<p> The increase in microbes resistant to a wide array of antibiotics has led to the need for the development of novel antimicrobials. However in order to develop new antimicrobials, novel pathways need to be targeted. Teichoic acid is an anionic polymer covalently attached to the cell wall of Gram-positive bacteria. Recent research has demonstrated that teichoic acid genes are indispensable to the viability of Bacillus subtilis. This makes teichoic acid biosynthetic proteins ideal candidates for the development of a new antimicrobial. Of the teichoic acid glycerol phosphate (tag) genes involved in the biosynthesis of teichoic acid in B. subtilis 168, a conditional deletion mutant of tagD, whose protein product encodes the proposed glycerol-3-phosphatecytidylyltransferase, has been previously constructed and was shown to have a lethal phenotype upon depletion of TagD. This was used in a microarray analysis to find genes that were transcriptionally up-regulated upon the depletion of TagD in B. subtilis 168. Ten candidate genes were selected from those up-regulated and used in the design of a novel, real-time, cell-based luminescent reporter system that responds to lesions in wall biosynthesis. Characterization of these reporter systems in tag gene deletion backgrounds and an examination of their response to antibiotics of various mechanism of action led to the identification of our candidate reporter system P ywac, a robust reporter of both lesions in teichoic acid and peptidoglycan synthesis. In a proof-of-principle screen, the use of Pywac as a reporter of lesions in the cell wall was validated. This reporter system is unique in that it combines conventional genetics with a high throughput capacity. It will not only be amenable for screening small molecules to find inhibitors that impinge on teichoic acid biosynthesis, but it can also be used to probe genetic interactions in B. subtilis. </p> / Thesis / Master of Science (MSc)

The objectives of this research were to: 1) evaluate the accuracy of the Molly cow model predictions of ruminal metabolism and nutrient digestion when simulating dairy and beef cattle diets, 2) advance representations of N recycling between blood and the gut and urinary N excretion in the model, 3) improve the representation of pH and to refit parameters related to ruminal metabolism and nutrient digestion in the model, 4) investigate how ruminal pH affects the microbial community, expression of carbohydrate-active enzyme transcripts (CAZymes), fiber degradation, and short chain fatty acid (SCFA) concentrations. To achieve the first objective, a total of 229 studies (n = 938 treatments) including dairy and beef cattle data, published from 1972 through 2016, were collected from the literature and used to assess the model accuracy and precision based on root mean squared errors (RMSE) and concordance correlation coefficients (CCC). Only slight mean and slope bias were exhibited for ruminal outflow of NDF, starch, lipid, total N, and non-ammonia N, and for fecal output of protein, NDF, lipid, and starch. However, ruminal pH was poorly simulated and contributed to problems in ruminal nutrient degradation and VFA production predictions. To achieve the second objective, representations including ruminal ammonia outflow, intestinal urea entry, microbial protein synthesis in the hindgut, and fecal urea N excretion, were added in the model. Total urea entry, gut urea entry, and urinary urea elimination rates collected from 15 published urea kinetics studies were used to derive related parameters. Significant improvements in predictions of variables describing ruminal N metabolism, blood urea metabolism and urinary N secretion were exhibited after the modifications. To achieve the third objective, a dataset assembled from the literature containing 284 peer reviewed studies with 1223 treatment means was used to derive parameter estimates for ruminal metabolism and nutrient digestions. After refitting the parameters, the model is even more robust in representing ruminal nutrient degradation compared to the initial model. Adding ammonia concentration as a driver to the pH equation increased the precision of predicted ruminal pH, and thereby, the precision of predicted VFA concentrations due to an improved representation of pH regulation of VFA production rates. To achieve the fourth objective, six cannulated Holstein heifers with an initial BW of 362 ± 22 kg (mean ± SD) were subjected to 2 treatments in a cross-over design. The treatments were 10 days of intraruminal infusions of both 1) distilled water (Control), and 2) a dilute blend of hydrochloric and