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Title: Genetic diversity of clinical and bovine non-o157 shiga toxin-producing escherichia coli (stec
Creator: Blankenship, Heather Marie
Date: 2019
Collection: Electronic Theses & Dissertations
Description: Shiga toxin-producing Escherichia coli (STEC) is a leading cause of foodborne infection resulting in 265,000 illnesses and more than 3,600 hospitalizations annually. Since its identification in 1982 associated with an outbreak of haemorrhagic colitis, serotype O157:H7 has been the primary focus of research and surveillance. However, the increasing incidence of other serogroups, or non-O157 STEC, that are associated with clinical illness has since surpassed the incidence of O157 and has raised...
Show moreShiga toxin-producing Escherichia coli (STEC) is a leading cause of foodborne infection resulting in 265,000 illnesses and more than 3,600 hospitalizations annually. Since its identification in 1982 associated with an outbreak of haemorrhagic colitis, serotype O157:H7 has been the primary focus of research and surveillance. However, the increasing incidence of other serogroups, or non-O157 STEC, that are associated with clinical illness has since surpassed the incidence of O157 and has raised questions about the genetic diversity of this pathogen population. Six serogroups, O26, O45, O103, O111, O121, and O145, have been denoted as "big six" non-O157 STEC serogroups since they are frequently associated with clinical outcomes.In this dissertation, 895 non-O157 STEC isolates recovered from patients in Michigan between 2001-2018 were analyzed using whole genome sequencing (WGS) to identify virulence gene profiles and apply new typing methods to better discriminate closely related strains. The recovery of a wide range of serogroups from cases presenting with symptoms ranging from mild diarrhea to hemorrhagic colitis, indicates that genetic diversity and variation may have an impact on disease outcomes. The number and richness of serogroups identified over the past 18 years has been steadily increasing and serogroup alone lacks the discriminatory capabilities to classify related isolates. Indeed, strains representing the same sequence types (ST) were often found to be unrelated by serogroup. Notably, some serogroups, STs, virulence gene profiles and alleles were associated with clinical outcomes and patient demographics. Contrast to national surveillance, cases between 11 and 29 years of age had the highest frequency of STEC infections in Michigan.Additionally, a subset of 44 non-O157 STEC recovered from Michigan patients between 2000 and 2006 were examined more comprehensively while making comparisons to 114 clinical STEC isolates from Connecticut to examine the impact of geographic location on risk factors for non-O157 STEC infections. Lastly, a subset of STEC isolates associated with outbreaks in Michigan were examined to identify the impact of WGS on identification of strain relatedness for surveillance compared to pulsed-field gel electrophoresis.While most of the work outlined in this dissertation focused on characterizing clinical non-O157 STEC isolates, a comparative analysis of cattle isolates was also performed since cattle are an important reservoir of STEC. Indeed, numerous outbreaks and illnesses have been traced back to contaminated cattle-based food products or fecal contamination of water and crops. The ability of STEC to persist in the cattle reservoir and farm environment may give rise to more pathogenic strains due to the accumulation of horizontally acquired genes. 66 STEC isolates recovered from a beef herd over four samplings were examined to identify the genetic diversity within the cattle population and longitudinal persistence. The ability of a strain to form a strong biofilm was associated with the ability to persist and be recovered at multiple sampling phases from the same animal. Further, to better understand the genetic diversity of STEC recovered from the cattle reservoir, an additional 12 STEC isolates from three bovine herds (n = 78) and 241 clinical O157 STEC isolates (n = 1,135) were included to identify shared profiles. The similarity in serogroups and virulence gene profiles warrant a continued surveillance of the cattle environment to better understand crossover events and the ability of strains to evolve into new virulent STEC lineages. The work described in this dissertation helped to elucidate the genetic characteristics important for clinical outcomes and identified targets for future surveillance to better understand lineages that may be important for disease.
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Title: Postmortem microbiome computational methods and applications
Creator: Kaszubinski, Sierra Frances
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Microbial communities have potential evidential utility for forensic applications. However, bioinformatic analysis of high-throughput sequencing data varies widely among laboratories and can potentially affect downstream forensic analyses and data interpretations. To illustrate the importance of standardizing methodology, we compared analyses of postmortem microbiome samples using several bioinformatic pipelines, while varying minimum library size or the minimum number of sequences per sample...
Show moreMicrobial communities have potential evidential utility for forensic applications. However, bioinformatic analysis of high-throughput sequencing data varies widely among laboratories and can potentially affect downstream forensic analyses and data interpretations. To illustrate the importance of standardizing methodology, we compared analyses of postmortem microbiome samples using several bioinformatic pipelines, while varying minimum library size or the minimum number of sequences per sample, and sample size. Using the same input sequence data, we found that pipeline significantly affected the microbial communities. Increasing minimum library size and sample size increased the number of low abundant and infrequent taxa detected. Our results show that bioinformatic pipeline and parameter choice significantly affect the resulting microbial communities, which is important for forensic applications. One such forensic application is the potential postmortem reflection of manner of death (MOD) and cause of death (COD). Microbial community metrics have linked the postmortem microbiome with antemortem health status. To further explore this association, we demonstrated that postmortem microbiomes could differentiate beta-dispersion among M/COD, especially for cardiovascular disease and drug-related deaths. Beta-dispersion associated with M/COD has potential forensic utility to aid certifiers of death by providing additional evidence for death determination. Additional supplemental files including tables of raw data and additional statistical tests are available in supplemental files online, denoted in the text as table 'S'.
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Title: Characterizing single-cell behavior of flagellated pathogenic bacteria in mucus and visco-elastic environments
Creator: Nguyen, Nhu Thi Quynh
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Flagellar motility is required for some enteric pathogens to colonize the intestine. Understanding how these pathogens overcome the mucus layer protecting the epithelial tissue is necessary for disease prevention. In this thesis project I examined bacterial motility in mucus to understand factors that facilitate mucus penetration and contrasted this with the motility in different visco-elastic materials.In this thesis, I used single cell tracking to characterize the flagellar motility of...
Show moreFlagellar motility is required for some enteric pathogens to colonize the intestine. Understanding how these pathogens overcome the mucus layer protecting the epithelial tissue is necessary for disease prevention. In this thesis project I examined bacterial motility in mucus to understand factors that facilitate mucus penetration and contrasted this with the motility in different visco-elastic materials.In this thesis, I used single cell tracking to characterize the flagellar motility of Vibrio cholerae and Salmonella enterica in different visco-elastic environments. First, I tested if V. cholerae and S. enterica were still motile in unprocessed pig intestinal mucus. Second, I studied factors that supported their motility in mucus. The first factor I investigated was the effect of pH on V. cholerae motility. I also studied the role of curvature in V. cholerae motility within mucus and some other visco-elastic environments, including liquid and agar. Last, I began investigation of the role of flagellar number in S. enterica motility. In these studies, I focused on analyzing the effective diffusion of bacteria and factors promoting the diffusion in each environment. My findings demonstrate that V. cholerae and S. enterica are able to swim in mucus, and that the torque, and the curvature of V. cholerae, and the flagellar number of S. enterica, play a significant role in bacteria motility in mucus and liquid. Moreover, my findings help elucidate the significance of motility in pathogenesis.
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Title: Characterization of map3773c, ferric uptake regulator protein, in iron metabolism of mycobacterium avium subsp. paratuberculosis
Creator: Miyagaki Shoyama, Fernanda
Date: 2020
Collection: Electronic Theses & Dissertations
Description: Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD) in ruminants, a chronic inflammation of the intestines characterized by persistent diarrhea that leads to malnutrition and muscular wasting (Rathnaiah et al., 2017). Unfortunately to date, reliable JD diagnostics are lacking. Culture of MAP from feces has been the most reliable method for diagnosis of JD, however MAP requires eight to sixteen weeks to produce colonies in culture, presenting a...
