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Iron is an essential nutrient for the proper functioning of many bacterial physiological processes. A deficiency of iron in bacteria can cause inhibition of cell growth and changes in morphology. Under iron restricted environments, microorganisms seek out different methods of acquiring iron. Our lab reported that RhizobiumLeguminosarum ATCC 14479, a gram-negative soil bacterium, is capable of utilizing heme as the sole source of iron. This work focuses on identifying and sequencing the hmuPSTUVoperon to assess its’ possible role in heme utilization in R. leguminosarum biovar trifoliiATCC 14479. We have confirmed the presence of this operon and have completed the sequencing of each gene involved in the operon. We have used in-silico analysis to assess the possible function of each gene. In the future, gene knockout will be performed to confirm the function of the hmuPSTUVoperon.

In the United States, symptoms caused by pathogenic strains of Escherichia coli are on the rise. A major source of these pathogenic strains is the E. coli in the digestive tract of cattle. The purpose of this project was to determine if E. coli are transferred between individuals of the same species and if interspecies transmission is possible. Proximity of cattle was also studied as a contributing factor to the transfer of E. coli. To accomplish this goal, E. coli isolates from cattle and cohabitating ground squirrels were compared through a new method of bacterial strain typing called pyroprinting. Bulls from the Cal Poly Bull Test were sampled every summer from May to September when around 200 bulls from ranches across California are housed together to be tested and eventually auctioned off. The impact of cattle origin (ranch, city) and habitation (pen) on E.coli isolate strain type were evaluated via pyroprinting . The cattle were studied to see if transfer was related to proximity of cohabitation. Since the complete population of intestinal E. coli could not be sampled, transfer could not be directly seen. The probability of sharing E. coli in each time point was used to infer transfer. There was an increase in the probability of sharing E. coli from the May sample date to the September date, indicating that some form of transfer was occurring. There was an even greater increase in the probability of sharing E. coli when the bulls were housed in close proximity. Lastly, ground squirrels cohabitating in the area were found to house some of the same strains as the cattle. This makes transfer between squirrels and cattle a possibility. Overall, this paper shows that the intestinal E. coli composition of bulls may be readily altered by the introduction of new bulls into a population.

Deep-sea hydrothermal vents are some of the most biologically productive ecosystems on Earth, yet receive little to no input of photosynthetically derived organic matter. The trophic system at hydrothermal vents is based primarily on the reduction-oxidation (redox) of inorganic chemicals by Bacteria and Archaea. However, the distributional patterns of the microorganisms that colonize deep-sea hydrothermal vent deposits and their link to the geologic setting are still not deeply understood. The goal of the studies presented in this thesis was to quantify the abundance, and distribution of major and understudied vent colonizing archaeal groups from globally distributed and geochemically distinct hydrothermal vent fields. The archaeal community composition was analyzed using quantitative PCR with lineage specific functional gene primers that target methanogens, and 16S rRNA gene primers designed or optimized from this study for the Thermococcales, Archaeoglobus, Ignicoccus and marine Nanoarchaeota. Overall, a general relationship was demonstrated between the geochemical differences of the hydrothermal vent fields and the archaeal community structure. The archaeal community assemblage varied dramatically from hydrothermal vents with different vent host rocks along the Mid-Atlantic Ridge and Eastern Lau Spreading Center. In contrast, two vent fields in the East Pacific, 9°N on the EPR and Guaymas Basin that are basalt and basalt-sediment hosted were found to have similar community composition. These observed differences may be driven in part by the metabolically available chemical energy as hydrogen oxidizing lineages of the methanogens and Archaeoglobus were found in higher abundance in the samples from vent field that had a high concentration of end-member hydrogen and the heterotrophic Thermococcales constituted a higher proportion of the archaeal community at the less enriched vent fields. Interestingly, the Nanoarchaeota and the genus of its only confirmed symbiont, Ignicoccus, were found to have an inconsistent proportional relationship, with the Nanoarchaeota comprising a larger proportion of the archaeal community at the ultramafic and fast spreading basalt vent fields and Ignicoccus at the ultra-slow spreading basalt and andesite hosted vent fields. There was also a more localized pattern identified within the hydrothermal vent deposit. The chemosynthetic lineages of the methanogens and Archaeoglobus constituted a higher proportion of the archaeal community in chimney samples compared to Thermococcales that was found in a higher proportion at horizontal flange samples. This archaeal proportional shift could be driven by energetic micro-niches within the vent deposit, as the chemolithotrophic lineages colonize the area closest to the venting source, and the heterotrophic Thermococcales dominate in more mature structures further from the venting source. Quantitative assessments of the archaeal community composition from this study provided added insight into the dynamic geologic influence on the archaeal lineages that colonize deep-sea hydrothermal vents, on a global and local scale.