phosphoric acids to achieve a pH reduction of 0.5 units (LpH). Statistical analyses indicated 19 bacterial genera and 4 protozoal genera were affected by low ruminal pH. We observed significant correlations between 54 microbes (43 bacterial and 11 protozoal genera) and 25 enzymes, of which 8 key enzymes participated in reactions leading to SCFA production, suggesting that the ruminal microbial community alters fiber catalysis and fermentation in response to altered pH through a shift in carbohydrate-active enzyme transcripts (CAZymes) expression. Overall, after the modifications and reparameterizations, 19.7 to 37.5% of RMSE with essentially no slope bias and minor mean bias were exhibited for of ruminal and fecal outflow of ADF, NDF, fat, and protein, suggesting the model is properly to represent nutrient degradation and digestion in the bovine. Considering ruminal microbes and CAZymes in predicting ruminal volatile fatty acid concentrations could explain more variance of observations. / Ph. D. / The purpose of this research was to improve ruminal nutrient metabolism and nutrient digestion representations in the Molly cow model. First, the model accuracy and precision were assessed using a dataset including 229 studies (n = 938 treatments) conducted with dairy and beef cattle. The model evaluation results indicated the mechanisms encoded in the model relative to ruminal and total tract nutrient digestion are properly represented. However, ruminal pH was very poorly represented in the model with a RMSE of 4.6% and a concordance correlation coefficient (CCC) of 0.0. Although VFA concentrations had negligible mean (2.5% of MSE) and slope (6.8% of MSE) bias, the CCC was 0.28 implying that further modifications with respect to VFA production and absorption are required to improve model precision. As identified by the residual analyses, the representations of N recycling between blood and the gut were improved by considering ruminal ammonia outflow, intestinal urea entry, microbial protein synthesis in the hindgut, and fecal urea N excretion in the model. Observations of total urea entry, gut urea entry, and urinary urea elimination rates were collected from 15 published urea kinetics studies were used to derive related parameters. After the modifications, prediction errors for ruminal outflows of total N, microbial N, and non-ammonia non-microbial N were 39.5, 27.8 and 35.9% of the respective observed mean values. Prediction errors of each were approximately 10% units less than the corresponding values before model modifications and fitting due primarily to decreased slope bias. The revised model predicted ruminal ammonia and blood urea concentrations with substantially decreased overall error and reductions in slope and mean bias. After that, ammonia concentration as a driver was added to the pH equation, and a dataset assembled from the literature containing 284 peer reviewed studies with 1223 treatment means was used to derive parameter estimates for ruminal metabolism and nutrient digestions. Refitting the parameters significantly improved the accuracy and precision of the model predictions for ruminal nutrient outflow (ADF, NDF, total N, microbial N, non-ammonia N, and non-ammonia, non-microbial N), ammonia concentrations, and fecal nutrient outflow (protein, ADF, and NDF). Therefore, the improved model can be used to simulate nutrient degradation and digestion in the bovine. Although minor mean and slope bias were observed for ruminal pH and VFA concentrations, the small values for concordance correlations indicated much of the observed variation in these variables remains unexplained. To further explain variance in ruminal metabolism and understand how ruminal pH affects the microbial community, expression of carbohydrate-active enzyme transcripts (CAZymes), fiber degradation, and short chain fatty acid (SCFA) concentrations, six cannulated Holstein heifers with an initial BW of 362 ± 22 kg (mean ± SD) were subjected to 2 treatments in a cross-over design. We observed 19 bacterial genera and 4 protozoal genera were affected by low ruminal pH, and significant correlations between 54 microbes (43 bacterial and 11 protozoal genera) and 25 enzymes, of which 8 key enzymes participated in reactions leading to SCFA production. In summary, after the modifications and reparameterizations, the model is even more robust to represent nutrient degradation and digestion in bovine compared to the initial model. More variance of observations of ruminal volatile fatty acid concentrations could be explained by considering ruminal microbes and CAZymes expressions in further study.

