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Title: Molecular epidemiology, pangenomic diversity, and comparative genomics of Campylobacter jejuni
Creator: Rodrigues, Jose Alexandre
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Campylobacter jejuni, the leading cause of bacterial gastroenteritis in the United States, is often resistant to commonly used antibiotics and has been classified as a serious threat to public health. Through this work, we sought to evaluate infection trends, quantify resistance frequencies, identify epidemiological factors associated with infection, and use whole-genome sequencing (WGS) as well as comparative phylogenomic and pangenomic approaches to understand circulating C. jejuni...
Show moreCampylobacter jejuni, the leading cause of bacterial gastroenteritis in the United States, is often resistant to commonly used antibiotics and has been classified as a serious threat to public health. Through this work, we sought to evaluate infection trends, quantify resistance frequencies, identify epidemiological factors associated with infection, and use whole-genome sequencing (WGS) as well as comparative phylogenomic and pangenomic approaches to understand circulating C. jejuni populations in Michigan. C. jejuni isolates (n=214) were collected from patients via an active surveillance system at four metropolitan hospitals in Michigan between 2011 and 2014. Among the 214 C. jejuni isolates, 135 (63.1%) were resistant to at least one antibiotic. Resistance was observed for all nine antibiotics tested yielding 11 distinct resistance phenotypes. Tetracycline resistance predominated (n=120; 56.1%) followed by resistance to ciprofloxacin (n= 49; 22.9%), which increased from 15.6% in 2011 to 25.0% in 2014. Notably, patients with ciprofloxacin resistant infections were more likely to report traveling in the past month (Odds Ratio (OR): 3.0; 95% confidence interval (CI): 1.37, 6.68) and international travel (OR: 9.8; 95% CI: 3.69, 26.09). To further characterize these strains, we used WGS to examine the pangenome and investigate the genomic epidemiology of this set of C. jejuni strains recovered from Michigan patients. Among the 214 strains evaluated, 83 unique multilocus sequence types (STs) were identified that were classified as belonging to 19 previously defined clonal complexes (CCs). Core-gene phylogenetic reconstruction based on 615 genes identified three clades, with Clade I comprising six subclades (IA-IF) and predominating (83.2%) among the strains. Because specific cattle-associated STs, such as ST-982, predominated among strains from Michigan patients, we also examined a collection of 72 C. jejuni strains from cattle recovered during an overlapping time period by WGS. Several phylogenetic analyses demonstrated that most cattle strains clustered separately within the phylogeny, but a subset clustered together with human strains. Hence, we used high quality single nucleotide polymorphism (hqSNP) profiling to more comprehensively examine those cattle and human strains that clustered together to evaluate the likelihood of interspecies transmission. Notably, this method distinguished highly related strains and identified clusters comprising strains from both humans and cattle. For instance, 88 SNPs separated a cattle and human strain that were previously classified as ST-8, while the human and cattle derived ST-982 strains differed by >200 SNP differences. These findings demonstrate that highly similar strains were circulating among Michigan patients and cattle during the same time period and highlight the potential for interspecies transmission and diversification within each host. In all, the data presented illustrate that WGS and pangenomic analyses are important tools for enhancing our understanding of the distribution, dissemination, and evolution of specific pathogen populations. Combined with more traditional phenotypic and genotypic approaches, these tools can guide the development of public health prevention and mitigation strategies for C. jejuni and other foodborne pathogens.
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Title: DEVELOPMENT OF 3D BIOACTIVE AND ANTIBACTERIAL SILICATE-BASED SCAFFOLDS FOR BONE TISSUE REGENERATION IN LOAD-BEARING APPLICATIONS
Creator: Marsh, Adam Christoph
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Current gold-standard approaches to addressing the needs of bone defects in load-bearing applications entail the use of either autographs or allographs. Both solutions, however, are imperfect as both autographs and allographs carry the risk of additional trauma, threat of disease transmission, and potential donor rejection respectively. Porous 3D scaffolds are attractive alternatives, illuminating a potential path towards achieving the ideal scaffold for targeting bone tissue regeneration in...