Fish of the genus Cottus were found infected with heterophyid metacercariae. Laboratory animals were infected with the metacercariae, and adult heterophid trematodes were recovered. These flukes were found to represent an undescribed species of the genus Euryhelmis. Various streams in the Willamette Valley and coastal areas were sampled for infected Cottus sp. Snails of the genera Oxytrema and Fluminicola were collected. Several possible definitive hosts were examined. A partial review of the subfamily Apophallinae and a complete review of the genus Euryhelmis are presented. Euryhelmis cotti n. sp. is placed in the subfamily Apophallinae, and its life cycle is partially described.

Studies investigating Escherichia coli strain diversity and demographics in human hosts are frequently inconsistent regarding sampling effort and methodology while current strain typing methods are often expensive or laborious. To rectify these inconsistencies, sampling effort was investigated by comparing the diversity of 15-isolate collections to 100-isolate collections from 3 human subjects. Temporal variation in E. coli strain diversity was also studied by collecting 15 isolates once every 6 months. Additionally, strain identification and diversity collected by different sampling methods (fecal swabs vs. anal swabs collected at different times around defecation) were compared to identify any inherent biases in sampling method. This study employed pyroprinting, a new inexpensive and simple strain typing method using pyrosequencing, to generate DNA fingerprints (or pyroprints) based on the Intergenic Transcribed Spacer sequences in the ribosomal RNA operon to differentiate E. coli strains. Differences in strain diversity were apparent when comparing sampling efforts. The sampling effort investigation suggested that certain subjects hosted very large and highly diverse E. coli strain populations such that even 100 isolates may not fully represent E. coli strain populations in human hosts. Instead, the sampling effort required to accurately represent strain demographics may depend on strain richness and evenness within each host. The temporal investigation yielded similar or greater strain abundance and diversity compared to other typing methods in the literature suggesting pyroprinting is a similarly discriminating tool. When agglomerated over time or by subject, no significant differences in diversity were observed between subjects or between sampling methods despite visible differences in strain richness and evenness.

Cultural eutrophication, the overproduction of phytoplankton biomass in response to increased nutrient inputs directly associated with human activities, is a major threat to the health of Chesapeake Bay. Strict regulations, which require a reduction in nutrient loading from all sources, have been a key component to restoration efforts. Water reclamation facilities (WRFs), which discharge effluent containing nitrogen (N) and other nutrients into receiving waters, have implemented upgrades in an effort to comply with regulations. These improvements have decreased the concentration of highly labile dissolved inorganic N (DIN), leaving behind significant concentrations of dissolved organic N (DON) whose bioavailability, and therefore its contribution to eutrophication, remains unclear. The concentration and composition of the N forms in effluent depend upon the characteristics of the influent entering the facility, the processes used to treat the wastewater, and the disinfection procedures employed prior to discharge. To investigate how these factors affect the composition of the effluent, samples were taken from four bench scale sequencing batch reactors designed to mimic commonly used treatment processes: nitrification only (NO), nitrogen removal (NR), biological nitrogen and phosphorus removal (BNPR), and biological nitrogen and phosphorus removal with additional chemical phosphorus removal (BNCPR). Effluent from each treatment process was also subjected to three disinfection procedures: no disinfection, ultraviolet radiation, and chlorination. To assess bioavailability, effluent from each of the treatment-disinfection combinations was added to natural water samples collected in the York River, VA. Results showed that total dissolved nitrogen (TDN) removal efficiencies of the treatments varied significantly from 12 to 98% and followed the trend NO < NR < BNPR < BNCPR. NO and NR produced effluent composed primarily of nitrate, while BNPR and BNCPR produced effluent composed primarily of DON. Bioassays showed that effluent from NO and NR stimulated phytoplankton growth, and that between 17 and 48% of effluent DON (EDON) was labile. Effluent from BNPR and BNCPR generally stunted or impeded phytoplankton growth and between 4 and 14% of EDON was labile. Overall, disinfection procedures had minor effects on effluent composition and bioavailability, indicating that the largest impacts on cultural eutrophication are made at the initial treatment level. This study provides results aimed at characterizing the composition of effluent resulting from both treatment and disinfection processes, eliminating influent as a variable. The data show that the discharge of NO and NR effluents would likely lead to eutrophication in both N and P limited receiving waters due to their high inorganic nutrient content and labile EDON. In contrast, the discharge of BNPR and BNCPR effluents, due to their low inorganic nutrient and relatively refractory DON concentrations, is less likely to contribute to eutrophication.

