Browsing by Subject "Bacteria"

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Development of Activity-Based Probes and Biochemical Methods for the Study of Penicillin-Binding Proteins in Live Bacteria
(2021-11) Shirley, Josh
Bacterial cells are surrounded by a polymeric, mesh-like structure known as the peptidoglycan, and is an essential component of all eubacteria. Multi-protein machinery complexes function to carry out highly orchestrated synthesis and remodeling of the peptidoglycan throughout cell growth and division. One of the key components of these machinery complexes is the class of essential bacterial enzymes known as the penicillin-binding proteins (PBPs). PBPs are a highly conserved class of membrane-associate enzymes that function to carry out the final steps of peptidoglycan biosynthesis. All PBPs have a highly homologous transpeptidation domain, which contains a conserved, catalytic serine, to enable cross-linking of adjacent stem-peptide chains within the peptidoglycan. The catalytic serine residue has been exploited by the β-lactam class of antibiotics for ~ 100 years. Despite the success of the β-lactams as the most clinically used class of antibiotics, significant gaps in knowledge regarding the PBPs remain. The specific roles and regulations of individual PBP homologs is poorly understood and this can be attributed to a lack of appropriate tools to enable these studies. Our group has undertaken a chemical biology approach to addressing this gap, through the development of activity-based probes and biochemical methods that enable the visualization of PBP activities in native environments. The work presented in this thesis is focused on the expansion of available tools and methods that we have at our disposable to study the PBPs within live cells of both Gram-negative and Gram-positive bacteria. Efforts focused on the expansion of a β-lactone library of activity-based probes demonstrated that bioorthogonal probes retained the same activity as previously synthesized fluorophore-conjugated molecules but have increased utility in protein pull-down experiments to investigate the protein-protein interactions of specific PBP homologs. Next, the development of a live-cell kinetics assay in Streptococcus pneumoniae has provided a novel means to determine the potency values of inhibitors against the entire complement of an organism’s PBPs in one assay. The data that will be generated from future work will enable quantitative structure-activity-relationship studies to be performed, which in turn will inform us on the rational design of future PBP-selective molecules. Finally, the development of a live-cell method to study inhibitors of the PBPs in non-hypersusceptible Gram-negative species provides a means to identify molecules that are selective for the PBPs in these species and enable the development of activity-based probes for PBP homologs in understudied bacteria. In sum, we present new tools and methods that when combined with existing strategies will provide a more complete understanding of how individual PBPs function within live cells, ultimately enabling us to identify targets for the next generations of antibiotics.
Ecological and evolutionary perspectives on bacterial resource use
(2014-08) Weisenhorn, Pamela
Bacterial metabolism mediates many biochemical transformations important to the stability and health of a diverse range of ecosystem types. In my dissertation, I examine the evolutionary and ecological context of a subset of bacterial metabolic pathways related to energy and metabolic precursor production that are crucial for bacterial growth. Specifically, I examine whether these pathways are conserved across a large, phylogenetically diverse set of organisms, whether related organisms respond similarly to differences in resource inputs, and whether knowledge of these pathways or phylogenetic relatedness can aid in the prediction of bacterial growth rates across a wide range of C substrates. While I found only a weak phylogenetic signal in the presence or absence of these pathways, there was strong evidence that constraints have limited the number of observed combinations of these pathways. Only 265 (6.5%) of the 4096 potential pathway combinations were found in this dataset of 8178 genomes. I propose this may suggest strong environmental selection acting to rapidly change pathway presence or absence, regardless of past evolutionary history. In order for this suggestion to be feasible, organisms must respond to their environment in a phylogeny-independent manner. To address this, I compared taxa response using 16S amplicon libraries from plots with substantial variation in C and N availability resulting from plant species identity in a long-term field experiment. I found an inconsistent response of soil bacteria at higher taxonomic levels to resource variation, in agreement with organisms responding to environment in a phylogeny-independent manner. I then cultured 56 bacterial isolates from these plots to examine the relative strength of phylogeny versus metabolic pathways in explaining growth responses of isolates across a range of substrates. Phylogenetic relatedness and similarities in energy metabolism each explained about 30% of the observed variation in patterns of bacterial growth, with about 50% overlap between the two approaches. Both phylogeny and energy metabolism are important in determining bacterial growth; however, environmental selection may lead to convergence towards a small number of ecotypes within a system despite high levels of phylogenetic diversity. The strength and consequences of such environmental optimization of metabolism warrant further study.
