Theses and Dissertations at Montana State University (MSU)
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Item Insect microbe interactions: honey bee antiviral defense mechanisms and characterization of Spiroplasma colonizing wheat stem sawfly(Montana State University - Bozeman, College of Agriculture, 2017) Brutscher, Laura Marie; Chairperson, Graduate Committee: Michelle Flenniken; Katie F. Daughenbaugh and Michelle L. Flenniken were co-authors of the article, 'Virus and DSRNA-triggered transcriptional responses reveal key components of honey bee antiviral defense' in the journal 'Scientific reports' which is contained within this thesis.; Curtis Fowler, David K. Weaver and Carl J. Yeoman were co-authors of the article, 'Identification and characterization of a Spiroplasma sp. (Ixodetis clade) associated with the wheat stem sawfly (Cephus cinctus)' submitted to the journal 'Microbial ecology' which is contained within this thesis.Insects play important roles in ecosystems throughout the world. There are many beneficial insects, including those that pollinate plants in diverse landscapes, while other insects are considered agricultural pests. Regardless of ecological role, insects are hosts for microbial symbionts and pathogens. Some microorganisms (e.g., viruses) are harmful to insect health, but many microbial symbionts aid in host biological processes. The projects herein describe the interplay between insects and microbes; specifically (1) honey bee host - virus interactions and (2) identification and characterization of a wheat stem sawfly-associated Spiroplasma. Honey bees (Apis mellifera) are pollinators of numerous agricultural crops and other plant species. Since 2006, there have been high annual losses of honey bee colonies in the U.S. (~33%) and throughout the world. Colony deaths are influenced by multiple factors including RNA virus infections. Honey bee antiviral defense involves several immune pathways, including dsRNA mediated responses, (i.e., RNA interference and non-sequence-specific dsRNA-triggered responses), but their relative importance in antiviral defense is not well understood. To investigate honey bee antiviral defense, bees were infected with model virus in the absence or presence of dsRNA, which reduced virus abundance. Transcriptome-level analysis determined hundreds of genes were differentially expressed in response to co-treatment of dsRNA and virus, including immune-related genes. RNAi-mediated gene knockdown of two putative antiviral genes increased virus abundance and supported their antiviral role. Additional investigation of these and other genes will improve our understanding of dsRNA-mediated antiviral defense in honey bees. In contrast, wheat stem sawflies (Cephus cintus) are major wheat pests in the Northwest United States. Strategies that target endosymbionts of sawflies could reduce wheat crop losses. Hereunto, the microbes that colonize wheat stem sawfly have not been explored. Targeted DNA sequencing determined sawflies were colonized by a Spiroplasma species that has greatest 16S rRNA sequence identity with Ixodetis clade species. Metagenomic sequencing identified several Spiroplasma encoded genes involved in metabolism, which may be important to the sawfly host. Further characterization of honey bee-virus interactions and the role of Spiroplasma in sawfly health may contribute to limiting threats to global crop production and will further scientific understanding of non-model insect-microbe interactions.Item Methanogenesis in low sulfate hot spring algal-bacterial mats(Montana State University - Bozeman, College of Agriculture, 1980) Sandbeck, Kenneth AndrewItem A comparison of some methane-oxidizing bacteria(Montana State University - Bozeman, College of Agriculture, 1960) Johnson, John LeRoyItem The toxic effect of ionic silver on the lower organisms(Montana State University - Bozeman, College of Agriculture, 1937) McElroy, JeanItem Molecular characterization of the bacterial flora of the murine female genital tract(Montana State University - Bozeman, College of Agriculture, 2001) McNamer, Deirdre KathleenThe female genital tract may harbor a potentially diverse microbial community that has not been thoroughly characterized, particularly in the murine system. A more complete knowledge of resident flora may enhance understanding of disease susceptibility and the persistence of infection which, in turn, may better guide research toward more efficacious treatments and protective measures. Organisms known to exist in this environment have largely been identified through cultivation. However, because the nutritional and environmental needs of previously uncultivated organisms cannot be anticipated, there are limitations to the use of this technique when attempting to characterize a community. Molecular techniques that take advantage of the conservation of ribosomal RNA have proven to be very useful for such efforts. The use of PCR in tandem with denaturing gradient gel electrophoresis (DGGE) allows for the characterization and comparison of microbial populations with relative ease and rapidity. This study applies these molecular techniques to the investigation of the microbial flora of the murine female genital tract. In addition, a comparison was made between cultivation and two different molecular techniques, DGGE and cloning. We found some overlap in the detection of organisms identified by the various methods, but some microbes were only detected by particular techniques. We further used DGGE to identify bacterial populations of this environment during the course of the natural estrous cycle, under the influence of exogenous hormones, following antibiotic therapy and in a Chlamydia trachomatis infection model. Our findings indicate that there is no change in the species present over the course of the estrous cycle or under the influence of administered hormones. However, because DGGE is not quantitative we were unable to determine if quantitative changes in the populations occurred. Our findings with the antibiotic treatment and Chlamydia infection model, though interesting, were not conclusive and require further investigation.Item The microbial dynamics of an epilithic algal-bacterial mat community in an oligotrophic, high alpine stream(Montana State University - Bozeman, College of Agriculture, 1981) Haack, Thomas KennethItem Recovery characteristics of bacteria injured in the natural