Show moreMycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease (JD) in ruminants, a chronic inflammation of the intestines characterized by persistent diarrhea that leads to malnutrition and muscular wasting (Rathnaiah et al., 2017). Unfortunately to date, reliable JD diagnostics are lacking. Culture of MAP from feces has been the most reliable method for diagnosis of JD, however MAP requires eight to sixteen weeks to produce colonies in culture, presenting a major hurdle to diagnosis (Bannantine et al., 2002). Currently, no current vaccine can protect animals against JD or prevent shedding of MAP (Garcia and Shalloo, 2015). These challenges establish a need for a better understanding of the MAP physiology during infection. Unlike other mycobacteria, MAP has a unique requirement for supplementation of an iron-binding siderophore (mycobactin J) for optimal growth in laboratory media. A whole genome sequence of MAP K10 revealed a truncation of the mbtA gene that was speculated to have led to its mycobactin dependency (Li et al., 2005). Zhu et al (2008) showed that MAP is able to transcribe all mycobactin synthesis genes in an intra-macrophage environment. Furthermore, several genes responsible for iron acquisition in infected tissues, including genes responsible for mycobactin biosynthesis have been shown to be upregulated in naturally infected tissues (Janagama et al., 2010). Its known that iron plays an important role in vital biological processes; however high intracellular concentration of free iron can lead to toxicity to the bacteria. As such, bacteria activate expression of specific group of genes that are controlled by a metal-sensing regulatory transcription factor. In 2009, Janagama and others identified and characterized the iron dependent regulator (IdeR) in MAP. In addition, in a MAP-specific genomic island, MAP carries three putative ferric uptake regulator (Fur) boxes, an iron regulated transcriptional control motif, (Stratmann et al., 2004). To date, nothing is known about the role of Fur in MAP. To elucidate the mechanisms of iron homeostasis in MAP, we investigated LSP15 using a transposon mutant MAP3776c. We demonstrated a phenotype for LSP15 genes in a culture model of cell entry and survival and suggest a function in epithelial cell pathogenesis, however further functional analysis with complementation by a MAP3776c::Tn strain would be necessary to confirm these findings. Additionally, full characterization of a Fur-like protein (MAP3773c) was performed. Using PRODORIC for computational analysis, 23 different pathways that were likely regulated by MAP3773c were identified. These findings were confirmed using a chromatin immunoprecipitation assay followed by high-throughput sequencing (ChIP-seq), that revealed 58 regions where Fur binds under iron replete and deplete conditions. From those, three were directed related to iron regulation MAP3638c (hemophore-like protein), MAP3736c (Fur box) and MAP3776c (ABC transporter). Using the Fur box consensus sequence, we confirmed binding specificity and Mn2+ availability by a chemiluminescent electrophoresis mobility shift assay (EMSA). A transcriptional profile of the parent MAP K10, deletion mutant of MAP3773c and the complemented strains was developed under iron replete and depleted conditions. However, under the current experimental conditions, we are unable to conclude if the lack of transcriptional responses in our study was indicative of a lack of FUR activity.
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Title: Establishment of novel Wolbachia symbiosis and characterization of sex determination genes in dengue mosquito vectors
Creator: Liang, Xiao
Date: 2020
Collection: Electronic Theses & Dissertations
Description: As one of the deadliest animals on earth, mosquitos transmit numerous diseases to humans, including dengue, Zika and malaria, which account for over one million human deaths every year. Due to insufficiency of traditional vector control strategies, significant efforts have recently been made to develop novel genetic approaches to either directly suppress mosquito populations or reduce mosquito’s ability to transmit pathogens to humans. One of them is based on the maternally transmitted...
Show moreAs one of the deadliest animals on earth, mosquitos transmit numerous diseases to humans, including dengue, Zika and malaria, which account for over one million human deaths every year. Due to insufficiency of traditional vector control strategies, significant efforts have recently been made to develop novel genetic approaches to either directly suppress mosquito populations or reduce mosquito’s ability to transmit pathogens to humans. One of them is based on the maternally transmitted intracellular symbiotic bacterium Wolbachia. Estimated to infect more than 60% of arthropods in nature, Wolbachia can spread through host populations by means of a reproduction-interfering referred to as cytoplasmic incompatibility (CI). By altering the host’s physiological environment, including immune priming or metabolic perturbation, Wolbachia can also confer antiviral resistance in mosquito vectors. Successful field trials have been conducted to release Wolbachia-infected mosquito males to induce incompatible matings for population suppression or spread Wolbachia into mosquito populations to reduce or block dengue transmission by population replacement. Both population suppression and replacement require for establishment of an artificial Wolbachia symbiosis in mosquito to make it incompatible with target populations. In order to develop a Wolbachia-based strategy for dengue/Zika control in Singapore and Mexico, I have established the transinfected line WB2. By comparing with another transinfected line WB1 which developed 15 years ago, I have demonstrated that wAlbB maintains a stable symbiosis with Ae. aegypti. Further assays show that Wolbachia induces strong resistance to dengue, Zika and Chikungunya viruses in WB2. WB2 line has now been released for field trials in both Mexico and Singapore. In order to improve Wolbachia-based mosquito control, transinfected mosquitoes must be optimized to display maximum pathogen blocking, the desired CI pattern, and the lowest possible fitness cost. Achieving such optimization, however, requires a better understanding of the interactions between the host and various Wolbabachia strains. Thus, we transferred the Wolbachia wMel strain into Ae. albopictus, resulting in a transinfected line, HM (wAlbAwAlbBwMel), no CI was induced when the triply infected males were crossed with the wild-type GUA females or with another triply infected HC females carrying wPip, wAlbA, and wAlbB, but removal of wAlbA from the HM line resulted in the expression of CI after crosses with lines infected by either one, two, or three strains of Wolbachia. These results show that introducing a novel strain of Wolbachia into a Wolbachia-infected host may result in complicated interactions between Wolbachia and the host and between the various Wolbachia strains, with competition likely to occur between strains in the same supergroup. In order to manage the potential risk of failure in population suppression in Singapore, I developed another Ae. aegypti carrying wMal. The transinfected line showed 100% maternal transmission. To facilitate developing a perfect sex separation approach for Wolbachia-based population suppression, I established the CRISPR/Cas9 approach to characterize the function of sex determination pathway genes in Ae. aegypti. By individually knocking out doublesex (dxl) and transformer-2 (tra-2), two essential genes in mosquito sex determination pathway, we show that dxl is not essential gene for female development while knockout of tra-2 results in male-biased sex ratio and absence of female mosquito with homozygous tra-2. These results indicate that the tra-2 is a potential sex determination target that can be explored to develop the female-specific lethality for mosquito sex separation.
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Title: THE EFFECT OF GROWTH-RESTRICTION ON THE MURINE GUT MICROBIOME
Creator: Quinn, Melissa
Date: 2020
Collection: Electronic Theses & Dissertations
Description: ABSTRACTTHE EFFECT OF GROWTH-RESTRICTION ON THE MURINE GUT MICROBIOME By Melissa QuinnINTRODUCTION. Growth restriction induced by undernutrition in early life increases the risk of developing chronic diseases in adulthood. We hypothesized growth restriction would alter the gut microbiome and metabolome across the lifespan, impairing vital growth signaling processes necessary for proper development, with a primary focus on muscular and hepatic Insulin-like Growth Factor (IGF-1) expression....