Ecological stoichiometry of bacterial assemblages
(2013-12) Godwin, Casey Michael
All organisms are faced with a chemical imbalance between their internal environment (cells, tissues, or body) and their external environment. Homeostasis is the ability to maintain an internal state that is different from the external environment and at least some degree of elemental homeostasis is required for metabolism and growth. Homeostasis is related to fitness since the degree of elemental imbalance between an organism's biomass and its resources controls the growth of populations, predicts the outcome of competition, and determines the relative rates of resource consumption, assimilation, and excretion of elements and energy. Since all organisms are composed of molecules that are comprised mainly of a common set of elements (carbon (C), hydrogen, oxygen, nitrogen (N), phosphorus (P), etc.), stoichiometric ratios of these elements in biomass (e.g. C:Pbiomass) and resources (C:Presources) can be used to diagnose the strength of imbalance and to assess the nutritional state of organisms. The strength of elemental homeostasis is variable within and among groups of taxa; some species and groups maintain strong homeostasis, but others adjust their chemical composition in response to their environment. Since ecosystems seldom contain only a single species, assemblages and communities can respond to elemental imbalance both through changes in the relative abundance of species and through simultaneous changes in the elemental content of the component species. The goals of this dissertation are to evaluate the role of resource competition and species shifts in the stoichiometry of assemblages and to understand the ranges of stoichiometric regulation and biomass chemistry within the bacterial assemblages of lakes. In chapter 1, I introduce the conceptual framework of `stoichiometric strategies' to align the gradient of stoichiometric regulation with physiological tradeoffs. Data from previously published studies on planktonic organisms show that the strength of homeostasis in a species is inversely proportional to the ratio of the two elements in its biomass when the denominator element is limiting. Under nutrient limitation, homeostatic species have lower biomass C:N, C:P, and N:P ratios than do species with flexible biomass stoichiometry. I show how a consumer-resource model with tradeoffs related to competitive ability for C and P couples homeostatic regulation to competitive ability. The result is a conceptual model in which assemblages are dominated by homeostatic species under low resource imbalance and by species with flexible stoichiometry when nutrients are strongly limiting. I test the stoichiometric strategies concept in chapter 2 by culturing assemblages of heterotrophic bacteria at a range of resource ratios and examining the strength of homeostasis in the dominant species. I found that low resource C:P ratios could select for homeostatic strains of bacteria and that higher resource C:P ratios yielded assemblages with flexible composition. In chapter 3, I use bacteria isolated from lakes to describe how homeostatic strains and flexible strains respond to imbalance in C and P. The strains exhibited substantial variation in stoichiometric regulation, but strong homeostasis was associated with higher C and P content and flexible stoichiometry was present only in strains with low P content. These experiments support the hypothesis that flexible biomass composition is competitively superior under P limitation. In the final chapter, I seek to characterize the range of cellular P content attainable by heterotrophic bacteria and determine how bacteria minimize their P content in response to P limitation. I show that bacteria can exhibit greater flexibility in P content than was known previously (< 0.01 to 3% of dry mass as P, biomass C:P of 30:1 to > 10,000:1) and that this flexibility is explained by a simultaneous increase in C content (13 to > 70 fmoles cell-1) and decrease in P content (0.62 to < 0.06 fmoles cell-1) under P limitation. These studies highlight the importance of physiological constraints and assemblage-level interactions to understanding the impact of stoichiometry on biogeochemical cycles. Additionally, the results of these experiments show that strains of bacteria differ dramatically in their elemental composition, stoichiometric regulation, and resource demands and that the assumptions of strong homeostasis and high nutrient content are not representative of bacteria in aquatic environments. Although aquatic heterotrophic bacteria serve as a useful system to address these questions, the constraints appear to be fundamental and these results are likely applicable to other groups of organisms.