Karst waters serve as important water sources in rural Appalachia and are well-connected to surface waters, making them susceptible to anthropogenic contamination, including by fecal indicator bacteria which represent a public health risk. This work designed and implemented a watershed-scale monitoring program for a 26 km2 sinking stream system in southwest Virginia to determine the fate and transport of E. coli in the system. This hydrologically complex watershed is predominantly agricultural and includes multiple key surface water sinks that enter Smokehole Cave and emerge at Smokehole Spring. Field campaigns at surface sites and within Smokehole Cave included bacteriological sampling, hydrologic measurement, and dye tracing. Field data was synthesized to: 1) examine variations in E. coli concentrations in the watershed during varying flows/seasonal conditions; and 2) calculate E. coli growth/decay coefficients for the karst system during different flow/antecedent conditions. E. coli concentrations at Smokehole Spring consistently peaked days after peak hydrologic stage. Flow conditions and storm event response were the largest drivers of E. coli transport through the system. Dye trace results revealed that water from sinks can be stored or move slowly through the karst system, resurging during storm events. E. coli was calculated to decay within the karst system, with a half-life of about 5-120 days which is longer than the travel time of water through the cave of approximately 0.5-2 days. Findings indicate that E. coli transport in Appalachian karst systems is hydrologically driven, roadside spring water collection is not recommended, and bacterial treatment is encouraged if performed. Targeted land-management practices should be explored to decrease E. coli loadings in karst waters. / Master of Science / Karst (cave) waters serve as important water sources in rural Appalachia and are well-connected to surface waters, making them susceptible to contamination from human or animal waste – a public health risk. A field monitoring program was conducted in an agriculturally impacted stream and cave system in southwest Virginia to determine how E. coli, a bacteria found in the waste of humans and other animals, moves through the system. There are several places where surface water sinks into the cave system, eventually entering Smokehole Cave and emerging at Smokehole Spring. Field data collection was performed at surface sites and within Smokehole Cave including sampling for E. coli, water flow measurements, and dye tracing. Field data was combined to 1) examine variations in E. coli concentrations during varying flows/seasonal conditions and 2) calculate E. coli growth/decay coefficients for the cave system during different flow and soil moisture conditions. It was found that E. coli concentrations at Smokehole Spring consistently peaked days after the water depth. Flow conditions and storm events were the largest drivers in E. coli movement through the system. Dye trace results revealed that water from sinks can be stored or moves slowly through the cave system and resurges during storm events. E. coli was found to decay within the cave system. Findings indicate that E. coli movement in Appalachian cave systems is driven by storm events, roadside spring water collection is not recommended, and bacteria treatment is recommended if performed. Cave-specific land-management practices are recommended to keep E. coli from entering cave waters.

<p>Biodegradation of petroleum hydrocarbons by microorganisms is one of the most effective methods used to remediate environmental systems. However, much of what is known is based on the ability of (mostly bacterial) species to degrade hydrocarbons under enrichment conditions in a laboratory setting. In order to refine biodegradation as a remediation method, there is a critical need to understand the dynamics and mechanisms of microbial communities under <em>in situ </em>conditions. The goal of this dissertation was to provide insight and knowledge into the function of microbial communities in petroleum-contaminated environments using a combination of DNA, lipid and isotopic analyses. Microbial biomass, community structure, carbon sources were assessed at two study sites: (1) a former industrial facility contaminated by PAHs and (2) coastal salt marshes impacted by the <em>Deepwater Horizon</em> oil spill.</p> <p>Isotopic analyses of soils collected from the PAH-contaminated site revealed that microbial carbon sources were derived from vegetation and/or natural organic matter present in soils matter rather than PAHs. Similarly, microbial community structure remained consistent across samples and there were no observed shifts in phylotype diversity with increasing levels of PAHs. Bioaccessibility assays revealed that a large fraction of soil-borne PAHs at the site are not bioavailable to microorganisms; thus, highlighting the importance of environmental factors to <em>in situ</em> biodegradation.</p> <p>Biodegradation of <em>Deepwater Horizon </em>spilled oil was detected in salt marsh sediments such that petroleum-derived carbon was a primary carbon source for indigenous microbial communities in the months following the spill. Likewise, pyrosequencing of all three microbial domains showed an increase in the relative of abundance of taxonomic groups known to include hydrocarbon-degrading species, such as <em>Sphingomonadales</em>. These results suggest that Gulf of Mexico marsh sediments have considerable biodegradation potential and that natural attenuation may be feasible remediation strategy in this region.</p> / Doctor of Philosophy (PhD)

