Eutrophication of freshwater lakes has led to an increase in the occurrence of harmful cyanobacterial blooms, and it is expected that a warming climate will further exacerbate the frequency and duration of such blooms. Microcystin is a cyanobacterial hepatotoxin that is found worldwide, and poses a serious threat to the ecological communities in which it is found as well as to those who use these waters for drinking, recreation, or as a food source. Although microcystin is known to accumulate in fish and other aquatic biota, the prevalence of microcystin in fish tissue and the human health risks posed by microcystin exposure through fish consumption remain poorly resolved. Very few studies have quantified microcystin (a broadly present cyanotoxin) in water from East African lakes, despite the large human and animal populations that rely on these lakes for both water and food, and to date there is very little information available on the accumulation of microcystin in fish from these lakes. A comprehensive set of water and fish samples was collected on a monthly basis between September 2008 and February 2009 from several lakes in Uganda. The study sites included two embayments in northern Lake Victoria (Murchison Bay and Napoleon Gulf), Lake Edward, Lake George, Lake Mburo, and the crater lakes Saka and Nkuruba. The large lakes sampled all support substantial commercially important fisheries, while the smaller lakes support subsistence fisheries that provide a critically important source of protein and income for riparian communities. Microcystin concentrations in water were determined in addition to chlorophyll and nutrient concentrations, phytoplankton community composition, mixing dynamics and light conditions. At all study sites except Lake Nkuruba, microcystin concentrations in water regularly exceeded the WHO guideline for microcystin in drinking water of 1.0 µg/L. Microcystis spp. emerged as the cyanobacterial taxa that is primarily responsible for microcystin production in these lakes, and as such, microcystin concentrations were closely linked to environmental factors that favour the development of high Microcystis biomass, including high nutrient concentrations, as well as shallow mixing depth which acts to increase mean mixed layer light intensity. Because of the importance of understanding the underlying food web when considering the accumulation and trophic transfer of a compound, stable carbon and nitrogen isotope analysis was used to characterize the food webs at the previously mentioned Ugandan study sites as well as in the East African great lake Albert. Omnivory was found to be common at all study sites, and based on δ13C values, the food webs in these lakes were strongly based on pelagic primary production, with no strong evidence of substantial benthic contribution to these food webs, likely as a result of reduced benthic primary productivity in these generally low-transparency eutrophic lakes. The distribution and trophic transfer of mercury was also characterized in the Ugandan study lakes (including Lake Albert) in order to provide a contrast for the trophic transfer of microcystin in the same lakes. Furthermore, relatively little is known about the behaviour of mercury in tropical hypereutrophic lakes, and the study sites included in the current study provided an opportunity for the exploration of this topic. Consistent biomagnification of mercury was observed at all study sites; however, mercury concentrations in fish were generally low, and would not be expected to pose a risk to consumers. Mercury dynamics were strongly linked to lake trophic status, with biomagnification rates significantly lower at the hypereutrophic study sites than at the mesotrophic and eutrophic study sites. I found evidence that growth and possibly biomass dilution can reduce mercury concentrations at the base of the food web, while growth dilution of mercury at consumer trophic levels might effectively reduce the biomagnification rate of mercury in these hypereutrophic lakes. Microcystin was prevalent in fish muscle tissue from all study sites and at all trophic levels. In contrast to mercury, for which consistent biomagnification was observed, neither biomagnification nor biodilution was observed for microcystin; and concentrations were relatively consistent throughout the fish food web, including in top predators, indicating that efficient trophic transfer of microcystin is occurring in these lakes. Microcystin concentrations in fish from several study sites followed seasonal trends that were similar to those observed for microcystin concentrations in water at these sites, suggesting that fish can rapidly respond to changes in microcystin concentrations in water through accumulation and depuration of this toxin. Microcystin concentrations in water and fish from all Ugandan study sites (including Lake Albert) in addition to data from two temperate eutrophic embayments (Maumee Bay in Lake Erie, and the Bay of Quinte in Lake Ontario) were compiled and used to estimate potential microcystin exposure to human consumers of both water and fish from these study sites. Microcystin was pervasive in water and fish from both the tropical and temperate study sites. Also, these results establish that fish consumption can be an important and even dominant source of microcystin to humans, and can cause consumers to exceed recommended total daily intake guidelines for microcystin. These results highlight the need to consider potential exposure to microcystin through fish consumption in addition to water consumption in order to adequately assess human exposure and risk.

