Thesis (Ph. D.)--Oregon State University, 2006. / Printout. Includes bibliographical references (leaves 223-233). Also available on the World Wide Web.

Thesis (PhD)--Montana State University--Bozeman, 2010. / Typescript. Chairperson, Graduate Committee: David M. Ward. Includes bibliographical references.

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

Biogeochemical processes in the marine biosphere are important in global element cycling and greatly influence the gas composition of the Earth’s atmosphere. The nitrogen cycle is a key component of marine biogeochemical cycles. Nitrogen is an essential constituent of living organisms, but bioavailable nitrogen is often short in supply thus limiting primary production. The largest input of nitrogen to the marine environment is by N2-fixation, the transformation of inert N2 gas into bioavailable ammonium by a distinct group of microbes. Hence, N2-fixation bypasses nitrogen limitation and stimulates productivity in oligotrophic regions of the marine biosphere. Extensive blooms of N2-fixing cyanobacteria occur regularly during summer in the Baltic Sea. N2-fixation during these blooms adds several hundred kilotons of new nitrogen into the Baltic Proper, which is similar in magnitude to the annual nitrogen load by riverine discharge and more than twice the atmospheric nitrogen deposition in this area. N2-fixing cyanobacteria are therefore a critical constituent of nitrogen cycling in the Baltic Sea. In this thesis N2 fixation of common cyanobacteria in the Baltic Sea and the direct fate of newly fixed nitrogen in otherwise nitrogen-impoverished waters were investigated. Initially, the commonly used 15N-stable isotope assay for N2-fixation measurements was evaluated and optimized in terms of reliability and practicality (Paper I), and later applied for N2-fixation assessments (Paper II–IV). N2 fixation in surface waters of the Baltic Sea was restricted to large filamentous heterocystous cyanobacteria (Aphanizomenon sp., Nodularia spumigena, Dolichospermum spp.) and absent in smaller filamentous cyanobacteria such as Pseudanabaena sp., and unicellular and colonial picocyanobacteria (Paper II-III). Most of the N2-fixation in the Northern Baltic Proper was contributed by Aphanizomenon sp. due to its high abundance throughout the summer and similar rates of specific N2-fixation as Dolichospermum spp. and N. spumigena. Specific N2 fixation was substantially higher near the coast than in an offshore region (Paper II). Half of the fixed nitrogen was released as ammonium at the site near the coast and taken up by non-N2-fixing organisms including phototrophic and heterotrophic, prokaryotic and eukaryotic planktonic organisms. Newly fixed nitrogen was thereby rapidly turned-over in the nitrogen-depleted waters (Paper III). In colonies of N. spumigena even the potential for a complete nitrogen cycle condensed to a microcosm of a few millimeters could be demonstrated (Paper IV). Cyanobacterial colonies can therefore be hot-spots of nitrogen transformation processes potentially including nitrogen gain, recycling and loss processes. In conclusion, blooms of cyanobacteria are instrumental for productivity and CO2 sequestration in the Baltic Sea. These findings advance our understanding of biogeochemical cycles and ecosystem functioning in relation to cyanobacterial blooms in the Baltic Sea with relevance for both ecosystem-based management in the Baltic Sea, and N2-fixation and nitrogen cycling in the global ocean. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.</p>

Streptococcus uberis is a Gram-positive, catalase-negative member of the family Streptococcaceae and is an important environmental pathogen primarily responsible for a significant amount of bovine intramammary infections. This thesis describes the sequencing and comparison of multiple strains from clinical and sub-clinical infections. Following de novo assembly, these are compared to the single reference strain (0140J). The assemblies of strains sequenced with two technologies (Illumina and SOLiD) were compared. From these assemblies, annotation allowed the comparison of gene content, the pan and core genomes and gene gain/loss of coding sequences associated with clustered regularly interspaced short palindromic repeats (CRISPRs), prophage and bacteriocin production. Identification of sequence variants allowed identification of highly conserved and highly variant genes. Inferred intraspecies and interspecies (host-S. uberis) protein-protein interaction networks revealed pathways of bovine proteins enriched with potentially interacting pathogen proteins. These identified known and predicted pathways and also novel interaction partners. This was the first “whole-genome” comparison of multiple S. uberis strains isolated from clinical vs non-clinical intramammary infections including the type virulent vs non-virulent strains. These data allowed the first insight into potential evolutionary forces behind virulence differences.