Show moreCurrent gold-standard approaches to addressing the needs of bone defects in load-bearing applications entail the use of either autographs or allographs. Both solutions, however, are imperfect as both autographs and allographs carry the risk of additional trauma, threat of disease transmission, and potential donor rejection respectively. Porous 3D scaffolds are attractive alternatives, illuminating a potential path towards achieving the ideal scaffold for targeting bone tissue regeneration in load-bearing applications, usurping autographs to become the new gold-standard. To unlock the full healing potential of 3D scaffolds, such scaffolds must be multifunctional such that (1) their mechanical performance meets the requisite requirements as dictated by the mechanical performance characteristics of interest for native bone tissue, (2) they stimulate the necessary biological responses for bone tissue regeneration, and (3) they exhibit antibacterial characteristics to combat the threat of infection. To date, no reports document 3D scaffolds exhibiting all three performance characteristics. The aim of this dissertation, therefore, is to deliver 3D scaffolds that are mechanically competent, possess and exhibit inherent and advanced antibacterial characteristics, and are successful at providing the needed biological characteristics for bone tissue regeneration. To achieve this, this dissertation implements a multidisciplinary approach, utilizing comprehensive structural characterization across a wide range of scales to elucidate process – performance relationships to execute scientifically driven modifications to engineer and deliver a 3D scaffold to successfully target bone tissue regeneration in load-bearing applications. A silver-doped bioactive glass-ceramic (Ag-BG) composition was selected as the material for scaffold synthesis due to its inherent and attractive antibacterial and biological performance characteristics. Two fundamentally different processing approaches were utilized for synthesizing Ag-BG scaffolds: the polymer foam replication technique and fused filament fabrication (FFF). The Ag-BG scaffolds studied herein were found to exhibit advanced antibacterial performance characteristics against methicillin-resistant Staphylococcus aureus (MRSA), a common pathogen implicated in osteomyelitis development, able to combat MRSA both in planktonic and biofilm forms. Ag-BG scaffolds demonstrated the ability to form an apatite-like layer when immersed in simulated body fluid (SBF), an indicator that Ag-BG scaffolds will induce the necessary mineralization for bone tissue regeneration, in addition to exhibiting attractive cell viability, proliferation, and differentiation characteristics when studied in vitro. The mechanical performance of Ag-BG scaffolds reported herein saw progressive improvements in each iteration of Ag-BG scaffold synthesis, achieving desirable mechanical competency and reliability as a result of the multidisciplinary approach formulated. In addition to the exploration of developing 3D antibacterial and biological silicate-based scaffolds capable of targeting bone tissue regeneration in load-bearing applications, foundational work towards the development of class II hybrid scaffolds comprised of gelatin methacryloyl (GelMA) and Ag-BG for targeting softer tissue regeneration. The novel syntheses applied to the successful molecular coupling of GelMA and Ag-BG presents an attractive class II hydrogel showing great promise as a compatible ink for 3D bioprinting cell-laden scaffolds capable of targeting tissue regeneration of more sophisticated systems.
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Title: Characterization of the human gut resistome, microbiome, and metabolome during enteric infection
Creator: Hansen, Zoe A.
Date: 2022
Collection: Electronic Theses & Dissertations
Description: The human gut environment is replete with host-microbe and microbe-microbe interactions that shape human health. This system is also a known reservoir for antimicrobial resistance (AMR). The ubiquity of AMR is alarming, as greater than 2.8 million antibiotic-resistant infections and 35,000 deaths occur annually in the United States. Multiple human pathogens have demonstrated reduced susceptibility to various antibiotics, including enteric pathogens such as Campylobacter, Salmonella, Shigella,...
Show moreThe human gut environment is replete with host-microbe and microbe-microbe interactions that shape human health. This system is also a known reservoir for antimicrobial resistance (AMR). The ubiquity of AMR is alarming, as greater than 2.8 million antibiotic-resistant infections and 35,000 deaths occur annually in the United States. Multiple human pathogens have demonstrated reduced susceptibility to various antibiotics, including enteric pathogens such as Campylobacter, Salmonella, Shigella, and STEC, which cause millions of foodborne infections each year. The increasing incidence of antibiotic resistant enteric infections substantiates a need to further characterize these pathogens’ role in the curation and dissemination of AMR across environments. In this dissertation, a total of 223 human stools were assessed using shotgun metagenomics sequencing to investigate gut microbiome changes associated with enteric infection. Sixty-three stools were collected from patients suffering from enteric infection between 2011-2015 by the Michigan Department of Health and Human Services (MDHHS). Sixty-one of these patients submitted a follow-up sample between 1- and 29-weeks post-infection, and 99 healthy household members also submitted stools to serve as controls. In Chapter 2, a subset of patients infected with Campylobacter spp. and their related controls were investigated to assess the gut resistome, or collection of all antimicrobial resistance genes (ARGs) and their genetic precursors, related to infection. This examination revealed significantly higher ARG diversity in infected patients compared to healthy controls. Specifically, levels of multi-drug resistance (MDR) were greatly increased during infection. Three case clusters with distinct resistomes were identified; two of these clusters had unique ARG profiles that differed from those of healthy family members. In Chapter 3, a larger subset of 120 paired samples (60 infected vs. 60 recovered) were investigated to further characterize resistome and microbiome fluctuations related to infection and recovery. Again, infected patients harbored greater resistome diversity; however, recovered individuals displayed higher diversity in their microbiota composition. Despite their lower overall microbial diversity, patients with acute infections showed an increase in the abundance of members of Enterobacteriaceae, with specific expansion of the genus Escherichia. Host-tracking analysis revealed that many Enterobacteriaceae carried ARGs related to MDR and biocide resistance, a finding with broad implications for the ecology of resistance during infection. The fourth chapter explored metabolic capacity of gut microbial communities. In addition to metabolic pathway prediction, untargeted metabolomics was performed via LC/MS for 122 paired samples. Pathway annotation suggested that infected individuals contain greater microbial functional capacity, but metabolomics indicated greater overall metabolite diversity among recovered patients. Infection was associated with enhanced nitrogen and amino acid metabolism pathways. Although many metabolites remain uncharacterized, their presence or absence among individuals suggest their importance during and after infection. Altogether, the findings of this dissertation further characterize ecological consequences related to enteric infection in the human gut. Specifically, this research illustrates the importance of enteric infection in the dissemination and persistence of resistance determinants. Moreover, the expansion of Enterobacteriaceae and the evident increase in nitrogen- and amino acid-related metabolism during infection represent potential targets for future intervention practices.
