Selected physicochemical properties of Little Lick Creek, in Hartford City, Indiana, were determined in the fall of 1969. Subsurface samples from five stations, two upstream and two downstream from a discharge point: at the. Minnesota Mining and Manufacturing Companywere analyzed. A complex effluent containing a dye entered the creek from the paper plant. Dissolved oxygen and biochemical oxygen demand determinations of the water downstream from the discharge displayed changes in water quality of the creek. The water temperature rose significantly after the discharge water entered the creek. A cell count study of the Cyanophyta of the creek was also determined.Little Lick Creek was considered polluted below the paper products plant discharge based on the observed changes in the water tested. Cyanophyta populations were apparently reduced by the effluent.Ball State UniversityMuncie, IN 47306

Coibamide A is a methyl-stabilized cyclic depsipeptide with a lariat side chain that was isolated from a marine cyanobacterium as part of an International Cooperative Biodiversity Groups program based in Panama. Previous testing of this potent and selective growth-inhibitory agent in the National Cancer Institute (NCI) in vitro 60 human cell line panel revealed a "COMPARE-negative" profile indicative of a unique mechanism of action. Presented herein is a collection of studies characterizing the mechanism of action of coibamide A and cataloguing the cytotoxicities of putative coibamide A and related structures from efforts at its total synthesis. We report that coibamide A induces apoptotic and non-apoptotic cell death in human U87-MG and NCI-SF-295 glioblastoma cells, respectively, which can occur independently of a rapid and sustained mTOR-independent autophagic response. Loss of cell viability from coibamide A exposure was concentration-dependent and time-sensitive, characterized by extensive cytoplasmic vacuolization and an absence of apoptotic morphology and DNA fragmentation prior to cell rounding and detachment from the substratum. Coibamide A also induces a cytostatic effect mediated by a G1 phase specific cell cycle arrest and inhibits glioma cell invasion but not migration. Lastly, structure activity relationships suggest that linearization, loss of N-methylation and disjoining of the cyclic and side chain structures of coibamide A are not well-tolerated modifications to retain activity. / Graduation date: 2013 / Access restricted to the OSU Community at author's request from Jan. 8, 2013 - Jan. 8, 2014

No description available.

The cyanobacterium Synechococcus elongatus builds a circadian clock on an oscillator comprised of three proteins, KaiA, KaiB, and KaiC, which can recapitulate a circadian rhythm of KaiC phosphorylation in vitro. The molecular structures of all three proteins are known, and the phosphorylation steps of KaiC, the interaction dynamics among the three Kai proteins, and a weak ATPase activity of KaiC have all been characterized. A mutant of a clock gene in the input pathway, cikA, has a cell division defect, and the circadian clock inhibits the cell cycle for a short period of time during each cycle. However, the interaction between the circadian cycle and the cell cycle and the molecular mechanisms underlying it have been poorly understood. In addition, the subcellular localization of clock proteins and possible localization dynamics, which are critical in the timing circuit of eukaryotic clock systems and might also shed light on the interaction between circadian cycle and cell cycle, have remained largely unknown. A combination of genetics, cell biology, and microscopy techniques has been employed to investigate both questions. This work showed that the cell division defect of a cikA mutant is a function of the circadian clock. High ATPase activity of KaiC coincides with the inhibition of cytokinesis by the circadian clock. CikA likely represses KaiC's ATPase activity through an unknown protein, which in cikA's absence stimulates both the ATPase and autokinase activities independently of KaiA or KaiB. SasA-RpaA acts as an output in the control of cell division, and the localization of FtsZ is the target, although it still remains to be seen how RpaA, directly or indirectly, inhibits FtsZ localization. The project also showed that clock proteins are localized to the cell poles. KaiC is targeted to the cell pole in a phosphorylation-dependent manner. KaiB and CikA are also found at the poles independently of KaiC. KaiA likely only localizes to the cell pole during the dephosphorylation phase, which is dependent on both KaiB and KaiC, specifically on the phosphorylation of KaiC at S431. Overall, significant progress was made in both areas and this project sheds light on how the circadian oscillator operates in cyanobacterial cells and interacts with another fundamental cellular function.

Thesis (M.Sc.)--Memorial University of Newfoundland, 2000. / Bibliography: p. 158-173.