Engineered Nanomaterial Interactions with Bacterial Cells
(2016-05) Gunsolus, Ian
Nanomaterials occur naturally in a variety of forms. They exist, for example, in the aerosols produced from sea spray and in the particulates produced from incomplete combustion of hydrocarbons. In the latter 20th century, development of instruments such as the scanning tunneling microscope and atomic force microscope have allowed us to directly see and to manipulate nanoscale matter. Armed with these instrumental capabilities and a desire to push the limits of our ability to create and manipulate matter, we have begun to engineer nanomaterials for our own use. Today, nanomaterials are used as additives in numerous commercial products to improve performance and/or reduce cost. Examples include silver nanomaterials in fabrics to inhibit microbial growth and titanium dioxide nanomaterials in outdoor paints to reduce weathering. Less often, nanomaterials serve a primary function in product performance; one important example of this is the use of nanoscale mixed metal oxides as cathode materials in lithium-ion batteries, used in some electric vehicles. The increasing commercial use of engineered nanomaterials increases direct human contact with nanoscale matter beyond that which formerly occurred naturally. Taking a proactive view of these developments, a small group of researchers began, in the early 2000s, to assess the implications of nanomaterial exposure on human health, giving rise to the field of nanotoxicology. In recent years, the field has expanded its focus beyond human health to include environmental health, recognizing that the waste streams resulting from the production, use, and disposal of products containing nanomaterials serve as new sources in natural environments. The goal of environmental nanotoxicity research, of which my dissertation research is a part, is to promote the sustainable use of engineered nanomaterials by assessing their environmental toxicity and informing their design in order to minimize environmental impact. As a project rooted in chemistry, my dissertation focuses in particular on identifying molecular structures, both nanomaterial and biological, that can be used to predict and control the environmental impact of nanomaterials. My research focuses on characterizing the interactions of commercially relevant nanomaterials with microorganisms, which play fundamental roles in healthy ecosystems. The bacterium Shewanella oneidensis MR-1, grown in culture, was used throughout my research as a model, albeit greatly simplified, of microorganism communities in natural environments. This particular bacterium was chosen due to the worldwide distribution of its genus, Shewanella, and its ability to survive in many environments, including aerobic, anaerobic, low-temperature, and high-salinity environments. Using this drastically simplified model greatly facilitates isolation of experimental variables, which would be much more difficult to achieve in the extremely chemically complex environment of soil or water samples collected from nature. This, in turn, greatly facilitates hypothesis testing. However, experimentation using samples obtained directly from nature is also necessary to develop a complete understanding of nanomaterial behavior in the environment. My research specifically addresses the following questions: What impact does natural organic matter (a ubiquitous component of natural sediments, soils, and water bodies) have on nanoparticle toxicity to bacteria in aquatic environments? How can we visually observe nanomaterial interactions with bacteria, both of which are near or below the diffraction limit of light, under hydrated conditions? Which structures on the bacterial cell surface primarily interact with nanomaterials? By what mechanism(s) might nanoscale battery cathode materials be toxic to bacteria, and how can we design less-toxic materials? The five major outcomes of my research, briefly summarized below, are presented in detail in Chapters 2-6. To address the first question (Chapters 2 and 3), I investigated the interactions between silver nanoparticles (also silver ions -- produced under aerobic conditions by the dissolution of silver nanoparticles) and natural organic matter. Natural organic matter is a complex mixture of polysaccharides, proteins, nucleic acids, and lipids and is produced through the decomposition of vegetative and microbial matter. Engineered nanoparticles entering natural environments, including soils, sediments, and water bodies, will inevitably encounter natural organic matter. Previous research has demonstrated that nanoparticle transport, persistence, and toxicity are influenced by interactions with natural organic matter. However, some reports conflict with these results and have demonstrated little or no impact