging from 4.9 kg N ha-1 wk-1 to 48.8 kg N ha-1 wk-1. A lysimeter study quantified leaching nitrate loss using the same weekly nitrogen rates, however, only six treatments existed. All plots were rated at weeks 4, 8 and 10 for cover, color, and density. A greenhouse study was implemented to evaluate potential nitrogen use for a single bermudagrass sprig, during the early post-sprigging stage. In 1998 and 1999, it appears that typical weekly nitrogen rates of 48.8 kg N ha-1 are in excess. Similar results were found using 48.8 kg N ha-1 to 97.6 kg N ha-1 over the first four week period and 195.2 kg N ha-1 to 244.0 kg N ha-1 during the entire grow-in. A 25 to 50 percent reduction in nitrogen application when compared to the typical 390.4 kg N ha-1. Lysimeter results revealed large quantities of nitrate lost via leaching. The 390.4 kg N ha-1 lost 34.7 and 44.6 percent of applied nitrogen in 1998 and 1999, respectively. Furthermore, similar turfgrass cover, color, and density were achieved using 80 percent less nitrogen during the grow-in period on sand-based media. The greenhouse study suggests that during the early post-sprigging period individual sprigs do not require large quantities of soluble nitrogen even on sand-based media. Results indicated that individual sprigs did not utilize 48.8 kg N ha-1 wk-1 during early post-sprigging. Similar results with respect to shoot weight, shoot length, and root weight were found using 40 percent less applied nitrogen. However, greater nitrogen rates may be necessary as plant density and root development increase later in the establishment period. / Master of Science