Modern microbialites are carbonate-precipitating microbial mats and represent the closest living analogues to ancient stromatolites. These ancient carbonate formations are the oldest fossil evidence of life on Earth; however, our comprehension of their relationship to early earth ecosystems relies heavily on understanding the formation of modern microbialites. Research regarding these formation processes has suggested that chemical constraints of CaCO 3 precipitation vary on sub-millimeter spatial scales within the living microbial community. In an attempt to shed light on the importance of these chemical microenvironments, this study focused on understanding the spatial distribution of the organisms and processes involved in the formation of modern microbialites. This was accomplished by isolating five visually distinct layers from the upper 2 – 3 cm of an actively forming microbialite found in the freshwater system of Cuatro Ciénegas, Mexico. Each layer was analyzed using genomic, molecular organic, and stable isotopic techniques. Bacterial diversity was determined by 16S rRNA gene analyses, lipid biomarker content was detected by GC-MS, and carbon isotope composition of organic matter and CaCO 3 were used as indicators of specific microbial processes. Results of the 16S rRNA gene analysis showed that there is little overlap in the community composition of individual layers. Approximately 90% of the ribotypes identified in the microbialite were unique to a single layer. Furthermore, the relative accretion of CaCO 3 at each layer was used to connect the distribution of organisms and processes with two specific zones of CaCO 3 precipitation. The first zone of CaCO3 accretion, which accounted for approximately 55% of total CaCO 3 accumulation, is found in the surface two layers of the microbialites and dominated by photoautotrophic cyanobacteria and algae. The second zone of CaCO 3 precipitation, found at the interior (layers 4 and 5), is composed primarily of heterotrophic proteobacteria and dominated by sulfate-reducing !-proteobacteria. The lipid content of the microbialite reflected the community structure as determined by genomics. Numerous photosynthetic biomarkers were detected and decreased in abundance with depth, indicating the important function of heterotrophic degradation. Additionally, the detection of sulfurized phytol compounds in layer 5 highlighted an important mechanism for the preservation of biogenic signatures, and reflected both the abundance of phototrophic organisms and sulfatereducing bacteria. In combination, these interdisciplinary analyses provided an understanding of microbial community composition and metabolism while indicating the spatial relationship to CaCO 3 formation and the preservation of distinct biochemical signatures. !

A previously un-described filamentous non-heterocystous cyanobacterium was isolated from the South China Sea. Molecular phylogenetic analyses together with morphological observations suggested that this organism should be assigned a new specific epithet. It was designated as Leptolyngbya nodulosa Li et Brand. Filaments were enclosed in a sheath, and were unusual in their flattened appearance as seen in cross section and in the frequent occurrence of a void space (as seen by transmission electron microscopy) between the trichome and the sheath. A distinctive feature of L. nodulosa was the presence, under low light intensities, of previously un-described multicellular structures (nodules). A L. nodulosa nodule consists of a portion of a filament folded and twisted into a distinct unit surrounded by a firm continuous sheath. Nodules were highly variable in size and shape, and occurred at irregular intervals along the filament. They disappeared from filaments of cultures grown at relatively high light intensities. L. nodulosa cultures could be grown indefinitely in media devoid of any source of combined nitrogen. Acetylene reduction assays showed that L. nodulosa cultures fix dinitrogen in the dark period of a diurnal cycle under microoxygenic conditions. The addition of DCMU ([3-(3,4-dichlorophenyl)-1,1-dimethylurea], an inhibitor of Photosystem II) to a culture of L. nodulosa induced much higher rates of dinitrogenase activity and altered the cycle of activity such that most acetylene reduction occurred during the light. Measurements of dinitrogenase activity in the presence of chloramphenicol (an inhibitor of protein synthesis) indicated that dinitrogenase is synthesized in darkness and destroyed in the subsequent light period. In the presence of DCMU, a much higher dinitrogenase activity is measured, but in this case only in the light. Neither the dark-mediated dinitrogenase in the absence of DCMU nor the light-mediated activity in the presence of DCMU could be sustained for more than two days without a photoperiodic light/dark cycle. Dinitrogenase activity occurred only in non-axenic cultures of L. nodulosa. A single nifH gene, with an identical sequence in axenic and non-axenic cultures, was isolated. The requirement of heterotrophic bacteria for dinitrogenase activity in L. nodulosa is not yet understood.