of natural organic matter on nanoparticle behavior (e.g., colloidal stability). This conflict may result from a lack of attention paid to differences in the chemical composition of natural organic matter derived from various natural sources. The chemical heterogeneity of natural organic matter in various natural environments is significant, but researchers have often considered it to be a standard “class” of molecules that has common patterns of interaction with nanoparticles. My research, conducted in collaboration with Drs. Philippe Bühlmann and Maral Mousavi at the University of Minnesota—Twin Cities, sought to more specifically define the characteristics of natural organic matter that influence the behavior of silver nanoparticles and ions in natural aquatic environments. This research revealed that natural organic matter adsorption to silver nanoparticles and binding to silver ions depend greatly on the concentration of sites with high affinity for silver (e.g., sites rich in S and N). This result was affirmed by subsequent experiments with Shewanella, wherein silver nanoparticles and ions were less toxic only when first exposed to natural organic matter with this high binding affinity. This research also demonstrated a novel application of ion-selective electrodes in real-time monitoring of the dissolution kinetics of silver nanoparticles and the kinetics of natural organic matter binding to silver ions. This approach represents a significant improvement over the previous state-of-the-art (i.e., inductively-coupled plasma optical emission spectroscopy/mass spectrometry), which was limited to observing total silver concentration only (rather than distinguishing complexed and free forms of silver) and could be applied only at discrete time-points rather than being used for continuous measurements. To address the problem of visually observing nanomaterial interactions with bacterial cells (Chapter 4), I developed a novel and facile method to fluorescently stain bacterial cell surfaces for super-resolution fluorescence microscopy (SRFM). SRFM is uniquely capable of visualizing biological samples with high (sub-diffraction-limited) resolution under hydrated conditions. Electron microscopy, the current gold standard for high-resolution imaging, achieves higher resolution than SRFM but requires that samples be dehydrated and embedded in resin, procedures that can significantly alter the sample from its native state. Despite this advantage over electron microscopy, SRFM has been underutilized due to the complex fluorescent labeling strategies required. Current strategies based on genetic encoding of fluorescent proteins and fluorescent small-molecule labels require significant development time and are not generalizable across bacterial types (i.e., gram-positive and gram-negative bacteria). The fluorescent labeling strategy I developed uses only commercially available reagents and can be used to label both gram-positive and gram-negative bacterial cells. Utilizing the imaging instrumentation and resources at the Pacific Northwest National Laboratory, Richland, WA and with the collaboration of Dr. Galya Orr’s laboratory, super-resolution images of the gram-negative Shewanella oneidensis and the gram-positive Bacillus subtilis were acquired using two SRFM techniques (structured-illumination microscopy and stochastic optical reconstruction microscopy). In addition, structured-illumination microscopy was performed to visualize Shewanella oneidensis exposed to fluorescent cadmium selenide/zinc sulfide core-shell quantum dots under hydrated conditions. This method achieved sufficient resolution to determine that quantum dots were bound to the cell surface without translocating across the cell membrane. Research to further characterize the site of bacterial cell-nanomaterial interactions was motivated in part by the aforementioned SRFM imaging of Shewanella oneidensis exposed to quantum dots. My goal was to determine which surface membrane species mediated the interaction of the quantum dots with the bacterial cells. I hypothesized that lipopolysaccharides, abundant molecules in the outer leaflet of gram-negative bacterial cell membranes and extending from the membrane surface into the surrounding solution, was the critical species. Lipopolysaccharides form a highly cross-linked, hydrated barrier that helps protect the lipid membrane from damage caused by antimicrobial peptides, hydrophobic antibiotics, and surfactants. Using ethylenediaminetetraacetic acid to release divalent cation crosslinkers between adjacent molecules, I reduced the concentration of lipopolysaccharides in the outer membrane of live Shewanella oneidensis cells. After exposing cells with either intact or depleted lipopolysaccharides to gold nanoparticles, I quantified nanoparticle-to-cell association using