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Title: Characterization of Broadly Conserved AvcID Toxin-Antitoxin System and Its Mechanism to Inhibit Phage by Disrupting Nucleotide Metabolism
Creator: Hsueh, Brian Yifei
Date: 2022
Collection: Electronic Theses & Dissertations
Description: ABSTRACTCHARACTERIZATION OF BROADLY CONSERVED AVCID TOXIN-ANTITOXIN SYSTEM AND ITS MECHANISM TO INHIBIT PHAGE BY DISRUPTING NUCLEOTIDE METABOLISMByBrian Yifei HsuehThe prevalence of antiphage defense systems, which have recently been shown to be located on mobile genetic elements in bacteria, have sparked interest to understand the coevolutionary arms race of bacteria and bacteriophage (phage). Bacteria and phages have coexisted for billions of years, and phages are widely distributed in...
Show moreABSTRACTCHARACTERIZATION OF BROADLY CONSERVED AVCID TOXIN-ANTITOXIN SYSTEM AND ITS MECHANISM TO INHIBIT PHAGE BY DISRUPTING NUCLEOTIDE METABOLISMByBrian Yifei HsuehThe prevalence of antiphage defense systems, which have recently been shown to be located on mobile genetic elements in bacteria, have sparked interest to understand the coevolutionary arms race of bacteria and bacteriophage (phage). Bacteria and phages have coexisted for billions of years, and phages are widely distributed in different environmental niches populated by their bacterial hosts, including the human intestine and marine environment. The evolutionary pressure imposed by phages have led bacteria to evolve diverse strategic systems to protect themselves from phage predation, including CRISPR-Cas, restriction-modification, and abortive infection. Recent studies have begun to reveal that toxin-antitoxin (TA) system are associated with antiphage defense systems. Vibrio cholerae El Tor, the causative agent of current cholera pandemics, has acquired two unique genomic islands of unknown origins, known as Vibrio Seventh Pandemic Islands 1 & 2 (VSP-1 & 2). It is hypothesized that the acquisition of VSP islands increase environmental fitness of El Tor. While both islands encode approximately 36 open reading frames, yet many remain largely uncharacterized. In this work, I characterize a novel TA antiphage system encoded on VSP-1 of V. cholerae, here named AvcID. Chapter 2 describes the biological function of AvcD toxin by which it possesses deoxycytidylate deaminase (DCD) activity and produces dUMP as the final product. Further experiments identify the AvcI antitoxin as a small RNA and determine that it post-translationally inhibits the activity of AvcD. Moreover, AvcD consists of two domains—a N-terminal P-loop NTPase and a C-terminal DCD—and mutations in conserved features of each domain abrogate its activity. AvcD is widely conserved across kingdoms, and virtually all bacteria that encode AvcD also have AvcI homologs. Notably, chromosomal AvcID can solely be activated by transcriptional shutoff in V. cholerae, demonstrating that AvcID is a type III TA system. Unlike canonical type III TA systems, in which the toxin is an endoribonuclease, the AvcD toxin is a deaminase. Importantly, the AvcID system provides antiphage defense in Escherichia coli that lacks this system by corrupting nucleotides for phages to utilize to reduce coliphage replication efficiency. In Chapter 3, I explore the activation mechanism of the AvcID system as well as the consequences to phages after encountering AvcID. During infection, virtually all lytic phages induce transcription shutoff of the host by hijacking host transcription machinery to make virion progeny. I uncover that phage-induced transcriptional shutoff leads to turnover of labile AvcI antitoxin and concomitantly activates the deaminase activity of AvcD, leading to a disruption of nucleotide levels. This disruption of nucleotide levels is shown in both susceptible phages (ex. T5) and resistant phages (ex. T7). Through an unknown mechanism, AvcID also increases the abundance of defective phages that are susceptible to AvcID. In summary, this work has made contributions in the field of TA systems and its association with the antiphage defense paradigm by uncovering the biological function and mechanism in response to phage infection.