Bt-maize is engineered to express insecticidal toxins derived from the bacterium Bacillus thuringiensis and has been shown to be very effective against pests like Busseola fusca. However, resistance of this pest against Bt-maize has developed and spread throughout South Africa. This study was inspired by the lack of knowledge over the microorganisms associated with the gut of these insects as they play a vital role in insect growth and development. Microbial-derived enzymes may have a role during an insect‟s adaption in different environmental conditions and to new diets. Previous studies suggest (1) that gut bacteria are required for B. thuringiensis-induced mortality in most Lepidoptera species and (2) that the toxicity of B. thuringiensis depends on microbial community interactions within the gut. The aim of this study was to determine the microbial diversity present in the midgut of B. fusca larvae occurring in maize. Busseola fusca larvae were collected from 30 sites throughout South Africa and dissected to collect their midgut contents. Serial dilutions were made of the contents and spread plated onto nutrient agar after which morphotypes were identified. One-hundred and five morphotypes were identified; DNA were extracted from the selected morphotypes and subjected to PCR analysis followed by secquencing. Sequencing results revealed the dominance of Enterococcus spp., specifically Enterococcus casseliflavus and Enterococcus gallinarum, Klebsiella spp., espesially Klebsiella pneumoniae and Klebsiella oxytoca and Bacillus spp. such as .B. thuringiensis and B. subtilis. Other organisms isolated, included Achromobacter spp., Brevudimonas spp., Caulobacter spp., Enterobacter spp., Halomonas spp., Ochrobactrum spp., Pantoea spp., Pseudomonas spp., Serratia spp., Stenotrophomonas spp., Arthrobacter spp., Brevibacterium spp., Leucobacter spp., Microbacterium spp., Planomicrobium spp. and Staphylococcus spp. The microbial diversity of larvae collected at the respective sampling sites were determined with the Shannon diversity index. The data were compared to several factors regarding the sampling sites. No significant differences were observed between the microbial diversities isolated at the respective sites. This may imply that the microbial community within B. fusca larvae are relative consistent throughout the maize production area. It is important to understand the distribution and structure of gut microbial communities within insects and whether the gut community is influenced by the geographical distribution of the insects. A better understanding of the distribution of the insects and community structure of their gut microbiota may aid in the development of better insect control strategies. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

Bt-maize is engineered to express insecticidal toxins derived from the bacterium Bacillus thuringiensis and has been shown to be very effective against pests like Busseola fusca. However, resistance of this pest against Bt-maize has developed and spread throughout South Africa. This study was inspired by the lack of knowledge over the microorganisms associated with the gut of these insects as they play a vital role in insect growth and development. Microbial-derived enzymes may have a role during an insect‟s adaption in different environmental conditions and to new diets. Previous studies suggest (1) that gut bacteria are required for B. thuringiensis-induced mortality in most Lepidoptera species and (2) that the toxicity of B. thuringiensis depends on microbial community interactions within the gut. The aim of this study was to determine the microbial diversity present in the midgut of B. fusca larvae occurring in maize. Busseola fusca larvae were collected from 30 sites throughout South Africa and dissected to collect their midgut contents. Serial dilutions were made of the contents and spread plated onto nutrient agar after which morphotypes were identified. One-hundred and five morphotypes were identified; DNA were extracted from the selected morphotypes and subjected to PCR analysis followed by secquencing. Sequencing results revealed the dominance of Enterococcus