a novel flow cytometry method developed in this work. This method exploited the high light-scattering cross section of gold nanoparticles as well as fluorescent labeling of cells to rapidly screen cells for gold nanoparticle association with high throughput. To more precisely assess lipopolysaccharide-nanoparticle interactions, parallel experiments using supported lipid bilayers were conducted by Dr. Kurt Jacobson in the laboratory of Dr. Joel Pedersen at the University of Wisconsin—Madison. The association between gold nanoparticles and supported lipid bilayers containing lipopolysaccharides was quantified using quartz crystal microbalance with dissipation. Use of supported lipid bilayers enabled greater control over lipopolysaccharide concentration and length than was possible using whole cells. Our combined results showed that lipopolysaccharide density and length determine the extent and distance of nanoparticle interaction with the gram-negative bacterial cell outer membrane. This work provides a basis for predicting the extent of interaction between nanoparticles and gram-negative bacteria, whose constituent lipopolysaccharides vary in length and density, and for engineering nanoparticles with enhanced or reduced bactericidal activity. The environmental implications of nanomaterial use in lithium-ion batteries is the subject of the final experimental chapter of my thesis, Chapter 6. This research, performed in collaboration with Mimi Hang from the laboratory of Dr. Robert Hamers at the University of Wisconsin—Madison, focused on nanoscale lithium nickel manganese cobalt oxide (NMC), currently used as a cathode material in the batteries of some commercially available electric vehicles. The goal of this research was to characterize the impact of NMC exposure on Shewanella oneidensis and to use this knowledge to propose a modified material design that reduces potential biological and environmental impacts. Our results show that exposure to NMC reduces bacterial growth and respiration and that this effect is attributable to leaching of metal ions (in particular Ni and Co species) from NMC in aqueous environments. Subsequently, we synthesized a series of Mn-enriched (and Ni- and Co-depleted) NMC species and characterized their impact on Shewanella oneidensis. Manganese enrichment significantly reduced but did not eliminate NMC’s toxicity. Ongoing research is focused on developing new synthetic strategies to limit metal ion leaching, including capping NMC with an insoluble layer, such as lithium iron phosphate. In summary, this research has identified several molecular-level phenomena that govern engineered nanomaterial interactions with bacterial cells, which are key members of natural ecosystems. By contributing to a more complete and fundamental understanding of engineered nanomaterial behavior in the environment, the author hopes this research will promote the sustainable and responsible use of engineered nanomaterials.
Fluoride and Gallein Inhibit Polyphosphate Accumulation by Oral Pathogen Rothia dentocariosa - Data Sharing Archive
(2023-01-25) Kumar, Dhiraj; Mandal, Subhrangshu; Bailey, Jake V.; Flood, Beverly E.; Jones, Robert S.; rsjones@umn.edu; Jones, Robert, S; Collaboration between Earth and Environmental Science and School of Dentistry
This raw data set supports publication found in Letters in Applied Microbiology: The uptake and storage of extracellular orthophosphate (Pi) by polyphosphate (polyP) accumulating bacteria may contribute to mineral dissolution in the oral cavity. To test the effect of potential inhibitors of polyP kinases on Rothia dentocariosa, gallein (0, 25, 50, 100 µM) and fluoride (0, 50, 100 ppm) were added to R. dentocariosa cultures grown in brain heart infusion broth. At late log growth phase (8h), extracellular Pi was measured using an ascorbic acid assay, and polyP was isolated from bacterial cells treated with RNA/DNAases using a neutral phenol/chloroform extraction. Extracts were hydrolyzed and quantified as above. Gallein and fluoride had minor effects on bacterial growth with NaF having a direct effect on media pH. Gallein (≥25 µM) and fluoride (≥50 ppm) attenuated the bacterial drawdown of extracellular Pi 56.7% (p <0.05) and 37.3% (p <0.01). There was a corresponding polyP synthesis decrease of 73.2% (p<0.0001) from gallein and 83.1% (p<0.0001) from fluoride. Attenuated total reflectance Fourier transform infrared spectroscopy validated the presence of polyP and its reduced concentration in R. dentocariosa bacterial cells following gallein and fluoride treatment. R. dentocariosa can directly change extracellular Pi and accumulate intracellular polyP but the mechanism is attenuated by gallein and NaF.
An In Vitro assessment of the setting expansion of gray and white mineral trioxide aggregate.
(2010-08) Meade, Brian M.