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Title: ELUCIDATING THE POTENTIAL ROLE OF ARYL HYDROCARBON RECEPTOR IN THE PATHOGENESIS OF CAMPYLOBACTER JEJUNI.
Creator: Ahmed, Husnain
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Campylobacter jejuni is a leading cause of human foodborne gastroenteritis in the US, with an incidence rate of 13.6 diagnosed cases per 100,000 individuals. The most frequent cause of C. jejuni infection in the US is the consumption of chicken contaminated during processing. Macrolide antibiotics such as azithromycin and ciprofloxacin are the drug of choice to treat C. jejuni infection in human populations. However, the over-use of antibiotics has led to the emergence of antimicrobial...
Show moreCampylobacter jejuni is a leading cause of human foodborne gastroenteritis in the US, with an incidence rate of 13.6 diagnosed cases per 100,000 individuals. The most frequent cause of C. jejuni infection in the US is the consumption of chicken contaminated during processing. Macrolide antibiotics such as azithromycin and ciprofloxacin are the drug of choice to treat C. jejuni infection in human populations. However, the over-use of antibiotics has led to the emergence of antimicrobial-resistant C. jejuni strains and reduced treatment efficacy. The development of antimicrobial resistance traits in C. jejuni isolates has augmented the need to develop innovative strategies to treat drug-resistant C. jejuni infections in human and animal populations.Members of the genus Lactobacillus are commonly used as probiotics, however the mechanisms by which they provide protective health effects remain elusive. In the first study, we described a novel mechanism by which L. murinus attenuates pro-inflammatory responses in the human intestinal epithelial cells. The results showed that L. murinus activates aryl hydrocarbon receptor (AHR) to decrease the secretion of IL-8 in response to exogenous stimulation by TNF-alpha in the human intestinal epithelial cells. Furthermore, activating the AHR with its defined ligand also reduced the secretion of IL-8 upon TNF-alpha stimulation. These results suggest that AHR can a novel target for inflammatory bowel disease (IBD) treatment. Furthermore, these results suggest that L. murinus can be a novel probiotic for treating IBD. In the 2nd study, we determined the effect of prophylactic inoculation of L. muirnus on the pathogenesis of C. jejuni in the BALB/c IL-10-/- mice. A total of 41 BALB/c IL-10-/- mice were used in this study. 11 mice were sham inoculated, 10 mice received only L. murinus, 10 mice received only C. jejuni, and 10 mice in the test group received both L. murinus and C. jejuni such that L. murinus was inoculated 32 days before C. jejuni infection. In addition, 30 days post-C. jejuni challenge mice were sacrificed and assessed for gut pathology. Fecal samples were also collected to access bacterial colonization levels in the gut through routine culture techniques and 16S sequence analysis. Both positive control group for C. jejuni and test groups mice developed severe colitis. 16S analysis of fecal DNA revealed that bacterial diversity in the test and positive control group for C. jejuni was significantly less (P<0.001) than in the Lactobacillus only and negative control group. These results suggest that prophylactic administration of L. murinus does not protect BALB/c IL-10-/- mice from developing disease following C. jejuni infection. Overall, this dissertation contains identification of a novel mechanism of action of L. murinus. The results provide insights for the identification of novel targets to treat C. jejuni disease without using antibiotics. This dissertation provides a basis for the future studies to further dissect the role of the AHR in the pathogenesis of C. jejuni.
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Title: CARBON-MEDIATED ECOLOGICAL AND PHYSIOLOGICAL CONTROLS ON NITROGEN CYCLING ACROSS AGRICULTURAL LANDSCAPES
Creator: Curtright, Andrew James
Date: 2022
Collection: Electronic Theses & Dissertations
Description: The sustainable intensification of agriculture relies on the efficient use of ecosystem services, particularly those provided by the microbial community. Managing for these ecosystem services can improve plant yields and reduce off-site impacts. For instance, increasing plant diversity is linked to positive effects on yield, and these beneficial effects are often mediated by the microbial community and the nutrient transformations it carries out. My dissertation has aimed to elucidate the...