Show moreABSTRACTTHE EFFECT OF GROWTH-RESTRICTION ON THE MURINE GUT MICROBIOME By Melissa QuinnINTRODUCTION. Growth restriction induced by undernutrition in early life increases the risk of developing chronic diseases in adulthood. We hypothesized growth restriction would alter the gut microbiome and metabolome across the lifespan, impairing vital growth signaling processes necessary for proper development, with a primary focus on muscular and hepatic Insulin-like Growth Factor (IGF-1) expression. METHODS. A cross-fostering, protein-restricted nutritive model (8% protein) was used to induce undernutrition during gestation (GUN) or lactation (PUN). At 21 days of age (PN21), all mice were weaned to a control diet (CON; 20% protein), isolating undernutrition to specific windows of early life. Fecal samples were collected weekly PN18-PN80 to determine longitudinal programming effects of growth restriction on the gut microbiome (CON N=5, GUN N=6, PUN N=6) and metabolome. Fecal sample DNA was extracted for amplification of the bacterial 16S rRNA genes using PCR, and then the amplicons were sequenced with the Illumina pipeline and analyzed using the Qiita bioinformatics software. Cecum samples were also collected at PN21 (CON N=4, GUN N=6, PUN N=5) and PN80 (CON N=5, GUN N=6, PUN N=6) for microbiome analysis. Liver samples were collected at PN21(CON N=12, GUN N=6, PUN N=7) and PN80 (CON N=13, GUN N=9, PUN N=11) and analyzed along with the cecum for metabolomics via tandem mass spectrometry (LC-MS/MS) and analyzed with the Global Natural Products Social Molecular Networking (GNPS) bioinformatics software. IGF-1 expression in the liver and gastrocnemius (CON N=15, GUN N=12, PUN N=13) was analyzed via a Total Protein NIR western blot to establish a connection between the gut microbiome, tissue metabolome and organ growth. RESULTS. The Beta-Diversity of the fecal microbiome was significantly separated by treatment group using Weighted UniFrac measures (PERMANOVA p=0.0001). Differences in the microbiome were not evident through analysis at the Phylum level (Firmicutes/Bacteroidetes ratio) but were instead driven by longitudinal alterations in the abundance of specific genera and species in PUN. Linear mixed model (LMM) analysis revealed PUN having significantly higher abundance of specific bacteria compared to GUN and CON across the lifespan including: Bacteroides uniformis, B. acidifaciens, B. ovatus, Bifidobacterium sp. and Clostridium sacchrogumia. Rikenellaceae was the only microbe that was significantly lower in abundance in the PUN group over time compared to GUN and CON. Additionally, the PUN metabolome was significantly altered compared to GUN and CON, primarily characterized by reduced: essential amino acids (EAAs: methionine, phenylalanine and tyrosine), riboflavin (B2), primary bile acids, and decreased Dehydroepiandrosterone (DHEA); and increased acylcarnitines and fecal peptides. NIR Western blot analysis revealed significantly lower IGF-1 expression in the liver at PN21 in GUN (p=0.0012) and PUN (p<0.001) as well as overall lower expression in the muscle in PUN (p=0.037) and GUN (p=0.007) compared to CON. CONCLUSION: The gut microbiome and metabolome are altered by early life growth restriction at PN21 and through adulthood. Elevated sugar-fermenting bacteria in the PUN group represent gut microbiome immaturity and delayed development. Temporary metabolic alterations of early life growth restriction are seen in decreased primary bile acids and increased synthesis of liver acylcarnitines, both of which are indicative as adaptations of the pups being calorie-restricted as a result of the low-protein fed dam. More permanent outcomes of growth restriction were evident by increased peptide excretion over the lifespan, significantly decreased methionine and riboflavin – which prevented protein synthesis to occur during early life development, and overall decreased muscle IGF-1 expression and DHEA levels in the PUN mice. Many of the metabolic pathways permanently altered by growth restriction are seen in the liver, making this organ an important site for future research on the development of treatment modalities that can limit growth restriction induced chronic disease.
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Title: Genetic and chemical biology studies of Mycobacterium tuberculosis pH-driven adaptation
Creator: Dechow, Shelby J.
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Mycobacterium tuberculosis (Mtb) endures robust immune responses by sensing and adapting to its host environment. One of the first cues the bacterium encounters during infection is acidic pH, a characteristic of its host niche – the macrophage. Targeting the ability of Mtb to sense and adapt to acidic pH has the potential to reduce survival of Mtb in macrophages. A high throughput screen of a >220,000 compound small molecule library was conducted to discover chemical probes that inhibit Mtb...
Show moreMycobacterium tuberculosis (Mtb) endures robust immune responses by sensing and adapting to its host environment. One of the first cues the bacterium encounters during infection is acidic pH, a characteristic of its host niche – the macrophage. Targeting the ability of Mtb to sense and adapt to acidic pH has the potential to reduce survival of Mtb in macrophages. A high throughput screen of a >220,000 compound small molecule library was conducted to discover chemical probes that inhibit Mtb growth at acidic pH. From this screen, AC2P20 was identified as a chemical probes that kills Mtb at pH 5.7 but is inactive at pH 7.0. Through a combination of transcriptional profiling, mass spectrometry, and free thiol abundance and redox assays, I show that AC2P20 likely functions by depleting intracellular thiol pools and dysregulating redox homeostasis. Findings from this study have helped define new pathways involved in Mtb’s response to acidic pH using a chemical genetic approach.Upon sensing acid stress, Mtb can adapt accordingly by entering a nonreplicating persistent state, resulting in increased tolerance to host immune pressures and antibiotics. During growth in vitro, when given glycerol as a sole carbon source, Mtb responds to acidic pH by arresting its growth and entering a metabolically active state of nonreplicating persistence, a physiology known as acid growth arrest. To answer how Mtb regulates and responds to acidic pH, I performed genetic selections to identify Mtb mutants defective in acid growth arrest. These selections identified enhanced acidic growth (eag) mutants which all mapped to the proline-proline-glutamate ppe51 gene and resulted in distinct amino acid substitutions: S211R, E215K, and A228D. I demonstrated that expression of the PPE51 variants in Mtb promotes significantly enhanced growth at acidic pH showing that the mutant alleles are sufficient to cause the dominant gain-of-function, eag phenotype. Furthermore, I performed single carbon source experiments and radiolabeling experiments showing that PPE51 variants preferentially uptake glycerol at an enhanced rate, suggesting a role in glycerol acquisition. Notably, the eag phenotype is deleterious for growth in macrophages, where the mutants have selectively faster replication but reduced virulence in activated macrophages as compared to resting macrophages. This supports that acid growth arrest is a genetically controlled, adaptive process that could act as a potential targetable physiology in future TB therapeutics. My work with the carbonic anhydrase inhibitor, ethoxzolamide, sought to combine genetic and chemical biology to better understand pH-adaptation in Mtb. Ethoxzolamide is a potent inhibitor of Mtb carbonic anhydrase activity and the PhoPR regulon, suggesting a previously unknown link between carbon dioxide and pH-sensing. We hypothesized that the production of protons from carbonic anhydrase activity could be modulating PhoPR signaling. Mtb has three carbonic anhydrases (CanA, CanB, and CanC), and by using CRISPRi and gene knockout, I show that CanB is required for pathogenesis in macrophages, but I did not observe a function in controlling PhoPR signaling. However, transcriptional profiling at different pH and CO2 concentrations show that PhoPR is induced by high CO2 and also revealed a core subset of CO2 responsive genes independent of PhoPR or acidic pH regulation. Overall, these studies defined new functions for thiol- and redox-homeostasis, glycerol uptake, and CO2-concentration in regulating Mtb adaptation to acidic environments and provide new targets for the development of acidic pH-dependent therapeutics.