spp., specifically Enterococcus casseliflavus and Enterococcus gallinarum, Klebsiella spp., espesially Klebsiella pneumoniae and Klebsiella oxytoca and Bacillus spp. such as .B. thuringiensis and B. subtilis. Other organisms isolated, included Achromobacter spp., Brevudimonas spp., Caulobacter spp., Enterobacter spp., Halomonas spp., Ochrobactrum spp., Pantoea spp., Pseudomonas spp., Serratia spp., Stenotrophomonas spp., Arthrobacter spp., Brevibacterium spp., Leucobacter spp., Microbacterium spp., Planomicrobium spp. and Staphylococcus spp. The microbial diversity of larvae collected at the respective sampling sites were determined with the Shannon diversity index. The data were compared to several factors regarding the sampling sites. No significant differences were observed between the microbial diversities isolated at the respective sites. This may imply that the microbial community within B. fusca larvae are relative consistent throughout the maize production area. It is important to understand the distribution and structure of gut microbial communities within insects and whether the gut community is influenced by the geographical distribution of the insects. A better understanding of the distribution of the insects and community structure of their gut microbiota may aid in the development of better insect control strategies. / MSc (Environmental Sciences), North-West University, Potchefstroom Campus, 2015

One of the largest environmental liabilities facing the Canadian and international mining industry includes the effects of acidic drainage to water resources. This thesis sought to determine biogeochemical mechanisms of rare earth element and yttrium (REY) enrichment in mine drainage, linkages between REYs and microbial populations, and whether REYs were present in water or biofilm at mineable quantities or toxic levels. Water and co-occurring biofilm samples were collected from North and South American mining and control sites, and a passive water treatment system in Pennsylvania. REY concentrations within mineralized biofilm were observed to occur at borderline mineable quantities within biofilm in coal mine drainage (1,000 mg/kg dry weight total REYs), where REYs were bound predominately to particulate organic matter, manganese and iron, limiting their bioavailability. Within the passive treatment system, REYs showed the greatest maximum water-biofilm partitioning coefficients after Al and Fe, and a strong inverse relationship with aqueous REY concentration. Photosynthetic eukaryotes were observed to occur within biofilms that contained an abundance of neutrophilic iron oxidizing bacteria.

Mastitis and milk quality affect every dairy farmer across the globe. Sand bedded freestalls are the industry standard for cow comfort, welfare, and the control of environmental mastitis. Compost bedded packs may be a viable alternative to the sand bedded freestall. Compost bedded packs are maintained at a consistent level of moisture, nutrients, and aeration to favor compost microorganisms. Greater bacteria counts in bedding have traditionally been associated with increased mastitis rates and mastitis pathogens can be found in the pack and on the teats of cattle housed in even well managed compost bedded pack barns. In spite of this, herd SCC often remains low in well managed herds. The relationship between stress and comfort in the housing environment was a primary focus of this research. Cows housed in environments with low stress and high comfort may be better able to defend themselves against pathogens. Establishing changes in immune function in response to housing environment would improve milk quality by contributing to the knowledge of how mastitis-causing pathogens are contracted. An additional goal of this research was to determine the effect of compost bedded pack barns on thermoduric bacteria populations. Due to the increased temperatures associated with composting, thermoduric bacteria capable of surviving pasteurization are of potential concern in compost bedded packs. This research will investigate potential differences in thermoduric bacteria counts between compost bedded packs and sand bedded freestalls.

