Bacillus thuringiensis subsp. isrealensis (B.t.i.) produces a crystalline endotoxin specific for some larvae of mosquitoes that are vectors of the malaria parasite and other infectious diseases. Fragments were obtained from the 108 kb plasmid from B.t.i. strain 4Q2 which encodes several proteins comprising the delta-endotoxin. These DNA fragments were inserted into the hybrid cyanobacterial cloning vector, pTNTV, downstream from its powerful lambda promoter, and the chimaeras were transformed into Escherichia coli. Ampicillin resistant transformants were screened with radioactively labelled oligonucleotides whose sequences were determined from the published sequences of the B.t.i. 130 kDa polypeptide. Clones showing hybridization were used in bioassays to determine their level of toxicity to the fourth instar larvae of the Aedes aegypti mosquito. Twelve clones were found that demonstrated toxicity which was statistically significantly greater than that observed in controls. Plasmid DNA from some of these clones was isolated, cut with restriction endonucleases, and viewed through agarose gel electrophoresis to confirm that B.t.i. fragments had been inserted into the vector. Future work will investigate the expression of these cloned toxin genes in transformable cyanobacteria and will determine their subsequent activity against the fourth instar larvae of Aedes aegypti and Anopheles quadrimaculatus. / Department of Biology

A C. perfringens type A genomic library was constructed in E. coli by banking overlapping 6-10 kbp Hind III fragments of chromosomal DNA from the enterotoxin (CPE) positive strain NCTC 8239 into the pUC derived vector pHG165. The library was screened by colony hybridization with a degenerate 26 bp oligonucleotide probe, derived from the amino acid sequence CPE9_17A. complex mixture of plasmid DNA was isolated from the only hybridization positive clone. A second round of screening picked out a single plasmid, with an apparently altered copy number, pLWl, that carried the CPE gene, cpe, on a 6.8 kbp insert. A sequence deduced primer strategy for direct plasmid sequencing was initiated using a primer deduced in a similar manner to the 26 bp probe, obviating the need for prior mapping and subcloning of the insert. The amino acid sequence for the conceptual gene product of the single open reading frame differed only slightly from the known CPE sequence but lacked the C terminal residues. The biased cpe codon usage reflected the low %G+C content of the DNA. The %G+C content was even lower in the upstream region and possessed properties characteristic of bent DNA. The region 5' to the ATG translational start codon contained a Shine-Dalgarno sequence and several sequences with significant homology to the putative transcriptional control regions for the tetanus toxin gene. The N-terminal coding region contained a direct repeat of an upstream sequence that shared considerable homologies with the crossover point in site 1 of the Tn3 res region. Southern blot analyses of chromosomal and plasmid DNAs from several isolates indicated that the majority of strains were cpe-. The chromosomal location and architecture of cpe appeared identical in all cpe+ strains. A second copy, pLW2, of the 5' end of cpe, on a 4.5 kbp Pst I/Eco RI restriction fragment, was cloned during one of many unsuccessful attempts to clone the 3' end. A separate re-cloning experiment isolated several different clones that contained the 0.6 kbp Hind III located = 2.5 kbp 5' to the ATG codon of both cloned copies of cpe but none of them carried the CPE gene. The fragment was used as a DNA probe to show that it was present in high copy number in some strains of C. perfringens but completely absent from others. An hypothesis describing the possible involvement of a mobile genetic element in C. perfringens enterotoxin production offers explanations for the cloning of a complex mixture of plasmids, the apparent alteration in plasmid copy number, the identification of putative DNA crossover points, the failure to clone the 3' end of cpe and the isolation of a novel DNA fragment.

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.