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Title: INSIGHTS INTO ORGANOSULFUR ASSIMILATION IN STAPHYLOCOCCUS AUREUS
Creator: Lensmire, Joshua Michael
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Bacterial pathogens deploy sophisticated strategies to acquire vital nutrients from the host during infection. Staphylococcus aureus is a considerable human pathogen due to its capacity to cause numerous life-threatening diseases. As such S. aureus has an intricate metabolism that promotes proliferation in distinct host environments. However, little is known regarding the sulfur sources the pathogen scavenges from host tissues. Sulfur is an essential nutrient due to its extensive redox...
Show moreBacterial pathogens deploy sophisticated strategies to acquire vital nutrients from the host during infection. Staphylococcus aureus is a considerable human pathogen due to its capacity to cause numerous life-threatening diseases. As such S. aureus has an intricate metabolism that promotes proliferation in distinct host environments. However, little is known regarding the sulfur sources the pathogen scavenges from host tissues. Sulfur is an essential nutrient due to its extensive redox capacity and consequently, it is a critical component of many cofactors. Prior studies started to define sulfur sources S. aureus can use including the inorganic sulfur sources, sulfide and thiosulfate, and organosulfur sources, cysteine, cystine, and glutathione. While we understand some of the sulfur sources S. aureus can use, we do not know the genetic determinants facilitating assimilation. The present studies sought to explain how S. aureus imports and catabolizes organic sources of sulfur. First, we wanted to uncover the proteins allowing S. aureus to utilize cystine and cysteine as sulfur sources. The S. aureus homologues of characterized cystine transporters, TcyP and TcyABC, were experimentally validated as cystine and cysteine transporters. We expanded the sulfur sources S. aureus can utilize to include homocystine and N-acetyl cysteine and show that both TcyABC and TcyP support growth on N-acetyl cysteine while only TcyABC supports growth on homocystine. Finally, a tcyP mutant is impaired in murine heart and liver when competing with WT S. aureus suggesting import of TcyP substrates is important for heart and liver colonization. While a tcyP mutant is reduced in competition with WT in murine heart and liver colonization is not fully ablated signifying more sulfur sources must be catabolized. We next examined how S. aureus imports and catabolizes GSSG. To identify S. aureus GSSG utilization strategies, we used a chemically defined medium containing GSSG as the sulfur source and isolated mutants harboring transposon insertions within a putative ABC-transporter and -glutamyl transpeptidase that fail to proliferate. The mutants also do not grow in medium supplemented with GSH. Consistent with these findings, we named the locus the glutathione import system (gisABCD-ggt). Biochemical analysis of recombinant Ggt confirms in silico functional predictions by demonstrating that Ggt cleaves both GSH and GSSG. Though Gis mutants display wildtype virulence, we find that Gis-Ggt promotes competition with Staphylococcus epidermidis when GSH or GSSG is supplied as the sole sulfur source in vitro. S. aureus resides as a nasal commensal in 30% of the population and once inside the body can infect nearly every organ. Throughout the changing host environments, S. aureus must sense and acclimate to nutrient availability. We sought to define how sulfur starvation and growth on different sulfur sources changes the transcriptional profile of S. aureus. We described the transcriptional changes when WT S. aureus or a CymR, the sulfur transcriptional regulator, mutant were grown in sulfur replete and deplete conditions. We show sulfur starvation leads to significant expression changes including upregulation of iron acquisition encoding genes and oxidative stress encoding genes. Furthermore, we provide evidence showing upregulation of CymR dependent sulfur transporters when S. aureus is grown on GSH and thiosulfate both of which are conditions in which CymR repression should be occurring. Finally, this dissertation ends with areas of future exploration of sulfur source utilization in S. aureus. These avenues include examining nutrient sulfur available in distinct infection sites and expansion of the sulfur sources S. aureus can use. Overall, the work presented here substantially contributes to our understanding of what sulfur sources S. aureus imports and catabolizes and how different sulfur sources change the transcriptional states of the cell.
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Title: WILDFIRE IMPACTS ON SOIL CARBON POOLS AND MICROBIAL COMMUNITIES IN MIXED-CONIFER FORESTS OF CALIFORNIA
Creator: Adkins, Jaron
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Forest ecosystems are important reservoirs for long term carbon (C) storage. Forests of the western United States account for 20-40% of total U.S. carbon C sequestration, and nearly half of the total C in these forests is stored in soil. However, many forests in the western U.S are experiencing wildfire conditions that diverge from historical fire regimes. Prior to Euro-American settlement, California’s mixed-conifer forests typically experienced frequent surface fires of low to moderate burn...
Show moreForest ecosystems are important reservoirs for long term carbon (C) storage. Forests of the western United States account for 20-40% of total U.S. carbon C sequestration, and nearly half of the total C in these forests is stored in soil. However, many forests in the western U.S are experiencing wildfire conditions that diverge from historical fire regimes. Prior to Euro-American settlement, California’s mixed-conifer forests typically experienced frequent surface fires of low to moderate burn severity, but, due to the combined effects of altered forest structure and climate change, now experience fires that are larger and more severe than historical conditions. Fires have numerous direct and indirect effects on the soil biological, chemical, and physical characteristics that influence the soil C cycle. Understanding how altered soil characteristics influence the cycling and persistence of soil C, and how they vary with severity, is important for managing forests for C storage and for predicting fire-climate feedbacks. My dissertation work incorporates observational and manipulative experiments to understand the direct and indirect effects of burn severity on soil C cycling and microbial communities over the short to intermediate term, with a particular focus on the distribution of soil C between active and slow cycling pools. Soil C can be conceptualized as discrete pools of variable persistence in soil. The active C pool is quickly decomposed, contributing to the return of CO2 to the atmosphere, whereas the non-active C pool is more stable and contributes to long term C storage. I leveraged a burn severity gradient resulting from a wildfire in a California mixed-conifer forest to determine the structure and kinetics of these C pools at an intermediate time point in post-fire recovery (i.e. three years). I found that the size of the non-active C pool was smaller in burned areas than unburned areas, and the kinetic rate of the non-active C pool was negatively related to burn severity. I also characterized the soil microbial communities across this severity gradient and identified the environmental characteristics responsible for differences. I found that fungal-to-bacterial ratio and oligotroph-to-copiotroph bacteria ratio decreased with burn severity, and these effects were driven by differences in live and dead tree basal area, soil nutrients, and pH. Leveraging another burn severity gradient, I then determined whether differences in microbial communities and soil C pools were related one-year post-fire in a mixed-conifer forest. I again found lower non-active C pool kinetic rates, and higher abundances of copiotrophic bacteria in burned compared to unburned areas. Differences in soil C pool kinetics were related to tree basal area, soil nutrients, and bacterial communities. I determined the short-term impacts of fire on soil C pools and cycling using lab experiments in which I manipulated soil heating intensity and pyrogenic organic matter (PyOM) additions. I found that high intensity soil heating can decrease microbial biomass C (MBC) accumulation, whereas PyOM had minimal effects on MBC in the short-term. Finally, I found that the size of the active C pool increased with soil heating intensity, while the kinetic rate of the non-active C pool decreased; PyOM primarily increased the size of the non-active C pool. Taken as a whole, my research suggests that fire induces short-term soil C losses by increasing the size of the active C pool, but, over the intermediate-term, residual soil C is more persistent. Fire severity is predicted to increase globally throughout the 21st century, and my research contributes to understanding how forest C storage will be affected by disrupted wildfire regimes.