Beach advisories are issued at recreational beaches when the water quality exceeds regulatory limits for the indicator organism, enterococci. Elevated levels of enterococci have been observed at Hobie Cat Beach, the study marine beach site, located on Virginia Key, Florida. The study site represents a classic non-point source sub/tropical marine recreational beach area with high human and animal use, representative of many beaches worldwide in sub/tropical areas. The dissertation consisted of two separate but related studies: the first to identify environmental and geographic factors, and the second to evaluate the impact of known animal sources of enterococci. The first efforts were made to identify the geographic location of the source of enterococci to the beach waters and to assess the environmental factors that impact the variation in concentrations observed at the site. These environmental factors and conditions include: proximity to shoreline, tidal changes, impacts of runoff, and sunlight intensity. Enterococci were enumerated by traditional membrane filtration or the chromogenic substrate method. Overall, results showed that the source of enterococci to the study beach was geographically located within the inter-tidal zone. These results suggest that the wash-in of sediments and accompanying pore waters (where the pore water is the water filling the spaces between grains of sediment) from the inter-tidal zone play a major role in controlling enterococci levels within the water column. Wash-in occurs through both tidal fluctuations and runoff. The second effort evaluated non-point sources, including animals, which are known to contribute to elevated levels of enterococci in recreational marine beach waters. Specifically, feces from dogs, birds, and shrimp mounds were collected from the beach; additional bird fecal samples were collected from both a local zoo and bird rehabilitation center. Fecal samples were weighed gravimetrically, and enumerated for enterococci using traditional membrane filtration method. The total numbers of animals which frequented the site were obtained through camera image analysis and in-field visual counting surveys. The highest enterococci concentrations were observed in dog feces (avg. 7.4 x 10^6 CFU/g dry feces), then birds (avg. 3.3 x 10^5 CFU/g dry feces) and the lowest measured levels of enterococci were observed in shrimp fecal mounds (2.0 CFU/g dry feces on average). A comparison of the microbial load (CFU per fecal event) showed that 1 dog fecal event was equivalent to 6,940 bird fecal events or 3.2 x 10^8 shrimp fecal events. Given the abundance of animals observed on the beach, these study results suggest that dogs are the largest contributing source of enterococci to the beach site (6.3 x 10^11 CFU per day during weekends and 2.9 x 10^11 CFU per day during weekdays), with humans (4.6 x 10^9 CFU per day during weekends and 4.8 x 10^8 CFU per day during weekdays) and birds (2.7 x 10^8 CFU per day) serving as secondary contributors. Shrimp served as an insignificant source (1.9 x 10^4 CFU per day). When maximum daily contributions were considered, dogs contributed the highest proportion of enterococci (99.2%) compared to humans (0.72%), birds (0.04%), and shrimp (<0.04%). Beach management efforts at the study site should thus focus on requiring dog owners to properly dispose of dog feces deposited at the beach.

Microbes are prevalent in geologic settings and a growing body of research suggests the roles they play in geologic processes may be more important than previously thought, and therefore underestimated. This dissertation addresses the influence of microbes on the dissolution of limestone in karst settings by analyzing the stable carbon isotopes and geochemistry of air and waters from three unique cave and karst settings: West-Central Florida, the Everglades (southern Florida) and The Bahamas. In Florida, these parameters as well as air/water temperature, rainfall, and water-level fluctuations were monitored for 22 and 10 months. In the Bahamas, geochemical data were collected from at varying time-intervals from a variety of cave and surface water bodies. Results showed that microbial respiration in these environments is an important source of carbon dioxide, which contributes to the formation of carbonic acid, which appears to be the major dissolving agent at each of these sites. At the same time, microbially-mediated oxidation of both organic matter and minerals exerts a secondary dissolution control by providing additional acid and inorganic ions that dissolve rock and/or inhibit limestone precipitation. This dissertation also includes a chapter discussing the role of the USF Department Geology in the evolution of assessment for Spreadsheets Across the Curriculum (SSAC) project, which promotes quantitative literacy (QL) by teaching math in the context of other disciplines. Assessment occurred primarily in the Computational Geology course from 2005 to 2008 and showed that this teaching strategy fostered gains in math knowledge and positive math association. Simultaneously, instructors learned that pre-planning and adaptability was central to developing a successful assessment strategy, which, when combined with the heterogeneity of subjects each year, presents challenges in the yearly comparison of results. These conditions are common in educational settings, illustrating the impracticality of standardized assessment instruments and practices, and the importance of the extensive preparation required in identifying assessment goals and the best strategies for achieving them in a given setting.