Abstract summary not available
An Investigation of Nanoparticle Toxicity Mechanisms against Environmentally Relevant Bacteria and the Potential for Sustainable Agriculture Applications
(2019-05) Buchman, Joseph
Due to the unique physicochemical properties of nanoparticles, largely due to their high surface area-to-volume ratio, they are being increasingly used in consumer products. At any time during the manufacture, use, and ultimately, disposal of these products, there is a reasonable likelihood of nanoparticle release into the environment. Once released, their impact on the environment are less well-understood. Therefore, there is a growing emphasis to understand the impacts of nanoparticles on the environment, by understanding how the nanoparticles interact with ubiquitous organisms that have important ecological roles. Beyond looking solely at whether nanoparticle introduction will kill these organisms, the molecular-level mechanisms of their toxicity have been studied. By understanding the mechanisms, the goal is to be able to predict the toxicity of nanoparticles prior to their mass production, and to inform a more sustainable design and use of nanomaterials. Chapter One of this work reviews the understanding of molecular-level toxicity mechanisms to organisms in the environment, with an emphasis on beneficial bacteria. It also describes different strategies that have been employed to redesign nanoparticles that reduce the impact of these toxicity mechanisms. Chapter Two illustrates the importance of using more than one organism when doing studies of nanoparticle toxicity. Not all organisms respond equally, and there are some that are not impacted by a given nanoparticle type, so use of multiple species that cover a range of complexities improves the chances that a nanoparticle will not be incorrectly labeled as “nontoxic”. By using multiple organisms, those that are most impacted can also be identified for follow-on research to investigate the mechanism of toxicity. Chapter Three assesses the toxicity mechanism of an important nanomaterial often used in energy storage applications that is made of the complex oxide, lithium nickel manganese cobalt oxide, across a range of industrially-relevant stoichiometries. While for equimolar stoichiometries of this material, the importance of nickel and cobalt release has been implicated as the main toxicity driver to Shewanella oneidensis MR-1, this work demonstrates that even at increased nickel concentrations in the material, the toxicity remained the same due to increased material stability leading to a similar dissolution profile. For another important environmental organism, Daphnia magna, the toxicity did increase with increasing nickel content, indicating that a material redesign will not necessarily have the same impact on different organisms. Chapter Four investigates the impact of iron oxide nanoparticles to S. oneidensis, which showed that these nanoparticles improved bacterial survival, mostly due to the release of beneficial iron ions. Since changing bacterial populations can perturb an environment, a mesoporous silica coating was applied to the iron oxide nanoparticles to reduce their dissolution and their impact on the bacteria. While more understanding of the mechanisms by which nanoparticles can exhibit toxicity is being gained, there are many nanoparticles for which there is a low toxicity to organisms. In Chapter Five, we apply silica nanoparticles, which have been found to be largely nontoxic, to our plant model, Citrullus lanatus. Through dissolution, silica nanoparticles are capable of serving as a source of silicic acid, an important micronutrient, for plants. These nanoparticles benefit healthy plants by increasing their biomass and improving the overall fruit yield. This work demonstrates a way to apply nanoparticle toxicity knowledge to proactively utilize nanoparticles to improve sustainability in agriculture.
Measurement and modeling of denitrification in sand-bed streams of varying land use
(2013-02) Guentzel, Kristopher Steven
Processes that govern transport and transformation of aquatic nitrogen are of growing importance due to increases in anthropogenic nitrogen input from fertilizer application and fossil fuel combustion. Denitrification, the incremental reduction of soluble nitrate to gaseous end products, is the main pathway in which nitrogen is biologically removed from aquatic ecosystems. In this study denitrification is measured from sediment cores in five streams in central Minnesota, USA, using denitrification enzyme activity (DEA) assays as well as microbiological techniques including the amplification of nirS gene fragments through qPCR. Hydraulic and environmental variables are measured in the vicinity of the sediment cores to determine a possible mediating influence of fluid flow and chemical variables on denitrification activity. Denitrification rates measured using DEA analysis with amended nutrients ranged from 0.02-10.1 mg-N m-2 hr-1. Denitrification rates measured without amended nutrients were a factor of 5.35 less on average and ranged from 0.03-0.98 mg-N m-2 hr-1. The abundance of the denitrifier gene nirS was positively correlated with denitrification potential measurements (R2 = 0.79, P < 0.001) for most of the streams studied. NirS distribution in one of the sites, a field scale experimental stream called the Outdoor StreamLab, followed the spatial distribution of benthic organic matter closely along the sediment bed and through the sediment column. Predictive models to determine nitrate uptake via denitrification were derived from hydraulic, morphologic and water quality variables. The first used hydraulic data collected over three summers in the Outdoor StreamLab. A Gaussian-type function was fit to these data and was dependent on fluid flow and channel characteristics within the stream system. The second model was derived following dimensional analysis on data from the Outdoor StreamLab and four other natural streams of varying watershed and in-stream conditions. This predictive model integrated not only stream hydraulic data but also environmental, morphological and DEA measurements for nutrient-amended and unamended samples. The proposed model explained 75% and 60% of the variability in amended and unamended DEA rates, respectively. Results from this study verify that denitrification is ubiquitous across varying stream systems but is most dependent on the distribution of sediment organic matter and interstitial pore space as well as stream hydraulic characteristics.