Background: The leading cause of surface water impairment in United States’ rivers and streams is pathogen contamination. Although use of fecal indicators has reduced human health risk, current approaches to identify and reduce exposure can be improved. One important knowledge gap within exposure assessment is characterization of complex fate and transport processes of fecal pollution. Novel modeling processes can inform watershed decision-making to improve exposure assessment. Methods: We used the ecological model, Maxent, and the fecal indicator bacterium Escherichia coli to identify environmental factors associated with surface water impairment. Samples were collected August, November, February, and May for 8 years on Sinking Creek in Northeast Tennessee and analyzed for 10 water quality parameters and E. coli concentrations. Univariate and multivariate models estimated probability of impairment given the water quality parameters. Model performance was assessed using area under the receiving operating characteristic (AUC) and prediction accuracy, defined as the model’s ability to predict both true positives (impairment) and true negatives (compliance). Univariate models generated action values, or environmental thresholds, to indicate potential E. coli impairment based on a single parameter. Multivariate models predicted probability of impairment given a suite of environmental variables, and jack-knife sensitivity analysis removed unresponsive variables to elicit a set of the most responsive parameters. Results: Water temperature univariate models performed best as indicated by AUC, but alkalinity models were the most accurate at correctly classifying impairment. Sensitivity analysis revealed that models were most sensitive to removal of specific conductance. Other sensitive variables included water temperature, dissolved oxygen, discharge, and NO3. The removal of dissolved oxygen improved model performance based on testing AUC, justifying development of two optimized multivariate models; a 5-variable model including all sensitive parameters, and a 4-variable model that excluded dissolved oxygen. Discussion: Results suggest that E. coli impairment in Sinking Creek is influenced by seasonality and agricultural run-off, stressing the need for multi-month sampling along a stream continuum. Although discharge was not predictive of E. coli impairment alone, its interactive effect stresses the importance of both flow dependent and independent processes associated with E. coli impairment. This research also highlights the interactions between nutrient and fecal pollution, a key consideration for watersheds with multiple synergistic impairments. Although one indicator cannot mimic the plethora of existing pathogens in water, incorporating modeling can fine tune an indicator’s utility, providing information concerning fate, transport, and source of fecal pollution while prioritizing resources and increasing confidence in decision making. Methods We used the ecological model, Maxent, and the fecal indicator bacterium Escherichia coli to identify environmental factors associated with surface water impairment. Samples were collected August, November, February, and May for 8 years on Sinking Creek in Northeast Tennessee and analyzed for 10 water quality parameters and E. coli concentrations. Univariate and multivariate models estimated probability of impairment given the water quality parameters. Model performance was assessed using area under the receiving operating characteristic (AUC) and prediction accuracy, defined as the model’s ability to predict both true positives (impairment) and true negatives (compliance). Univariate models generated action values, or environmental thresholds, to indicate potential E. coli impairment based on a single parameter. Multivariate models predicted probability of impairment given a suite of environmental variables, and jack-knife sensitivity analysis removed unresponsive variables to elicit a set of the most responsive parameters. Results Water temperature univariate models performed best as indicated by AUC, but alkalinity models were the most accurate at correctly classifying impairment. Sensitivity analysis revealed that models were most sensitive to removal of specific conductance. Other sensitive variables included water temperature, dissolved oxygen, discharge, and NO3. The removal of dissolved oxygen improved model performance based on testing AUC, justifying development of two optimized multivariate models; a 5-variable model including all sensitive parameters, and a 4-variable model that excluded dissolved oxygen. Discussion Results suggest that E. coli impairment in Sinking Creek is influenced by seasonality and agricultural run-off, stressing the need for multi-month sampling along a stream continuum. Although discharge was not predictive of E. coli impairment alone, its interactive effect stresses the importance of both flow dependent and independent processes associated with E. coli impairment. This research also highlights the interactions between nutrient and fecal pollution, a key consideration for watersheds with multiple synergistic impairments. Although one indicator cannot mimic theplethora of existing pathogens in water, incorporating modeling can fine tune an indicator’s utility, providing information concerning fate, transport, and source of fecal pollution while prioritizing resources and increasing confidence in decision making.