Show moreThe sustainable intensification of agriculture relies on the efficient use of ecosystem services, particularly those provided by the microbial community. Managing for these ecosystem services can improve plant yields and reduce off-site impacts. For instance, increasing plant diversity is linked to positive effects on yield, and these beneficial effects are often mediated by the microbial community and the nutrient transformations it carries out. My dissertation has aimed to elucidate the mechanisms by which plant diversity improves agricultural production. In particular, I have focused on how changes to the amount and diversity of carbon (C) inputs affects soil microorganisms involved in the nitrogen (N) cycle. My work spans multiple scales of observation: from a global meta-analysis to mechanistic studies utilizing denitrification as a model system.In a global meta-analysis, I found that increasing plant diversity through intercropping yields a net increase in extracellular enzyme activity. This effect varied by plant species and soil type suggesting that increases in the quality of nutrient inputs mediates these positive effects on microbial activity. Then, I looked at how intercropping cover crops into corn affects soil nutrient pools and microbial activities in a field experiment. No effect of interseeding cover crops into corn was found on soil nutrient pools or microbial activities. However, by analyzing differences in relationships between nutrient pools and microbial activities at two locations throughout Michigan, I was able to describe how the availability of dissolved organic C (DOC) drives differences in microbial N-cycling processes. I then investigated how C availability drives activity in microbial hotspots within the soil by comparing differences in denitrification potential in bulk soil versus the rhizospheres of corn and interseeded cover crops. Here, I found that denitrification rates were increased in the rhizospheres of all plant types, and this effect varied depending on the species of plant. I was able to further differentiate the impact of DOC and microbial biomass C on the rhizosphere effect and found that C availability was the primary driver of differences in denitrification rates between rhizospheres. Since plants provide many different forms of C to soil microbes, it is important to understand how the chemistry of C inputs affects microbial activity. I used a series of C-substrate additions to determine how C chemistry affects denitrifiers. I found that amino acids and organic acids tended to stimulate the most nitrous oxide (N2O) production and reduction. Although management and site affected overall rates of denitrification, C-utilization patterns of microbes were mostly similar between locations. To identify the mechanisms responsible for these effects, I performed a final experiment to track how denitrifiers utilized different C compounds. The C substrates that stimulated the most complete reduce of N2O also were utilized with the lowest C-use efficiency (CUE). This suggests possible trade-offs between N2O reduction and CUE, with important implications for how to manage microbial communities. Overall, my work demonstrates that land management can impact microbial community activity by influencing the identity of soil C inputs. While the importance of increasing soil C inputs has been known, this dissertation supports the notion that the chemical identity of C inputs can exert significant controls on microbial activity. Moreover, by comparing microbial traits I highlight the importance of trade-offs in how microbially mediated C- and N cycling are coupled.
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Title: DISRUPTIONS IN HEPATIC ONE CARBON METABOLISM AND THE GUT MICROBIOME DURING THE PROGRESSION OF NON-ALCOHOLIC FATTY LIVER DISEASE
Creator: Fling, Russell Ryan
Date: 2022
Collection: Electronic Theses & Dissertations
Description: The etiology of non-alcoholic fatty liver disease is complex, with multiple contributing factors including dietary, environmental, gut microbiome and genetic mechanisms. Accumulating evidence suggests exposure to polychlorinated dibenzo-p-dioxins and similar compounds may increases risk for NAFLD development. These environmentally persistent dioxin-like compounds bind and activate the aryl hydrocarbon receptor, a transcription factor that regulates intestinal homeostasis, xenobiotic and...