The role of Enterococcus faecalis biofilm formation in the regulation of conjugation.
(2012-06) Cook, Laura Carol Case
Enterococcus faecalis has recently emerged as an important nosocomial pathogen. Pathogenicity of these organisms depends greatly on a few important aspect of enterococcal physiology. The ability of enterococci to form biofilms greatly enhances their virulence. Their innate resistance to many antibiotics and their ability to transfer these resistance genes through conjugation heightens their threat to human health. The work described in this thesis attempts to explain the roles of biofilm growth, conjugation, and cell communication in E. faecalis. To examine the role of biofilm growth on the E. faecalis transcriptome, RNAseq analysis was undertaken. We found that over 100 genes were measurably upregulated during biofilm growth while approximately 26 genes were downregulated. These data gives us important insights into the biology of enterococcal biofilms. In clinical settings, biofilms are likely locations for antibiotic resistance transfer events involving nosocomial pathogens such as E. faecalis. Conjugation is an important mode of horizontal gene transfer in bacteria, enhancing the spread of antibiotic resistance. In this work, I demonstrate that growth in biofilms alters the induction of conjugation by a sex pheromone in E. faecalis. Mathematical modeling suggested that a higher plasmid copy number in biofilm cells would enhance a switch-like behavior in the pheromone response of donor cells with a delayed, but increased response to the mating signal. Alterations in plasmid copy number and a bimodal response to induction of conjugation in populations of plasmid-containing donor cells were both observed in biofilms, consistent with the predictions of the model. The pheromone system may have evolved such that donor cells in biofilms are only induced to transfer when they are in extremely close proximity to potential recipients in the biofilm community. These results have important implications for development of chemotherapeutic agents to block resistance transfer and treat biofilm-related clinical infections.
A Source to Tap Investigation of Minnesota's Groundwater Supplies Used for Drinking Water
(2018-11) Galt, John
Groundwater is often a desirable drinking water source because it is generally free of suspended solids and microbial pathogens and thus requires minimal, if any, treatment prior to distribution. Epidemiological studies have shown, however, that consumption of untreated groundwater increases risk of gastrointestinal illness. Previous work in Wisconsin, USA reported the occurrence of pathogenic viruses in groundwater supplies and resulting health impacts but bacterial pathogens were not investigated. In this study, a high-volume (300 – 1500 L) dead-end ultrafiltration sampling method was used to capture and recover microbes from 16 public groundwater systems throughout the State of Minnesota. The systems were sampled at the wellhead or source, after treatment if any (i.e., two systems did not treat or disinfect before distribution), and from one location in the distribution system. DNA was extracted from the microbes recovered in these samples and used as template for quantitative PCR analyses targeting 14 genes corresponding to pathogenic bacteria, one gene for a DNA virus, and the 16S rRNA gene as a marker for total bacteria. All samples were negative for the targeted genes from Campylobacter jejuni, Shigella spp., and Adenovirus; Escherichia coli-specific genes were only detected in water from a non-potable well with a documented history of contamination. Genes markers for two genera, Legionella and Mycobacteria, that include species that are opportunistic pathogens, were detected in four of the 16 public groundwater supplies, with Legionella levels decreasing in disinfected systems while Mycobacteria levels tended to increase. Raw water 16S rRNA gene concentrations ranged from 10^5 – 10^8 gene copies/L, decreased to background levels after disinfection, then rebounded at the tap in the majority of cities. There was no significant difference in 16S rRNA gene concentrations from source-to-tap in the two non-disinfecting cities. Raw water samples contained diverse and previously uncharacterized organisms as revealed by DNA sequencing analyses, and beta diversity analyses suggest that community composition is driven by source water and/or disinfection. The results from this study suggest that groundwaters supplying public water systems in Minnesota are largely free of enteric pathogens but may contain opportunistic pathogens.