Trichodesmium is a globally significant marine diazotroph responsible for supplying new nitrogen to the oligotrophic regions in which it is found. Though it has been studied for decades, our understanding of the ways in which environmental factors can affect its nitrogen fixation rate remains limited. A continuous culture of Trichodesmium was established in which steady state growth and nitrogen fixation were maintained at dilution rates ranging from 0.27 to 0.67 d-1. Our results clearly show that, as growth rate increased, biomass decreased linearly and nitrogen fixation rate increased linearly. C:N:P ratios remained constant over the range of growth rates studied, suggesting a tight coupling between macronutrient uptake and the maintenance of balanced growth at steady state. We used cultures at steady state to determine the impact of nitrate exposure and uptake on nitrogen fixation. Nitrate inhibits nitrogen fixation by up to 70% in a concentration-dependent manner at initial nitrate concentrations less than 10?? Nitrate uptake accounted for as much as 86% of total N uptake and, at initial nitrate concentrations greater than 2.5 ??more than made up for the observed inhibition of nitrogen fixation. A field study of this diazotroph shows that nitrogen fixation scales with light intensity from a maximum at 50% surface irradiance. Estimated areal nitrogen fixation rates in the Gulf of Mexico, based on vertical abundance profiles and the relationship between nitrogen fixation and surface irradiance, are comparable to measurements made in other oligotrophic regions. Stable isotopic composition of the particulate organic matter and the zooplankton confirms that Trichodesmium nitrogen and carbon are moving into the food chain and are important to higher trophic levels. As much as 60% of the zooplankton carbon was derived from Trichodesmium. Our work established that this diazotroph is ecologically important in the water column of the Gulf of Mexico, with important implications for nitrogen and carbon cycling. Findings from our field and culture studies can be added to models used to quantify the importance of Trichodesmium nitrogen fixation on an oceanic scale.

The study of the in vitro circadian oscillator of the cyanobacterium Synechococcus elongatus has uncovered a complex interplay of its three protein components. Synchronization of the clock's central oscillatory component, KaiC, has been thought to be achieved through subunit shuffling at specific intervals during the clock’s period. By utilizing an established fluorescence-based analysis on completely phosphorylated and dephosphorylated mutants as well as wild-type KaiC, this study has shown that shuffling rates are largely unaffected by phosphorylation state. These findings conflict with previous reports and hence revise our understanding of this oscillator.

This research investigated the existence of <10 £gm nitrogen-fixing unicellular cyanobacteria in the South China Sea. The surveys covered the period from February 2004 to January 2005 and a total of seven cruises. The unicellular cyanobacteria that express orange-yellow from cellular phycoerythrin were observed under a fluorescence microscope. Their expressions in nitrogen-fixation were confirmed by the results from reverse transcription polymerase chain reaction (RT-PCR) and whole cell fluorescence immunolocalization of nitrogenase. The nifH gene sequences of the unicellular cyanobacteria collected from the South China Sea was with >90% identities of their nucleotides similar to the nifH gene sequences of unicellular diazotrophs from ALOHA (Hawaii) as well as Synechocystis sp. WH 8501, Cyanothece sp. ATCC 51142, Cyanothece sp. WH 8902, Cyanothece sp. WH 8904 and Synechococcus sp. RF-1. Positive reactions of fluorescence immunolocalization of nitrogenase were only observed in some, not all, of <10 £gm unicellular cyanobacteria, suggesting that cell counting alone can not be used to estimate nitrogen fixation rate. There was great seasonal and spatial variation in the unicellular cyanobacteria cell density. There was, however, no significant relationship between cell density and the investigated environmental factors. Cell density was high when temperature was high or where stratification index of water column was high, such as in summer or in basin in contrast to other seasons or the shelf-slope regions.