Show moreThe etiology of non-alcoholic fatty liver disease is complex, with multiple contributing factors including dietary, environmental, gut microbiome and genetic mechanisms. Accumulating evidence suggests exposure to polychlorinated dibenzo-p-dioxins and similar compounds may increases risk for NAFLD development. These environmentally persistent dioxin-like compounds bind and activate the aryl hydrocarbon receptor, a transcription factor that regulates intestinal homeostasis, xenobiotic and central metabolism. In a AhR-dependent manner, mice orally gavaged with 2,3,7,8-tetracholordibenzo-p-dioxin (TCDD) exhibit steatosis progressing to steatohepatitis with fibrosis akin to NAFLD progression. NAFLD and hepatocellular carcinoma (HCC) is also closely correlated with dysregulation of central metabolism e.g., hepatic one carbon metabolism (OCM), and gut dysbiosis contributing to NAFLD progression and worsening prognosis. This report investigates mechanisms involved in the dysregulation of the gut microbiome and OCM associated pathways relevant to NAFLD progression through comparisons of molecular analyses of TCDD-treated mice to human NAFLD and HCC. OCM describes the biosynthesis, homeostasis, and utilization of the cell’s main methyl donor, S-adenosylmethionine (SAM) including high flux anabolic biosynthesis of polyamines, phosphatidylcholine and creatine. In later stages of NAFLD, OCM is dysregulated with altered OCM gene expression as well as SAM and s-adenosylhomocysteine (SAH) levels. To assess TCDD-elicited effects on OCM, mice were orally gavaged with TCDD every 4 days for 28 days. Serum and livers collected at early (8 days) and late (28 days) time points were subjected to metabolomic analyses with integration of chromatin immunoprecipitation sequencing, transcriptomics and protein levels. Results from these studies suggest AhR-mediated repression of OCM required prolonged repeated TCDD-treatment and indirect effects elicited by AhR activation e.g., oxidative stress. Gut dysbiosis with disrupted enterohepatic bile acid metabolism is commonly associated with NAFLD and recapitulated in TCDD-treated mice. Similar to NAFLD, TCDD also increases systematic levels of secondary bile acids. These microbial transformed secondary bile acids are involved in modulation of host bile acid signaling pathways relevant to NAFLD. To investigate the effects of TCDD on the gut microbiota, the cecum contents of TCDD-treated mice were subjected to shotgun metagenomic sequencing. Taxonomic analysis identified dose-dependent increases in Lactobacillus species, notably Lactobacillus reuteri. Top enriched species were also associated with increased abundances of bile salt hydrolase sequences, responsible for the initial deconjugation reaction in secondary bile acid metabolism. L. reuteri levels were also attributed to enrichment of mevalonate-dependent isopentenyl diphosphate (IPP) biosynthesis genes, a pathway that was also elevated in cirrhosis patients. These results extend the role of Lactobacilli in the AhR/intestinal axis and NAFLD progression as well as highlight the similarities between TCDD-elicited phenotypes in mice to human NAFLD. Collectively, these studies evaluated TCDD-elicited mechanisms involved in disruptions in host and microbial metabolism, highlighting the AhR’s role in NAFLD progression.
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Title: MEMBRANE-LOCALIZED TRANSCRIPTION REGULATORS : UNDERSTANDING POST-TRANSLATIONAL REGULATION AND SINGLE-MOLECULE DYNAMICS OF TCPP IN VIBRIO CHOLERAE
Creator: Demey, Lucas Maurice
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Vibrio cholerae is a Gram-negative gastrointestinal pathogen that has evolved an elegant regulatory system to precisely time production of essential virulence factors. A key step in this regulatory system is the transcription of a soluble AraC-like transcription factor, ToxT. ToxR and TcpP, two membrane-localized transcription regulators (MLTRs), positively regulate toxT. Much work has contributed to our understanding of TcpP and ToxR regulation, yet major gaps remain in our knowledge of...
Show moreVibrio cholerae is a Gram-negative gastrointestinal pathogen that has evolved an elegant regulatory system to precisely time production of essential virulence factors. A key step in this regulatory system is the transcription of a soluble AraC-like transcription factor, ToxT. ToxR and TcpP, two membrane-localized transcription regulators (MLTRs), positively regulate toxT. Much work has contributed to our understanding of TcpP and ToxR regulation, yet major gaps remain in our knowledge of these MLTRs. MLTRs are unique one-component signal transduction systems because they respond to extracellular stimuli by influencing gene transcription from their location in the cytoplasmic membrane. In Chapter 2, I explore the prevalence and diversity of MLTRs within prokaryotes to enhance our understanding of TcpP and ToxR. I show that MLTRs are far more common among prokaryotes than previously anticipated and that MLTRs are an understudied class of transcription regulators. In Chapter 3, I describe the use of super-resolution single-molecule tracking to investigate how TcpP, a model MLTR, identifies the toxT promoter. I provide evidence that TcpP binds to the toxT promoter independent of ToxR, and TcpP transitions to a specific diffusion state. The data support the first biophysical model for how TcpP-like MLTRs locate their target promoters. TcpP is subject to a form of post-translational regulation known as regulated intramembrane proteolysis (RIP). RIP of TcpP results in its complete inactivation, resulting in loss of virulence factor production. TcpH inhibits RIP of TcpP under certain pH and temperature conditions. In Chapter 4, I describe the mechanism TcpH employs to inhibit TcpP RIP while V. cholerae is present in the mouse gastrointestinal tract. I demonstrate that the dietary fatty acid α-linolenic acid enhances inhibition. I also show that α-linolenic acid promotes TcpH-mediated inhibition of TcpP RIP by increasing association of both proteins with detergent-resistant membrane (DRM) domains. My work provides the first evidence that DRMs influence virulence factor transcription in V. cholerae and that a dietary fatty acid promotes V. cholerae pathogenesis.