Use of Machine Learning to Predict the Desiccation Tolerance of Bacteria
(2021-08) Clipsham, Maia
For efficient long-term storage and use of bacteria for environmental applications, understanding and identifying desiccation resistance in bacteria is key. In the past, desiccation tolerance was a common way of characterizing bacteria, so there is much data on the desiccation tolerance of a wide range of bacterial species. Since the advent of transcriptomics, multiple papers have been published on the expression level of genes during desiccation stress. Additionally, many reviews have described mechanisms and genes relevant to desiccation tolerance in bacteria, but an overarching framework for the prediction of desiccation survival in bacteria is lacking. Model building based on data collected from the literature has been used to successfully predict aerobic vs anaerobic phenotype, enzyme function and substrate specificity (Robinson et al., 2020; Jabłońska et al, 2019) Building on this wealth of previous research, machine learning was used to create a robust model that predicts desiccation tolerance given bacterial genomes. Validation and accuracy of the machine learning model was tested using a desiccation assay carried out over three months. To build the model, a literature review was conducted to find genes that were upregulated greater than two-fold during desiccation stress in bacteria. From the review, 2609 genes from 11 papers were found and condensed to 1082 non-homologous and non near-zero variance genes. A second literature search was conducted to identify bacterial species with a known desiccation response, either tolerant or sensitive, and a publicly available genome. Thirty-five desiccation tolerant and 33 desiccation sensitive genomes were chosen and then queried for the previously curated desiccation upregulated genes list. Approximately 176,800 genes were analyzed, and genes with non-zero variance were removed. The remaining 75,982 genes are included in the model (Rogozin et al., 2002). A random forest supervised machine learning approach was used to create a preliminary model for desiccation resistance. The genomes were split into 80% training data and 20% test data and the model was run 100 times with different seeds, 10-fold cross validation, and three repeats. The average accuracy for the 100 iterations of the model was 0.898 ± 0.0266, indicating the model could accurately predict the desiccation phenotype of the testing data 89.8% of the time. The experimental validation of the desiccation model looked at the viability of 28 bacteria, seven with documented desiccation phenotypes and 21 bacteria with no known desiccation phenotype. For all organisms tested the model had an accuracy of 0.75 demonstrating good model performance.
Wild Primate Gut Microbiota Protect Against Obesity
(2017-04) Sidiropoulos, Dimitrios, N; Clayton, Jonathan; Al-Ghalith, Gabe; Shields-Cutler, Robin; Ward, Tonya; Blekhman, Ran; Kashyap, Purna; Knights, Dan
The gastrointestinal tract hosts trillions of bacteria that play major roles in metabolism, immune system development, and pathogen resistance. Although there is increasing evidence that low dietary fiber in Westernized societies is associated with dramatic loss of natural human gut microbiome diversity, the role of this loss in obesity and inflammation is not well understood. Non-human primates (NHPs) can be used as model systems for studying the effects of diet and lifestyle disruption on the human gut microbiome. Captive primates are typically exposed to low-fiber diets and tend to have human-associated microbiota in place of their native microbiota. In order to explore interactions between the gut microbiota and dietary fiber, we transplanted captive and wild primate gut microbiota into germ-free mice and then exposed them to either a high- or low-fiber diet. We found that the group receiving low-fiber diet and captive primate microbiota became obese and had high levels of circulating inflammatory cytokines, while mice receiving high-fiber diet and wild primate microbiota remained healthy. Mice with the wild primate microbiota and low-fiber diet acquired intermediate levels of obesity, demonstrating an interaction between dietary fiber and the microbiota. These results show that the modern human gut microbiome interacts with low-fiber diets to cause inflammation and obesity, and suggest a possible clinical role for manipulation of the microbiota in the treatment of obesity.

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