Cyanobacteria are photosynthetic prokaryotes that live in aquatic environments. The cyanobacterium Synechococcus elongatus PCC 7942, (hereafter S. elongatus) coordinates its day and night behaviors via a circadian clock. The clock is entrained by light/dark cycles but continues to run in constant light conditions. The core circadian clock in S. elongatus is encoded by post-translational modifications of three Kai proteins, but the extent and mechanism of circadian gene expression are unknown. We provide the first unbiased characterization of circadian gene expression in S. elongatus, demonstrating that \(\sim 65\%\) of genes display oscillation in continuous light conditions, with some genes peaking in expression at subjective dawn and others at subjective dusk. We next sought to identify the mechanism by which such a large fraction of the genome could be rhythmically controlled. Through bioinformatic, correlative, and perturbation experiments, we find that circadian changes in chromosome topology/supercoiling are sufficient to drive rhythmic expression (Chapter 2). To further investigate how chromosome topology can control gene expression we performed a high resolution characterization of transcripts and RNA polymerase across the S. elongatus genome (Chapter 3). Bioinformatic analysis of transcription start sites suggests that the AT/GC content a particular region of the promoter is informative in defining the phase at which a transcript is maximally expressed. We find that these sequences are sufficient to drive circadian gene expression at a particular phase and that mutation of single nucleotides in this region can reverse the expression phase of a transcript (Chapter 4). To understand the role of chromosome dynamics in circadian gene expression and cyanobacterial physiology, we tagged and followed chromosomes over multiple cell divisions. We find that S. elongatus cells harbor multiple ordered copies of a single chromosome, and the organization of chromosomes in the cytoplasm facilitates equal segregation of chromosomes to daughter cells (Chapter 5).

published_or_final_version / Ecology and Biodiversity / Doctoral / Doctor of Philosophy

The growth of micro-organisms in foods is different from that in axenic liquid culture in laboratory media. In natural environments, including food, micro-organisms generally grow in mixed culture and in close proximity to each other, because of which antagonistic or synergistic interactions can occur. To elucidate the behaviour of bacteria within food matrices an understanding of the food structure is required, as foods are complex ecosystems on the micrometer scale. Most processed foods are emulsions and as such are highly structured heterogeneous environments. Antibody-linked probes can be used for the immuno-location of micro-organisms or their products within food matrices to demonstrate the sites at which growth occurs and elucidate the possible bacterial interactions with food components. The aim of the project was to raise antibodies to spoilage Pseudomonas species and to use the developed antibody-linked probes to follow psychrotrophic spoilage Pseudomonas within heterogeneous foods. By using antibody-linked probes the natural spoilage of milk and milk products can be followed along traditional lines examining extrinsic parameters but with the additional benefit that the major spoilage organisms can be located within the mixed natural flora. The use of antibodies in this way facilitated the study of a defined natural population and surmounted any adaptive problems associated with introduced organisms. An oil-in-water near-foodgrade model was developed to investigate the growth of Pseudomonas as it overcame some of the technical problems of using natural cream. Pseudomonas species, which grew as colonies within the near-food-grade model, were visualised using fluorescently-labeled antibody-linked probes. Pseudomonas used to raise the antisera were isolated from psychrotrophically spoiled food and characterised together with isolates retrieved from the environment. The phenotypic characterisation of Pseudomonas using classical biochemical tests and API 20NE test strips (BioMerieux) did not produce definitive identifications of the unknown isolates. Nutritional screening of the Pseudomonas isolates using commercially produced standardised test microtitration plates (Biolog MicroPlate TM), that contained 95 carbon sources, was carried out. The data produced from the test microtitration plates were analysed using numerical taxonomic methods. The relatedness of the Pseudomonas isolates was strongly influenced by the source from which the test isolates originated and did not definitively identify all of the unknown isolates tested. Molecular techniques, ribotyping and amplified ribosomal DNA restriction analysis (ARDRA), based on the genomic fingerprinting of the 16S rRNA gene were evaluated to aid the definitive identification of the Pseudomonas isolates but needed a more extensive data base to be useful. The difficulties encountered in phenotypically identifying food and environmentally isolated Pseudomonas species stems from the fact that the Pseudomonas genus is now classified according to its ribosomal DNA homology. The classification of the species within the Pseudomonas genus is still under review. Robust phenotypic criteria for the identification of all the species within the genus have not to date been defined. In this study, the association of phenotype with environmental source of isolation (whether characterised by nutritional studies or by antibody cross-reaction) demonstrates clearly that more appropriate phenotypic characterisation is required to allow identification schemes to reflect the underlying phylogeny of this group.