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Title: IMPACT OF AGRICULTURAL MANAGEMENT AND MICROBIAL INOCULATION ON SOYBEAN (GLYCINE MAX) AND ITS ASSOCIATED MICROBIOME
Creator: Longley, Reid
Date: 2022
Collection: Electronic Theses & Dissertations
Description: Soybean (Glycine max) is a globally important crop with uses as food, cooking oil livestock feed, and biodiesel. Soybean can be considered holobionts because they host diverse microbiomes which extend plant genotypes and phenotypes through various microbial functions such as nitrogen fixation and increased disease resistance. My research focused on assessing the impact of three agricultural management strategies on the soybean holobiont. Soybean cropping systems can be managed using various...
Show moreSoybean (Glycine max) is a globally important crop with uses as food, cooking oil livestock feed, and biodiesel. Soybean can be considered holobionts because they host diverse microbiomes which extend plant genotypes and phenotypes through various microbial functions such as nitrogen fixation and increased disease resistance. My research focused on assessing the impact of three agricultural management strategies on the soybean holobiont. Soybean cropping systems can be managed using various strategies, including conventional tillage, no-till, and organic management regimes. These management systems have been shown to impact the microbiomes of soybean-associated soils, however, their impacts on plant-associated microbiomes are still not well understood. In this study, I assessed the impact of conventional, no-till, and organic management treatments on soybean microbiomes at Michigan State’s Kellogg Biological Station Long-Term Ecological Research site (KBS LTER). I found that management impacted microbiome composition and diversity in soil, roots, stems, and leaves and that this impact persisted throughout the season. Additionally, when comparing the same soybean genotype grown in conventional and no-till management systems, tillage regime impacted the microbiome throughout the plant and the growing season. This effect impacted microbial taxa which are likely to be plant beneficial, including nitrogen fixing Bradyrhizobium. Another important management tool that is expected to impact plant-associated microbial communities is the application of foliar fungicides. While fungicides are known to protect plants from particular fungal pathogens, non-target impacts of fungicides on crop microbiomes, and the impact of management on microbiome recovery are not well understood. To address this knowledge gap, I assessed the impact of foliar fungicide application on the maize (Headline® fungicide, 2017) and soybean (Delaro® fungicide, 2018) microbiomes in conventional and no-till plots at the KBS LTER. I found that fungicide applications have a non-target impact on Tremellomycete yeasts in the phyllosphere and this impact was greater in soybean than maize. Co-occurrence network analysis and random forest modelling indicated that changes in fungal communities may lead to indirect impacts on prokaryotic communities in the phyllosphere. Importantly, this work demonstrated that phyllosphere communities of soybeans under no-till management had greater recovery from fungicide disturbance. This novel finding exemplifies how tillage regime can impact phyllosphere microbiomes and their responses to disturbance. Microbial inoculants in agriculture have long been used for biocontrol of pathogens, but there is also interest in their use to dampen the impacts of abiotic stress including drought. In this study, I tested whether inoculating soybeans with hub taxa identified through network analysis from no-till soybean root microbiome data from the KBS LTER could provide protection against water limitation. Soybean seedlings were enriched in consortia of hub bacteria and fungi and were grown in no-till field soil. Seedlings were then exposed to low-moisture stress, and plant phenotypes, plant gene expression, and amplicon sequencing of microbial DNA and cDNA were assessed throughout the stress period. Inoculation increased plant growth, nodule numbers, and led to increased expression of nodulation-associated genes. 16S sequencing of cDNA revealed higher levels Bradyrhizobium in inoculated samples. These results indicate that inoculation with hub microbes can benefit soybean plants, possibly through interaction with other microbes, interaction with the plant, or both. In summary, fungicide, tillage, and inoculation all impact the soybean microbiome, indicating that management choices impact the entire holobiont.
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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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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: THE EFFECT OF FREEZE-THAW CYCLES ON MICROBIAL RESILIENCE ALONG A CROP BIODIVERSITY GRADIENT
Creator: Liang, Brian Wan
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Freeze-thaw cycles (FTCs) are cyclical periods of soil disturbance that are increasing in number and intensity due to climate change effects on winter precipitation and temperature patterns and are not well characterized within an agroecosystem environment. First, I review the literature and discuss the effects of FTCs on soil properties, explore the nuances of characterizing FTCs in experiments, and assess the knowledge gaps of FTC studies in agroecosystems. I conducted a laboratory...