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Title: DYNAMIC DESERTS : THE SYNERGISTIC EFFECTS OF FUNGI, SUNLIGHT, AND NON-RAINFALL MOISTURE ON PLANT LITTER DECOMPOSITION IN DRYLANDS
Creator: Logan V, James Robert
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Drylands cover well over one third of the Earth’s land and are an important part of the global carbon cycle. Despite this, most models underestimate carbon turnover in arid and semi-arid systems, limiting our ability to predict how they will respond to changing climates. This is partly because many models are driven by rainfall, assuming little to no decay between precipitation events. Unlike in many wetter systems though, plant litter decomposition in drylands is largely controlled by non...
Show moreDrylands cover well over one third of the Earth’s land and are an important part of the global carbon cycle. Despite this, most models underestimate carbon turnover in arid and semi-arid systems, limiting our ability to predict how they will respond to changing climates. This is partly because many models are driven by rainfall, assuming little to no decay between precipitation events. Unlike in many wetter systems though, plant litter decomposition in drylands is largely controlled by non-rainfall processes including photodegradation and biotic decomposition supported by non-rainfall moisture (fog, dew, and water vapor; “NRM”). Despite their importance however, few studies have examined how these drivers interact with one another and with fungal communities to influence carbon turnover. In this dissertation, I demonstrate how photodegradation, NRM, and fungal decomposers interact to accelerate carbon turnover in drylands. To do this, I leveraged a natural gradient of NRM frequency in the Namib Desert that receives intense solar radiation. In one study, I used a reciprocal transplant design to show that moisture regime exerts a strong influence on litter-associated fungal communities and show that the relationship between NRM and litter decay rates depends on the composition of the decomposer community. In another study, I manipulated solar radiation for three years and found that photodegradation of the plant cuticle allows litter to absorb more water during NRM events, accelerating biotic decomposition. By examining litter-associated fungal communities under these same radiation treatments, I also show that fungi are largely insensitive to radiation stress and that photodegradation mainly affects decomposition rates in this system through photochemical changes in litter that increase subsequent biotic decomposition. Finally, to quantify the relationship between NRM and carbon turnover on multi-year timescales, I measured mass loss for 30 months along a moisture gradient spanning an order of magnitude of NRM frequency. By coupling these data with continuous meteorological measurements over the same period, I show that accounting for NRM and temperature sensitivity substantially improves the performance of a simple exponential litter decay model. These findings build on previous work demonstrating the importance of solar radiation and NRM as crucial drivers of litter decomposition and point a way forward for future studies to examine how these two processes may interact under future climate scenarios. Drylands are undergoing significant changes from anthropogenic climate change and understanding the drivers of litter decomposition allows us to better predict how these ecosystems are responding to global change.
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Title: CHEMICAL BIOLOGY AND GENETIC STUDIES TARGETING THE MYCOBACTERIAL CELL ENVELOPE
Creator: Williams, John Tison
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Mycobacterium tuberculosis is one of the leading causes of death due to a single infectious pathogen. The evolution and spread of drug resistant strains requires new antibiotics to control the TB pandemic. Over the last decade, the lipid flippase MmpL3 has been identified as a potential drug candidate based on its essential nature for cell viability and repeated identification as the lead target of small molecule inhibitors of Mtb growth. Using a combined untargeted and targeted whole cell...
Show moreMycobacterium tuberculosis is one of the leading causes of death due to a single infectious pathogen. The evolution and spread of drug resistant strains requires new antibiotics to control the TB pandemic. Over the last decade, the lipid flippase MmpL3 has been identified as a potential drug candidate based on its essential nature for cell viability and repeated identification as the lead target of small molecule inhibitors of Mtb growth. Using a combined untargeted and targeted whole cell phenotypic screen I identified novel inhibitors of this valued target. A combination of lipid profiling and an innovative competitive binding assay supported MmpL3 as the target of these inhibitors. Cross resistance profiling of MmpL3 inhibitors against twenty-four unique mmpL3 Mtb mutants demonstrated that the level of resistance is associated with the proximity of resistant mutants to essential residues for protein function. Further, these resistance profiles suggested that MmpL3 inhibitors fall into separate clades depending on their chemical scaffolds. The results of this screen led to the development of novel potent analogs for one of the identified MmpL3 inhibitors, HC2099. These analogs were active against clinically relevant drug resistant Mtb strains that cause treatment failure in patients. Active analogs were able to kill Mtb inside of infected macrophages, an infectious niche of Mtb, without inducing cytotoxicity against these important immune cells. One of these analogs, MSU-43085, was orally bioavailable and successfully inhibited Mtb growth in infected mice, supporting further development and highlighting the therapeutic potential of this series. High throughput screens are often used to identify new inhibitors of Mtb growth. However, prioritized hits form these screens often identify similar targets such as MmpL3, lipid synthesis enzymes, redox cyclers, as well as inhibitors of the electron transport chain. Follow up studies of these inhibitors are often time consuming, costly and result in the rediscovery of previously identified targets. While this is not necessarily detrimental to Mtb drug discovery, as these reoccurring targets have therapeutic potential. The continued prioritization of inhibitors for these common metabolic pathways potentially limits the identification of inhibitors for novel targets. Therefore, additional steps that identify inhibitors of these common pathways could reshape how high throughput screen hits are prioritized. By applying the targeted mutant screen used to identify MmpL3 inhibitors to a non-prioritized library of hits from a high throughput screen, we identified more than fifty new potential MmpL3 inhibitors. Using an iterative strategy of applying additional mutants of commonly identified targets, this strategy promises to lead to parallel follow-up studies of inhibitors with known and unknown mechanisms of action. The ability of Mtb to enter into quiescent states in response to host stresses is one of the leading causes for the extended time to cure and evolution of drug resistance. These states can be induced by several environmental stresses including acidic pH, hypoxia, and others. In an effort to study this adaptation in the rapidly growing mycobacterial species M. smegmatis, we identified a lethal sodium citrate phenotype. Transcriptional profiling and genetic screening of mutants tolerant to sodium citrate indicated that this phenotype was due to the combined action of both chelation and osmotic stresses. Cell viability could be reduced from sodium citrate killing by cation and osmoprotectant supplementation. From these experiments we propose a model that can be applied to study carbon source uptake and probe the role of genes identified from the forward genetic screen with unknown function
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Title: Microbial Community Dynamics within a Serpentinization-Influenced Aquifer : Characterization of Community Assembly Processes and Responses to Drilling-Induced Perturbations
Creator: Putman, Lindsay Irene
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Serpentinization is the hydrothermal alteration of ultramafic rock, which results in high pH, reducing fluids that are low in dissolved inorganic carbon, and have high levels of methane and hydrogen. While endemic microbial communities have been well-characterized from a variety of marine and terrestrial serpentinizing ecosystems, to date the ecological processes that contribute to microbial community assembly, community dynamics, and the impact of disturbances in serpentinizing environments...
Show moreSerpentinization is the hydrothermal alteration of ultramafic rock, which results in high pH, reducing fluids that are low in dissolved inorganic carbon, and have high levels of methane and hydrogen. While endemic microbial communities have been well-characterized from a variety of marine and terrestrial serpentinizing ecosystems, to date the ecological processes that contribute to microbial community assembly, community dynamics, and the impact of disturbances in serpentinizing environments have not yet been assessed. The work in this dissertation was performed at the Coast Range Ophiolite Microbial Observatory, CA, USA, where a series of wells were drilled in 2011 to access serpentinization-influenced fluids directly from the subsurface. Geochemical and 16S rRNA gene amplicon datasets were collected directly from these wells and a series of microcosm experiments were performed on fluids from the site. Samples collected over the course of six years were used to assess community assembly processes and the biogeochemical impacts of drilling fluid injection into the subsurface. A series of microcosm experiments were also performed to better understand the response of microbial populations to geochemical changes observed in situ following drilling. Results from this work will inform studies of biogeochemical dynamics relevant to modern and ancient Earth and extraterrestrial sites such as Mars. These data further our understanding of microbial community responses to environmental perturbations and provide information that will aid in the development of future drilling and monitoring projects focused on learning about life in the deep subsurface.