abstract: Some cyanobacteria, referred to as boring or euendolithic, are capable of excavating tunnels into calcareous substrates, both mineral and biogenic. The erosive activity of these cyanobacteria results in the destruction of coastal limestones and dead corals, the reworking of carbonate sands, and the cementation of microbialites. They thus link the biological and mineral parts of the global carbon cycle directly. They are also relevant for marine aquaculture as pests of mollusk populations. In spite of their importance, the mechanism by which these cyanobacteria bore remains unknown. In fact, boring by phototrophs is geochemically paradoxical, in that they should promote precipitation of carbonates, not dissolution. To approach this paradox experimentally, I developed an empirical model based on a newly isolated euendolith, which I characterized physiologically, ultrastructurally and phylogenetically (Mastigocoleus testarum BC008); it bores on pure calcite in the laboratory under controlled conditions. Mechanistic hypotheses suggesting the aid of accompanying heterotrophic bacteria, or the spatial/temporal separation of photosynthesis and boring could be readily rejected. Real-time Ca2+ mapping by laser scanning confocal microscopy of boring BC008 cells showed that boring resulted in undersaturation at the boring front and supersaturation in and around boreholes. This is consistent with a process of uptake of Ca2+ from the boring front, trans-cellular mobilization, and extrusion at the distal end of the filaments (borehole entrance). Ca2+ disequilibrium could be inhibited by ceasing illumination, preventing ATP generation, and, more specifically, by blocking P-type Ca2+ ATPase transporters. This demonstrates that BC008 bores by promoting calcite dissolution locally at the boring front through Ca2+ uptake, an unprecedented capacity among living organisms. Parallel studies using mixed microbial assemblages of euendoliths boring into Caribbean, Mediterranean, North and South Pacific marine carbonates, demonstrate that the mechanism operating in BC008 is widespread, but perhaps not universal. / Dissertation/Thesis / Ph.D. Microbiology 2010

abstract: Like most other phototrophic organisms the cyanobacterium Synechocystis sp. PCC 6803 produces carotenoids. These pigments often bind to proteins and assume various functions in light harvesting, protection from reactive oxygen species (ROS) and protein stabilization. One hypothesis was that carotenoids bind to the surface (S-)layer protein. In this work the Synechocystis S-layer protein was identified as Sll1951 and the effect on the carotenoid composition of this prokaryote by disruption of sll1951 was studied. Loss of the S-layer, which was demonstrated by electron microscopy, did not result in loss of carotenoids or changes in the carotenoid profile of the mutant, which was shown by HPLC and protein analysis. Although &#916;sll1951 was more susceptible to osmotic stress than the wild type, the general viability of the mutant remained unaffected. In a different study a combination of mutants having single or multiple deletions of putative carotenoid cleavage dioxygenase (CCD) genes was created. CCDs are presumed to play a role in the breakdown of carotenoids or apo-carotenoids. The carotenoid profiles of the mutants that were grown under conditions of increased reactive oxygen species were analyzed by HPLC. Pigment lifetimes of all strains were estimated by 13C-labeling. Carotenoid composition and metabolism were similar in all strains leading to the conclusion that the deleted CCDs do not affect carotenoid turnover in Synechocystis. The putative CCDs either do not fulfill this function in cyanobacteria or alternative pathways for carotenoid degradation exist. Finally, slr0941, a gene of unknown function but a conserved genome position in many cyanobacteria downstream of the &#948;-carotene desaturase, was disrupted. Initially, the mutant strain was impaired in growth but displayed a rather normal carotenoid content and composition, but an apparent second-site mutation occurred infrequently that restored growth rates and caused an accumulation of carotenoid isomers not found in the wild type. Based on the obtained data a role of the slr0941 gene in carotenoid binding/positioning for isomerization and further conversion to mature carotenoids is suggested. / Dissertation/Thesis / Ph.D. Microbiology 2011