The release of the neurotoxic alkaloid saxitoxin by cyanobacterial cells was previously thought to occur primarily after cell lysis, yet recent evidence also suggests active toxin export by membrane transporters. Transporter proteins associated with STX biosynthesis in Cylindrospermopsis raciborskii T3 (sxtF and sxtM) and Anabaena circinalis 131C (naDt) were predicted to be involved in the export of STX from cyanobacterial cells. The main aim of this project was to characterise the transporters associated with STX biosynthesis, by investigation of their genetic prevalence, functional substrates and specific regulation. An sxtM homologue was discovered in A. circinalis 131C, as part of an sxt cluster, and found to be uniquely associated with STX-producing strains. Bioinformatic and phylogenetic analysis showed that the translated sxt transporters clustered with the NorM prokaryotic MATE sub-family and membrane topology analysis predicted 12 membrane-spanning regions. To characterise the functional substrates of the putative STX-transporters, they were heterologously expressed in the antibiotic-sensitive E. coli strain KAM32. Expression of the sxt MATES complemented host sensitivity to the cationic fluroquinolone antibiotics, ciprofloxacin and ofloxacin. Disruption of gene homologues of naDt and the sxt MATE genes in Synechocystis sp. PCC6803 yielded mutant strains with increased sensitivity to the toxic organic cations, methyl viologen and acriflavine. Transcription of the putative STX transporters, and the putative STX biosynthesis gene sxtA, was studied in C. raciborskii T3 and A. circinalis 131C under alkali and Na+ stress. Alkali stress (pH 9) decreased total STX levels in A. circinalis 131C and was correlated with a down-regulation of the putative transport and biosynthetic genes. In C. raciborskii T3, alkali stress promoted higher extracellular but lower intracellular STX levels, which also correlated with large increases in transcription of the putative STX transport genes.

The role and function of hepatotoxins microcystin and nodularin produced by M.aeruginosa PCC 7806 and N. spumigena NSORlO respectively have yet to be elucidated. The mode of transcriptional regulation of these toxins, incorporating DNA binding proteins, was investigated, as an attempt to further understand the key control mechanisms acting on the toxins. The DNA binding proteins that control nitrogen and iron responsive transcription, NtcA and Fur, were identified from M. aeruginosa PCC7806 and N. spumigena NSOR10. Cloning and over-expression in E. coli was followed by mobility shift assays to determine binding characteristics of NtcA and Fur to the promoters, mcyA/D and ndaA/C, those regions that control the toxin encoding gene clusters in M. aeruginosa PCC 7806 and N. spumigena NSOR10, respectively. The results from these studies suggested a role for iron and nitrogen in the transcriptional control of microcystin and nodularin. biosynthesis. As NtcA and Fur classically act to regulate nitrogen and iron dependent genes, a link may be made to the putative function and control of microcystin and nodularin. By identifying the transcription factors NtcA and Fur in these genera, a greater understanding of the link between nutrient levels in the environment and hepatotoxin production in cyanobacteria may be possible.

Thesis (M.S.)--Ohio University, August, 2003. / Title from PDF t.p. Includes bibliographical references (leaves 100-101).

Thesis (Ph. D.), Memorial University of Newfoundland, 1999. / Bibliography: p. 258-279.

Thesis (Ph. D.)--Biology, Georgia Institute of Technology, 2006. / Klausmeier, Christopher, Committee Member ; Montoya, Joseph, Committee Member ; Snell, Terry, Committee Member ; Sarnelle, Orlando, Committee Member ; Hay, Mark, Committee Chair.

Cancer-related diseases are mostly associated with reduced or inappropriate cell death. This thesis focuses on secondary cyanobacterial metabolites which induce apoptosis in human cancer cells in vitro and thus may serve as potential drug hits. Screening and selection of active natural extracts clearly precede activity-guided isolation of a bioactive compound itself. Summarizing the results of phenotypic screening of cyanobacterial extracts for inducers of apoptosis, I show that adjustment of measurement the activity of key apoptotic enzymes, caspases, per cell significantly enlarges the pool of detected hits. This could be of particular importance, since this correction is relevant for complex natural extracts as well as chemical libraries of pure compounds, and moreover applicable all the way from small-sized screens to high-throughput ones. Further, I investigated the apoptosis inducing activity of nocuolin A (NoA) a new cyanobacterial compound isolated and described by our group. NoA shows remarkable characteristics regarding its structure (1,2,3-oxadiazine heterocycle), biosynthetic origin and also its biological activity. It induces caspase-dependent apoptosis and shows potency against a panel of nine human cancer cell lines, which makes NoA a pharmaceutically interesting compound. I also bring the first insights into elucidation of its mode of action in cancer cells in vitro.

Morphology and phylogeny for 36 heterocytous cyanobacterial strains are studied. Discussion with morphological, ecological, and phylogenetically related taxa is given for each strain. Potentially new genera and species are found, one of them being proposed as a novel genus.