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Title: ENVIRONMENTAL DRIVERS AND EVOLUTIONARY CONSEQUENCES OF HORIZONTAL GENE TRANSFER IN SOIL BACTERIA
Creator: Kittredge, Heather
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Horizontal gene transfer (HGT) is a driving force in bacterial evolution and could drive rapid adaptation in bacterial communities. Natural transformation is one mechanism of HGT that allows bacteria to pick up extracellular DNA (eDNA) from the environment and integrate it into their genome. But the rate of HGT in natural environments, and the role this process plays in facilitating rapid adaptation remains unknown. As climate change threatens the stability of environments worldwide,...
Show moreHorizontal gene transfer (HGT) is a driving force in bacterial evolution and could drive rapid adaptation in bacterial communities. Natural transformation is one mechanism of HGT that allows bacteria to pick up extracellular DNA (eDNA) from the environment and integrate it into their genome. But the rate of HGT in natural environments, and the role this process plays in facilitating rapid adaptation remains unknown. As climate change threatens the stability of environments worldwide, understanding how quickly bacteria can adapt to novel environments is essential. My dissertation research characterizes the environmental drivers and evolutionary consequences of natural transformation in a highly transformable model soil bacterium Pseudomonas stutzeri.Despite decades of research on understanding HGT at the molecular level, less is known about the ecological drivers of HGT. To understand the soil conditions relevant for transformation, I first measured eDNA in the field over a short-term drying rewetting disturbance (Ch. 2). I found that eDNA increased in response to the rewetting disturbance but quickly disappeared from soil, suggesting a small portion of this eDNA could be transformed by bacterial cells recovering from the disturbance. To test the efficiency of transformation under the conditions in which eDNA disappeared, I created a novel microcosm system for quantifying transformation in soil (Ch. 3). Here, I inoculated soil with live antibiotic-susceptible, and dead antibiotic-resistant P. stutzeri. I then tracked the evolution of antibiotic resistance over a range of soil conditions and eDNA concentrations. Transformation drove the evolution of antibiotic resistance across a wide range of soil moistures and increased in response to larger inputs of dead cells (eDNA source), with antibiotic resistance repeatedly appearing in antibiotic free soil. Despite the prevalence of transformation across bacterial species, the evolutionary origins and consequences of transformation are still largely unknown. Transformation presumably provides a fitness benefit in stressful or continuously changing environments, but few studies have quantified changes in transformation in response to adaptive evolution. Here, I evolved P. stutzeri at different salinities and tested how the growth rate and transformation efficiency changed in response to salt adaptation (Ch. 4). Overall, the growth rate increased in response to adaptation, but the transformation efficiency declined, with only ~50% of the evolved populations transforming eDNA at the end of experiment – as opposed to 100% of ancestral populations transforming eDNA. Overall, my dissertation research elucidates the factors driving transformation in soil, setting the stage for future experiments to scale up estimates of transformation to the whole community level. I find that transformation occurs under most soil conditions and allows genetic variants to arise at low frequencies in the absence of selection. I also report novel experimental evidence that transformation efficiency can change dramatically, and in a highly variable manner, over just ~330 generations. Taken together, this body of research highlights a role for transformation in many natural systems of ecological significance, and points to dead cells as an important but often overlooked source of genetic diversity.
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Title: The Control of Phenotypic Diversity in Vibrio cholerae during the Transition between Motility and Attachment
Creator: Lee, John Seungwu
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Vibrio cholerae is a causative agent of human intestinal disease, cholera. It requires both flagellar-based motility and biofilm formation to colonize the small intestine. The secondary messenger molecule, c-di-GMP, plays a central role in controlling transition between motility and biofilm formation. However, the switch between these two lifestyles has been studied on an average population scale, overlooking the heterogeneous phenotypic response that can occur at a single-cell level. V....
Show moreVibrio cholerae is a causative agent of human intestinal disease, cholera. It requires both flagellar-based motility and biofilm formation to colonize the small intestine. The secondary messenger molecule, c-di-GMP, plays a central role in controlling transition between motility and biofilm formation. However, the switch between these two lifestyles has been studied on an average population scale, overlooking the heterogeneous phenotypic response that can occur at a single-cell level. V. cholerae infections are characterized by the co-occurrence of cells with motile and sessile behaviors, but the determinant of this phenotypic diversity remains poorly understood. We used single-cell tracking to examine the motile behaviors of two V. cholerae strains (El Tor C6706 and Classical O395) in response to direct manipulations of c-di-GMP concentration. Both motile and non-motile cells are present in a well-mixed batch culture with distributions of these phenotypes that change depending on the growth phase. We determined that the proportion of motile cells differs between El Tor and Classical strains because they maintain different levels of c-di-GMP. However, even in conditions that promote biofilm formation, V. cholerae still generates a sub-population of motile cells. C-di-GMP is known to inversely regulate assembly of mannose-sensitive haemagglutinin (MSHA) pili and flagella. We found that the most cells in clonal populations are flagellated, but MSHA piliation affords the variable opportunity to attach at single-cell level, driving behavioral switching between motile and sessile behaviors. Our results support the hypothesis that c-di-GMP regulates phenotypic diversity in V. cholerae, and it does so by simultaneous elaboration of MSHA pili and flagellum.
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Title: COMPUTATIONAL APPROACHES TO COMPLEX BIOLOGICAL PHENOMENA
Creator: Franklin, Joshua Luke
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Biological systems can be difficult to understand due to a vast array of interacting phenom-ena. The result is that some seemingly "easy" questions go unanswered. For example, we have long known that bacteria utilize many distinct flagellar configurations, but in most cases it remains unclear why they do so. We know that cell differentiation is critical to many biological processes, yet we still do not fully understand how such spatiotemporal patterning occurs. Despite mutation being one of...
Show moreBiological systems can be difficult to understand due to a vast array of interacting phenom-ena. The result is that some seemingly "easy" questions go unanswered. For example, we have long known that bacteria utilize many distinct flagellar configurations, but in most cases it remains unclear why they do so. We know that cell differentiation is critical to many biological processes, yet we still do not fully understand how such spatiotemporal patterning occurs. Despite mutation being one of the driving forces of evolution, we still have a hazy understanding of how organisms respond and adapt to high mutation rates. However, ad- vances in technology, modeling, and experimental techniques have enabled us to investigate the small and nuanced effects that can answer these questions. In this dissertation, I have used modern computational tools and statistical techniques to investigate evolutionary and behavioral processes. Agent-based models of evolution and flagellar inheritance have allowed me to investigate the evolution of mutational robustness and trade-offs associated with flagellar motility, respectively. By writing Bayesian mixed- effect models, I have been able to precisely quantify the metabolic cost of producing flagella and describe spatiotemporal patterns of cell differentiation within fruiting bodies of Myxo- coccus xanthus. Careful quantitative modeling of biological phenomena can help cut through the complexity of these systems. As computational power continues to grow and software continues to become more sophisticated, these computational approaches will both become more powerful and easier to use. Computational approaches will not replace experiments in most cases; instead, computational models can direct experiments, which can themselves direct new modeling efforts, in an iterative loop of progressing knowledge.