aquatic environment(Montana State University - Bozeman, College of Agriculture, 1974) Bissonnette, Gary K.Item Molecular analysis of diversity, gene expression and activity of mineral-associated bacteria(Montana State University - Bozeman, College of Letters & Science, 2005) Reardon, Catherine Leona; Chairperson, Graduate Committee: Gill G. GeeseyThis dissertation investigated the diversity and hydrogenase activity and gene expression of mineral-associated microorganisms. Surface-associated microbes have been shown to dominate diversity and activity in the environment, however molecular analysis of sediment-associated communities is hindered by both inaccessibility to the subsurface and co-extraction of inhibitory compounds. In order to analyze microbial communities in which the environmental conditions previously had precluded the use of traditional sediment extraction techniques, biofilm coupons (metal, mesh cylinders containing surrogate substrata) were used to recover microorganisms able to attach and compete in a biofilm. The community recovered from the contaminated site using hematite as a surrogate substratum was dominated by microbes most closely related to Alcaligenes sp. (metal-tolerant), Frateuria sp. (acidophilic), and Methylobacterium radiotolerans (radionuclide-tolerant) which together reflect the acidic, metal-, and radionuclide-contaminated environment. Hematite, as compared to other substrata, was shown to recover communities most closely representative of sediment communities inhabiting the saturated zone. Surface-associated cells have been shown to express greater activity than suspended populations and mineral-associated sulfate-reducing bacteria (SRB) mediate the formation of different secondary minerals as compared to suspended cells. In order to investigate the affect of surface-association on enzyme activity, hydrogenase enzyme activity was compared in hematite-associated and suspended populations of the SRB Desulfovibrio desulfuricans Essex 6. Hydrogenase activity of surface-associated populations was higher than that displayed by suspended cells. Hydrogenase likely affects the pH and pE of the conditions immediately surrounding the cell. The greater rate of activity may be one factor which contributes to the formation of a mineral phase not observed in the presence of suspended populations of this bacterium. In order to determine the portion of cells expressing hydrogenase in the surface-associated populations, in situ reverse-transcription PCR was applied to the hematite-associated cells and all cells were expressing the [NiFe] hydrogenase gene. This thesis demonstrates that environmental conditions of contaminated subsurface environments select for microorganisms able to tolerate or utilize the contaminants. Also, the hydrogenase activity of surface-associated populations is not representative of the suspended cells thus indicating the importance of studying attached populations where enzyme activity likely influences the conditions at the mineral-microbe interface.Item The detection, characterization, and cultivation of nonculturable Helicobacter pylori(Montana State University - Bozeman, College of Letters & Science, 2010) Richards, Crystal Lynette; Chairperson, Graduate Committee: Anne Camper; Susan C. Broadaway, Margaret J. Eggers, Emily Colgate, John Doyle, Barry H. Pyle, Anne K. Camper, and Timothy E. Ford were co-authors of the article, 'Detection of Mycobacteria, Legionella, and Helicobacter in drinking water and associated biofilms on the Crow Reservation, Montana, USA' in the journal 'Applied and environmental microbiology' which is contained within this thesis.; Kerry Williamson, Timothy E. Ford, and Anne K. Camper were co-authors of the article, 'Multiple processes govern switch to nonculturable state in H. pylori' in the journal 'Journal of bacteriology' which is contained within this thesis.; Brittany J. Buchholz, Timothy E. Ford, Susan C. Broadaway, Barry H. Pyle, and Anne K. Camper were co-authors of the article, 'Optimizing the growth of stressed Helicobacter pylori' in the journal 'Journal of microbiological methods' which is contained within this thesis.Transmission of a bacterial pathogen from host to host is a complex process that may involve survival of the pathogen outside the host for considerable lengths of time. The bacterium Helicobacter pylori causes severe gastritis and gastric ulcers, and infection can increase the risk of stomach cancer. The main mode of transmission is believed to be the oral-oral route, however other routes of transmission such as drinking water have been implicated but have not been proven due to difficulty in culturing this organism. In this dissertation, the environmental transmission of H. pylori was investigated using several approaches. A primary objective of this study was to determine if H. pylori could be detected in an environmental reservoir readily consumed by humans, such as drinking water. H. pylori was detected by PCR but not culture in drinking water and biofilms that were obtained from groundwater and municipal systems. H. pylori contamination was sporadic and not associated with measured environmental factors, such as pH or temperature. Growth curve analysis of laboratory grown H. pylori showed that the cells exhibited a switch from a spiral to coccoid morphology as they aged or were exposed to stressful culture conditions. However, results showed that cell morphology was not indicative of culturability, with spiral forms dominant in early nonculturable samples. Microarray analysis of the transition to a nonculturable state showed that cells under oxygen stress quickly modified their transcriptional activity while the cells exposed to nutrient deprivation had nearly undetectable changes in transcriptional activities. Resuscitation of the stressed cells showed that type of stress and length of exposure affected regrowth of H. pylori. The oxygen stressed cells increased virulence factor transcription while nutrient deprived cells decreased transcription of the same factors. This observation led to the conclusion that oxygen stressed and nutrient deprived cells are metabolically active but react differently to in vitro culture conditions with starved cells likely undergoing nutrient shock. Collectively these data suggest that H. pylori can persist and are metabolically active under stressful conditions posed by the environmental mode of transmission.