The circadian input kinase gene (cikA) was first identified from a Tn5 mutant of Synechococcus elongatus PCC 7942. A cikA null strain shows a striking phenotype related to circadian gene regulation: all sampled loci show a shortened circadian period and reduced amplitude of oscillation and a failure to exhibit a wild-type resetting of the phase of the rhythm after an environmental signal. This global defect in response to the environment suggests a key role for CikA in the circadian input pathways. Bioinformatics results classify CikA as a divergent member of the bacteriophytochrome family, suggesting a role in light signal transduction. In vitro analysis previously showed that CikA is a bona fide histidine protein kinase (HPK), and its kinase activity is regulated by the presence of other domains. Its own pseudo-receiver (PsR) domain is not the cognate receiver domain of its kinase HPK domain, and its GAF domain does not likely bind a bilin chromophore as do photoreceptive phytochromes. Recent results suggested that CikA may function as a redox-sensor. In this study, we examined the function of each domain of CikA using different mutant cikA alleles, and determined their phenotypes with respect to complementation of a null mutant and overexpression in both wild type and cikA null strains. All domains except the featureless N-terminus were required for CikA function. Overexpression of all mutant alleles that encoded the PsR domain, whether or not the HPK was functional, caused a dominant arrhythmia phenotype. In the absence of PsR, overexpressed variants did not cause arrhythmia, but affected the amplitude and period of oscillation. The results suggest a model in which the PsR domain regulates kinase activity and mediates interaction with other input pathway components to allow CikA to reach the correct cellular position to fulfill its function. Cellular localization assays showed CikA can interact with a complex and showed a polar localization pattern, whereas its variant without PsR showed uniform distribution in the cell. In summary, CikA is an autoregulated kinase in which the PsR domain regulates activity of the HPK domain and also serves as an interaction module to lead the CikA to a specific cellular position.

The study of the in vitro circadian oscillator of the cyanobacterium Synechococcus elongatus has uncovered a complex interplay of its three protein components. Synchronization of the clock's central oscillatory component, KaiC, has been thought to be achieved through subunit shuffling at specific intervals during the clock's period. By utilizing an established fluorescence-based analysis on completely phosphorylated and dephosphorylated mutants as well as wild-type KaiC, this study has shown that shuffling rates are largely unaffected by phosphorylation state. These findings conflict with previous reports and hence revise our understanding of this oscillator.

Thesis (Ph. D.)--University of Hawaii at Manoa, 2003. / Includes bibliographical references (leaves 190-210). Also available via World Wide Web.

Thesis (Ph. D.)--University of Hawaii at Manoa, 2003. / Includes bibliographical references (leaves 190-210).

Haemophilus influenzae type b (Hib) was a main cause of bacterial meningitis. Together with Neisseria meningitidis and Streptococcus pneumoniae, it occurred with comparable frequency prior to the introduction of conjugated polysaccharide vaccines against Hib. Despite the fact that the introduction of conjugated vaccines against Hib has virtually eradicated infection in many areas of the globe, this pathogen still causes many infections in developing countries and a number of cases of infection have been reported in fully vaccinated children in developed countries. Recent studies have focused on the mechanisms by which these pathogens invade the central nervous system through the blood brain barrier (BBB). One study revealed that the outer membrane protein OmpP2 of H. influenzae interacted with the 37/67-kDa non-integrin laminin receptor (LamR) of human brain microvascular endothelial cells (HBMECs). In this study, OmpP2 of H. influenzae was expressed and purified and its interactions with purified recombinant LamR (rLamR) were analysed. OmpP2, which is predicted to contain eight extracellular loops, was expressed in two parts: OmpP2Δ1-4, lacking loops 1-4, and OmpP2Δ5-8, lacking loops 5-8. The protein fragments were purified and their interaction with rLamR was investigated by ELISA. The LamR binding site of OmpP2 was found to be restricted to loops 1-4. Therefore, ompP2 derivatives encoding OmpP2 lacking loops 1, 2, 3 and 4 individually, were constructed. The OmpP2 derivatives were expressed and tested in rLamR-binding assays to determine which of these loops is required for LamR binding. Only OmpP2∆L2 showed dramatically decreased binding to rLamR. Accordingly, loop 2 of OmpP2 may play an important role in the interaction of H. influenzae with LamR. Therefore, ompP2 and ompP2ΔL2 null mutants were generated in H. influenzae strain Rd KW20 and utilized in subsequent characterization experiments to facilitate a study of the potential role of OmpP2 and its second loop in the pathogenesis of H. influenzae. The interaction of H. influenzae and its derivative mutants to LamR was investigated using a combination of molecular and immunological techniques, including ELISA, whole cell lysate pull-down assays, invasion and association assays and flow cytometry. H. influenzae cells either lacking OmpP2 or expressing OmpP2∆L2 showed significantly reduced rLamR-binding compared to the wild type. Furthermore, synthetic peptides based on the loop 2 sequence coupled to micro-beads mediated adherence of the beads to HBMECs. The amino acid sequence of OmpP2 loop 2 was found to be highly conserved in Hib isolates, but not in non-typeable H. influenzae, which rarely cause invasive disease. The potential roles of OmpP2 and Loop 2 in H. influenzae pathogenesis are discussed.

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