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Title: METABOLIC AND RESPIRATORY PATHWAYS CONTROLLING VIBRIO CHOLERAE COLONIZATION
Creator: Van Alst, Andrew John
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Vibrio cholerae is an enteric pathogen of the human small intestine that proliferates to high cell density during human infection. Although not typically classified as a virulence factor, metabolism is a cornerstone for fitness in the host environment. In this work, I explore the essential role of aerobic metabolism, including oxidative respiration, for successful colonization of V. cholerae in the infant mouse model. Oxidative respiration is the most efficient energy generating metabolic...
Show moreVibrio cholerae is an enteric pathogen of the human small intestine that proliferates to high cell density during human infection. Although not typically classified as a virulence factor, metabolism is a cornerstone for fitness in the host environment. In this work, I explore the essential role of aerobic metabolism, including oxidative respiration, for successful colonization of V. cholerae in the infant mouse model. Oxidative respiration is the most efficient energy generating metabolic pathway in living organisms and supports the rapid proliferation of V. cholerae in the small intestinal environment. Despite knowledge that oxygen diffuses from the host epithelium into the gut lumen, the role of oxygen in supporting colonization and proliferation of V. cholerae had not been explored prior to the work presented here in Chapters 2 and 3.In Chapter 2, by targeting the pyruvate dehydrogenase (PDH) complex, an enzyme required to convert pyruvate to acetyl-CoA under aerobic conditions, I show that aerobic metabolism through the PDH complex is required for population expansion in the infant mouse. As the gut was predominantly considered anaerobic and exists in a state of low oxygen tension, I also examined the contribution of anaerobic metabolism to infant mouse colonization. By targeting cognate pyruvate formate-lyase (PFL) that similarly converts pyruvate to acetyl-CoA, but only under anaerobic conditions, I determined that anaerobic respiration is dispensable for colonization. In Chapter 3, I directly test the importance of aerobic and anaerobic respiration by targeting the complete set of terminal oxidases and terminal reductases encoded by V. cholerae. Using a modified Multiplex Genome Editing by Natural Transformation (MuGENT) approach, I generated strains denoted Aero7 and Ana4. Aero7 is a functionally strict anaerobe derivative of V. cholerae, lacking all four terminal oxidases (cbb3, bd-I, bd-II, and bd-III), whereas Ana4 lacked functionality in each of the four terminal reductase complexes (fumarate, trimethylamine-N-oxide, nitrate, and biotin sulfoxide reductases). Disruption in the oxidase complexes in strain Aero7 severely attenuated V. cholerae colonization in the infant mouse, however, no attenuation was observed for Ana4. These data supported our findings in Chapter 2 that aerobic, but not anaerobic metabolism was critical for V. cholerae growth in the infant mouse. Furthermore, I determined that the bd-I oxidase, and to a lesser extent the cbb3 oxidase, support oxidative respiration during infection with bd-II and bd-III oxidases being dispensable for colonization. In summation, aerobic metabolism through the PDH complex and the terminal reduction of oxygen by the bd-I oxidase are essential to V. cholerae colonization of the infant mouse. Through this work, I uncovered a role for oxidative metabolism for V. cholerae colonization. These findings expand our knowledge of V. cholerae biology and pathogenicity in the gastrointestinal tract and implicate oxygen as a critical electron acceptor that shapes the progression of enteric infections.
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Title: Ecological and evolutionary consequences of exometabolites for microbial interactions
Creator: Chodkowski, John Luke
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Interspecies interactions have fundamental roles in shaping microbial communities. Microbial community members can produce and release a diverse set of extracellular small molecules, collectively referred to as exometabolites. Interspecies interactions that result from exometabolites can alter the response and behaviors of microbial community members. This dissertation work demonstrates the establishment of a synthetic community system that facilitates the study of exometabolite-mediated...
Show moreInterspecies interactions have fundamental roles in shaping microbial communities. Microbial community members can produce and release a diverse set of extracellular small molecules, collectively referred to as exometabolites. Interspecies interactions that result from exometabolites can alter the response and behaviors of microbial community members. This dissertation work demonstrates the establishment of a synthetic community system that facilitates the study of exometabolite-mediated interspecies interactions. This system was then used to understand the consequences of exometabolite-mediated interactions in a 3-member synthetic microbial community over stationary phase using a multi-omics approach. Lastly, an experimental evolution showed the consequences of long-term exometabolite interspecies interactions on the evolution of antibiotic resistance. This work advances knowledge on the dynamic response and behaviors of microbial community members' during non-growth states. This work also demonstrates how bacterial-bacterial interactions in a simple environment can lead to the emergence of antibiotic resistance.
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Title: Environmental Microbial Surveillance : From Source Tracking in Watersheds to Pathogen Monitoring in Sewersheds
Creator: Flood, Matthew Thomas
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Understanding of the connections between water and health, through the use of water quality monitoring, surveys and surveillance, can help to address the impacts of anthropomorphic changes on the environment. This study sought to understand these connections through the water quality monitoring within watershed basins as well as pathogen surveillance within sewersheds. Specifically, this dissertation sought to 1) understand the sources of pollution and their connections with land use in the...
Show moreUnderstanding of the connections between water and health, through the use of water quality monitoring, surveys and surveillance, can help to address the impacts of anthropomorphic changes on the environment. This study sought to understand these connections through the water quality monitoring within watershed basins as well as pathogen surveillance within sewersheds. Specifically, this dissertation sought to 1) understand the sources of pollution and their connections with land use in the various subsections of watersheds; 2) to find a cost-effective way to surveil the spread of SARS-CoV-2 using wastewater surveillance; and 3) to understand the differences in wastewater surveillance between communities. Water quality monitoring using microbial source tracking (MST) was performed with a survey of five mixed-use watersheds in Michigan. Through the use of spatial clustering, it was found that temporal contamination was primarily driven by precipitation and its associated variables (e.g., streamflow, turbidity, overland flow), while spatial contamination is driven by land uses (e.g., septic tank density, tile drain proportions, and tillage). Additionally, porcine fecal contamination was more often correlated with nutrients in streams than either bovine or human contamination. The development of a cost-effective workflow for the detection and quantification of SARS-COV-2 in wastewater was undertaken. Wastewater from communities around Michigan were collected and analyzed along with viral surrogates for SARS-CoV-2 to investigate different workflow options. The Pseudomonas phage Phi6 was seeded in different wastewater matrices to test concentration and recovery by ultrafiltration-based method and polyethylene glycol (PEG) precipitation. The PEG method provided better virus recovery than the ultrafiltration-based methods as measured using RT-ddPCR. The comparison of two communities (A and B) wastewater results for SARS-CoV-2 analyzed against case data was undertaken. These results were significantly correlated with cases in both communites, but the level of correlation differed based on spatial (e.g., zipcode vs county level cases) and temporal (e.g., date of symptom(s) onset vs. the referral date for cases) resolution. Wastewater surveillance was more representative of higher spatial resolution (zipcode data) of cases in both communities. When examining the temporal resolution of the communities, community B’s wastewater results were more closely tied to the onset of symptoms and not the case referral date. The ability to monitor indicators of pollution in watersheds and surveil etiological agents of disease in sewersheds provide non-intrusive methods for evaluating the potential risks and current burdens to community health. The first part of the work could be considered “downstream” monitoring identifying sources and potential exposures with the goal of reducing waterborne disease. While “upstream” monitoring was used for identifying the disease trends in the community and was focused on public health measures to prevent transmission. This project contributed novel methods, results and analysis providing valuable knowledge ultimately addressing the role of monitoring strategies to protect public health.