abstract: Molybdenum (Mo) is a key trace nutrient for biological assimilation of nitrogen, either as nitrogen gas (N2) or nitrate (NO3-). Although Mo is the most abundant metal in seawater (105 nM), its concentration is low (<5 nM) in most freshwaters today, and it was scarce in the ocean before 600 million years ago. The use of Mo for nitrogen assimilation can be understood in terms of the changing Mo availability through time; for instance, the higher Mo content of eukaryotic vs. prokaryotic nitrate reductase may have stalled proliferation of eukaryotes in low-Mo Proterozoic oceans. Field and laboratory experiments were performed to study Mo requirements for NO3- assimilation and N2 fixation, respectively. Molybdenum-nitrate addition experiments at Castle Lake, California revealed interannual and depth variability in plankton community response, perhaps resulting from differences in species composition and/or ammonium availability. Furthermore, lake sediments were elevated in Mo compared to soils and bedrock in the watershed. Box modeling suggested that the largest source of Mo to the lake was particulate matter from the watershed. Month-long laboratory experiments with heterocystous cyanobacteria (HC) showed that <1 nM Mo led to low N2 fixation rates, while 10 nM Mo was sufficient for optimal rates. At 1500 nM Mo, freshwater HC hyperaccumulated Mo intercellularly, whereas coastal HC did not. These differences in storage capacity were likely due to the presence in freshwater HC of the small molybdate-binding protein, Mop, and its absence in coastal and marine cyanobacterial species. Expression of the mop gene was regulated by Mo availability in the freshwater HC species Nostoc sp. PCC 7120. Under low Mo (<1 nM) conditions, mop gene expression was up-regulated compared to higher Mo (150 and 3000 nM) treatments, but the subunit composition of the Mop protein changed, suggesting that Mop does not bind Mo in the same manner at <1 nM Mo that it can at higher Mo concentrations. These findings support a role for Mop as a Mo storage protein in HC and suggest that freshwater HC control Mo cellular homeostasis at the post-translational level. Mop's widespread distribution in prokaryotes lends support to the theory that it may be an ancient protein inherited from low-Mo Precambrian oceans. / Dissertation/Thesis / Ph.D. Geological Sciences 2011

Masters of Science / South African dams and reservoirs are increasingly showing the propensity to support sustained populations of Cyanobacteria (blue green algae). These photosynthetic bacteria occur throughout the world and can rapidly form blooms in eutrophic water systems. The occurrence of these photosynthetic bacteria, in our dwindling drinking water source dams, poses a serious, economic, as well as a health, threat to and arid country like South Africa due to is potential to produce of toxic metabolites like Microcystins and Nodularins (MCN). MCN's are cyclic peptides toxins, harmful to humans and animals, and its toxicological mechanism is based on a strong inhibition of protein phosphatises in the liver. This may lead to severe liver damage and increased tumour development. Rural communities consuming untreated water in South Africa are most at risk due the high toxicity of MCN’s at low doses.We endeavour to develop an immunosensor for the detection of Microcystins and nodularins using anti-sheep IgG antibody labelled with horseradish peroxidase (HRP) immobilised on a modified glassy-carbon polymer surface. The immunosensor will be applied to water samples for MCN’s as a group of compounds recognised by the ADDA moiety common to all MCN congeners. The immunosensor will provide immediate confirmation and quantification of MCN’s in situ. A competitive Enzyme Linked Immuno-Sorbant Assay (ELISA) and High Performance liquid Chromatography (HPLC) will be used to validate results of our immunosensor. Elisa's are widely used as a screening test method for MCN's. The antibody-antigen specificity forms the bases for the recognition of target compound (MCN's) by antibodies which bind to a compound which is labelled with a colour indicator, and quantified by spectrophotometry.

Cyanobacterial secondary metabolites are a rich source of bioactive compounds with potential utilization in pharmacology. The aim of this study was to evaluate the effect of the novel compound nostatin A isolated from the cyanobacterium Desmonostoc muscorum. Project was focused on the extraction, purification and characterization of the cytostatic effect caused by this novel compound and its naturally occurred structural analogues. The cytostatic activity of nostatin A and its analogs was evaluated in HeLa cell line. Experiments based on microscopy, flow cytometry and HPLC-HRMS techniques were performed in order to clarify the cytostatic effect of nostatin A in HeLa cells and its mechanism of the action.