White Island is a highly active volcano with an acidic, S-rich hydrothermal system in the Bay of Plenty, North Island, New Zealand. In this acidic environment a series of subsurface Fe-rich layers are ubiquitous in the crater sediments at shallow depth and are capable of modifying the flow and gas flux dynamics in the system. The mineralogy of the subsurface Fe-rich layer(s) and the processes leading to their formation are unknown. Here the mineralogy and formation of the subsurface Fe-rich layers in relation to the surface and subsurface environment(s) within the Main Crater at White Island are assessed. Based on geochemical analyses, subsurface Fe-rich crusts are composed of a mix of jarosite and goethite, cementing crater fill sediments into cohesive layers. Saturation index (SI) and Eh/pH assessments identify that fluids evolved at White Island are undersaturated with respect to the mineral phases present in the Fe-rich subsurface layers. Formation of the Fe-rich subsurface layers is most likely related to the transition between atmospheric gases and/or meteoric water mixing with hydrothermal fluids. This transition zone creates an environment conducive to forming jarosite and goethite forming in the same layer. Additionally, subsurface sediments including the Fe-rich layers show a consistent organic carbon isotopic signature of -23 ‰. Microscopic investigations confirm diatoms and microbes are present in the subsurface Fe-rich layers. The full extent of microbial activity in relation to the Fe-rich layers at White Island still requires further investigation. Based on chemical extractions for isotopic analyses, Fe-rich layers are shown to preserve δ¹³C signatures indicative of microbial life. Interface zones such as those identified in the hydrothermal environment at White Island can create metal-rich deposits and habitable/preservative microbial environments as well as affecting the macroscopic dynamics of volcanic and epithermal systems.

Climate models forecast lower and less frequent precipitation in the next 50 years. This is especially pronounced in the central United States, where Texas is expected to lose a week’s worth of rain every summer. Water availability is a primary driver of carbon flux in terrestrial ecosystems – controlling photosynthesis and organic matter decomposition. Thus, under proposed rainfall shifts, understanding the potential ecosystem response is key to predicting the future of terrestrial productivity. Terrestrial nutrient cycling is also driven by microbial saprotrophs, which are the chief decomposers of organic matter. Understanding the microbial response to rain shifts is key in predicting the ecosystem response. Research supports both microbial community specialization to local environment, and that the microbial communities may have the ability to rapidly acclimate to environmental change. To address this question of microbial response, we used a steep natural rainfall gradient along the Edwards Plateau in central Texas. The Edwards Plateau is an ideal field site in which to test these ideas because nearly identical grassland habitat and soils are found across its entirety, while mean annual precipitation ranges from 45 cm to 91 cm. To understand how soil microbial communities varied as a result of historical rainfall differences, we divided the gradient into four isoclines based on precipitation (46-56 cm, 56-66 cm, 66-76 cm, and 76-86 cm), and examined soil and soil microbial community characteristics at three sites in each isocline. We further used soils from the same sites for a reciprocal soil moisture experiment, where we asked how soil microbial communities responded to altered moisture conditions. Using a full factorial design, soils from each site in each isocline were exposed to one of four soil moisture treatments: soil moisture from the ‘home’ isocline and the three other ‘away’ isoclines. The moisture treatments were maintained for one year. Microbial respiration was measured at regular intervals throughout the experiment; fungal hyphal abundance and inorganic nitrogen were measured at the final harvest. The soils collected from the gradient decreased in both soil moisture and hyphal abundance from the wet to the dry end of the gradient, but there was no trend in inorganic nitrogen. In the reciprocal moisture experiment, microbial CO2 respiration was affected by both home isocline and soil moisture treatment. Drier sites had a narrower response to wetter treatments and did not achieve the same activity as wetter sites regardless of soil moisture treatment. In contrast, soils from the wettest isocline experienced severe reductions in activity with drying, with activity at the driest moisture treatment below that found in soils that were from the driest isocline. These patterns are consistent with some degree of local specialization, which may constrain the ability of microbial communities to rapidly acclimate to altered precipitation regimes. This experiment did not include immigration, however, and shifts in community composition in the presence of dispersal may be able to counteract local specialization. Given expected future increases in drought intensity microbial decomposition activity is likely to decrease and local specialization may create a lag in acclimation to the new condition. Thus, local specialization of microbial communities should be considered when predicting ecosystem responses to future climate change and their potential feedbacks to ecosystem productivity and carbon storage. / text