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Title: ECOPHYSIOLOGY OF (PERI)ORAL BACTERIA AND IMPACT OF OTIC COLONIZATION
Creator: Jacob, Kristin Marie
Date: 2022
Collection: Electronic Theses & Dissertations
Description: The middle ear is typically assumed to be sterile in health due to its secluded location, closed off from external forces by the tympanic membrane (ear drum) and from the naso/oropharynx by a collapsed Eustachian tube. However, the periodic opening of the Eustachian tube to the naso/oropharyngeal space, which releases pressure across the eardrum and drains otic fluids, could introduce bacteria. Previous studies have tested for the presence of bacteria in the uninfected otic cavity using...
Show moreThe middle ear is typically assumed to be sterile in health due to its secluded location, closed off from external forces by the tympanic membrane (ear drum) and from the naso/oropharynx by a collapsed Eustachian tube. However, the periodic opening of the Eustachian tube to the naso/oropharyngeal space, which releases pressure across the eardrum and drains otic fluids, could introduce bacteria. Previous studies have tested for the presence of bacteria in the uninfected otic cavity using samples collected via invasive surgeries (through or around the eardrum). Findings from these studies are controversial due to contradictory results between studies, lack of critical experimental controls, and sampling of participants with underlying ailments (i.e., cochlear implant surgery) that could impact the microbiology of the otic mucosa. The studies reported herein bypass these limitations by using samples of otic secretions collectively non-invasively (through the mouth) in a cohort of healthy young adults. This dissertation describes cultivation-dependent methods to investigate the microbiology of the middle ear in health. The study used an IRB-approved protocol (#17-502) to collect otic secretions in order to 1) sequence their microbiome (contribution by Dr. Joo-Young Lee) and 2) recover in pure culture otic bacteria for further characterization (my contribution). As controls, we also collected buccal (top palate and inside of cheeks) and oropharyngeal swabs from each participant. Of the collected secretions, samples from 19 individuals were used for culture independent studies, while samples from the remaining 3 participants were subjected to culture dependent studies. 16S rRNA-V4 sequencing detected a diverse and distinct microbiome in otic secretions comprised primarily of strictly anaerobic bacteria belonging to the phyla Bacteroidetes, Firmicutes and Fusobacteria, and to a lesser extent facultative anaerobes (Streptococcus). I recovered from the otic, oropharyngeal, and buccal secretions 39 isolates of predominantly facultative anaerobes belonging to Firmicutes (Streptococcus and Staphylococcus), Actinobacteria (Micrococcus and Corynebacterium), and Proteobacteria (Neisseria) phyla, and used partial 16S rRNA amplicon sequences to demonstrate the distinct phylogenetic placement of otic streptococci compared to the oral ancestors (Chapter 2). This finding is consistent with the ecological diversification of oral streptococci once in the middle ear microenvironment. The recovery of streptococci and transient migrants (Staphylococcus, Neisseria, Micrococcus and Corynebacterium) from otic secretions prompted us to study the adaptive responses that give the streptococcal migrants a competitive advantage during the colonization of the middle ear (Chapter 3). For these studies, I sequenced and partially assembled the genomes of the otic isolates and used the full length 16S rRNA sequences for taxonomic demarcation at the species levels. Phylogenetic analyses demonstrated the oral ancestry of the otic streptococci, which retained from the otic adaptive traits critical for growth and reproduction in the middle ear mucosa (biofilm formation, mucolytic and proteolytic activity, robust growth under redox fluctuations, and fermentative production of lactate, a key metabolic intermediate in the otic trophic webs). These adaptive traits give oral streptococci a colonization advantage over competing (peri)oral migrants such as Staphylococcus. Furthermore, the otic streptococci inhibited the growth of otopathogens, including Staphylococcus aureus. These antagonistic interactions give streptococci a competitive advantage during the colonization of the middle ear and suggest a role for these commensals in promoting mucosal health. The ability of staphylococcal migrants to breach the middle ear mucosal barrier and cause infections prompted us to study the environmental factors that facilitate the spreading of staphylococci from the nasal to the middle ear mucosa. Allergies, respiratory maladies (cold, flu), or (peri)oral bacterial infections (sinus, adenoids, tonsils, etc.) lead to inflammation of the Eustachian tube and changes in the rheological properties of the otic mucus that increase the risk of infections. Thus, we examined the spreading of staphylococci on mucus-like viscous surfaces (semisolid agar plates). In Chapter 4, I show that mucins, the mucosal glycoproteins that control the viscosity and wettability of the mucus layer, induce the rapid spreading and dendritic expansion of clinical isolates closely related to S. aureus and Staphylococcus epidermidis but not of Staphylococcus hominis. Mucin glycosylation controlled the hydration of the mucoid surface and the ability of the cells to spread rapidly, in a process that was dependent on the secretion of surfactant-active, phenol-soluble modulins via the agr-quorum sensing two-component system. These results provide a plausible explanation for the rapid spreading of staphylococcal otopathogens from the nasopharynx to the middle ear through a swollen, and mucin-rich Eustachian tube. The work described in this dissertation provides much needed understanding of the adaptive responses that allow (peri)oral bacteria to colonize the middle ear. The studies add to the accumulating evidence that the middle ear mucosa is not sterile but rather harbors a commensal microbiota in health. These commensal community shares many metabolic similarities with ancestors in oral biofilms and retain adaptive traits critical for growth in the otic mucosa and inhibition of otopathogens. Additionally, this work identifies environmental factors that could contribute to staphylococcal virulence, broadening the understanding of newly identified motility phenotypes in the genus that could provide novel pharmaceutical targets.
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Title: UNDERSTANDING DRIVERS OF PLANT MICROBIOME IN MICHIGAN AGRICULTURE : STUDIES OF THE APPLE ROOT ZONE AND COMMON BEAN SEEDS
Creator: Bintarti, Ari Fina
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Plant-associated microbial communities are crucial for plant health and fitness, and may enhance plant tolerance to various environmental stresses. As global climate change threatens crop production and increases demands on sustainable agriculture, harnessing the plant microbiome has become one potential strategy to address these issues. Thus, it is fundamental to understand the relative contributions of both the host plant as well as the environment in shaping the plant microbiome. Moreover,...
Show morePlant-associated microbial communities are crucial for plant health and fitness, and may enhance plant tolerance to various environmental stresses. As global climate change threatens crop production and increases demands on sustainable agriculture, harnessing the plant microbiome has become one potential strategy to address these issues. Thus, it is fundamental to understand the relative contributions of both the host plant as well as the environment in shaping the plant microbiome. Moreover, the response of plant microbiomes to stress and any consequences of microbiome stress responses for the host plants are poorly understood, though this information is critical to achieve a basis of knowledge for plant microbiome engineering. My research aimed to contribute to this knowledge by investigating the factors that structure root- and seed-associated microbial communities of two valuable crops for Michigan’s agricultural economy: apple and common bean. The first chapter of my dissertation aimed to assess the biogeography of bacterial, archaeal, fungal, and nematode communities in the root zone of apple trees, and to determine their relationships with each other and their changes over natural abiotic gradients across orchards. I also assessed the influence of plant cultivar on microbiome structure in the root zone. I found that root zone microbiome community structure was strongly affected by geographic location and edaphic properties of soil. The next chapter of my dissertation investigated the variability of seed endophyte community of common bean (Phaseolus vulgaris L.). My results showed that plant-to-plant variability under controlled growth conditions exceeded within-plant variability among seeds from different pods. My study developed protocols and added insights to the growing toolkit of approaches to understand the plant-microbiome engagements that support the health of agricultural and environmental ecosystems. The last chapter assessed the responses of common bean seed endophytes to drought stress in the field across two growing locations and four genotypes of common bean. To summarize, this work advances foundational knowledge of the seed microbiome as a critical component of the plant microbiome, and in the context of two key crops for Michigan agriculture.
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