Show moreFreeze-thaw cycles (FTCs) are cyclical periods of soil disturbance that are increasing in number and intensity due to climate change effects on winter precipitation and temperature patterns and are not well characterized within an agroecosystem environment. First, I review the literature and discuss the effects of FTCs on soil properties, explore the nuances of characterizing FTCs in experiments, and assess the knowledge gaps of FTC studies in agroecosystems. I conducted a laboratory experiment using soils from a crop rotational diversity gradient and froze them at three distinct FTC frequencies. My results indicate that increased crop rotational diversity did not moderate FTC disturbance effects at any frequency level. Increased FTC frequencies generally increased soil organic C losses as CO2, decreased ammonium (NH4+), increased nitrate (NO3-) pools, and increased extracellular enzyme activities (EEA). The respiratory burst after each freezing period was the predominant contributor to differences by FTC in cumulative CO2 respiration by the end of the incubation. Interestingly, the medium FTC frequencies facilitated the highest EEA for select enzymes with minimal reductions in microbial biomass. This suggests that microbes and their EEA are impacted too severely with high frequency FTCs to maintain function. My study revealed that the novel microbial communities and soil processes found along a crop rotational diversity gradient are not resilient against climate change effects of FTCs in soils. Accelerated soil organic C loss and nutrient turnover are expected to occur throughout agroecosystems that experience increased FTCs.
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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: 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: 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: 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: UNDERSTANDING THE ROLES OF INTERKINGDOM MICROBIAL INTERACTIONS, MICROBIAL TRAITS, AND HOST FACTORS IN THE ASSEMBLY OF PLANT MICROBIOMES
Creator: Liber, Julian Aaron
Date: 2021
Collection: Electronic Theses & Dissertations
Description: The community of organisms that associate with plants are vital to both the survival of the host plant but also the diseases which may kill it. The processes by which this community, called the microbiome, assemble and function can contribute to the traits of the host, including plants that humans rely on for food, resources, and ecosystems services. This thesis focuses on understanding the assembly of microbiomes at the scale of microbe-microbe interactions and traits of individual microbes,...
Show moreThe community of organisms that associate with plants are vital to both the survival of the host plant but also the diseases which may kill it. The processes by which this community, called the microbiome, assemble and function can contribute to the traits of the host, including plants that humans rely on for food, resources, and ecosystems services. This thesis focuses on understanding the assembly of microbiomes at the scale of microbe-microbe interactions and traits of individual microbes, as well as how characters of the host may change this process. I first address this by examining the in vitro and in planta interactions within small synthetic communities of root-inhabiting bacteria and fungi and with the plant host and viral disease of the host. While intermicrobial interactions in vitro were not predictive of in planta interactions, adding host disease or additional organisms to the system altered the assembly process. I then show the development and applications of the CONSTAX2 classifier, a taxonomic assignment tool for metabarcoding studies, which offers improved accuracy and ease of use for conducting metabarcoding studies exploring the diversity and structure of microbial communities. Last, I present a study testing which factors affected the composition of forest fungal communities to understand the ecology of litter-inhabiting fungi and improve methodologies for sampling leaf-associated fungal communities. The factors affecting the assembly of plant microbiomes are complex and varied but connecting individual interactions to community composition and ultimately function may improve our abilities to predict and manage microbiome processes.
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Title: INFLUENCE OF AQUATIC MICROBES ON MOSQUITO OVIPOSITION BEHAVIOR AND LIFE HISTORY
Creator: Receveur, Joseph
Date: 2021
Collection: Electronic Theses & Dissertations
Description: Mosquito-microbe interactions have major roles in mediating mosquito life histories and the capacity of mosquitoes to transmit pathogens to humans. While essential to better understanding mosquito life histories, our knowledge of how microbes influence oviposition behavior and development remains limited, especially in complex polymicrobial communities. This dissertation tested the relationship between aquatic microbial communities and mosquito oviposition behavior. Field studies were...
Show moreMosquito-microbe interactions have major roles in mediating mosquito life histories and the capacity of mosquitoes to transmit pathogens to humans. While essential to better understanding mosquito life histories, our knowledge of how microbes influence oviposition behavior and development remains limited, especially in complex polymicrobial communities. This dissertation tested the relationship between aquatic microbial communities and mosquito oviposition behavior. Field studies were conducted in Michigan to investigate how different leaf-litter derived microbial communities (and modifications of the microbes present in those communities) effected mosquito oviposition and survivorship. Additionally, field (Ouidah, Benin) and laboratory assays were used to determine the effect of a bacterial toxin (mycolactone, produced by Mycobacterium ulcerans) on other aquatic microbes and the downstream impacts of those interactions on mosquito oviposition. Ae. japonicus japonicus (Theobald) mosquitoes preferred red oak (Quercus rubra) leaf-derived leachates over other leaf species tested (sycamore Platanus occidentalis, and honeysuckle Lonicera maackii). Leachates displayed distinct bacterial and fungal communities with sterilization of these communities causing a reduction in oviposition. Larval mosquito growth and survivorship were influenced by both leaf type and leachate concentration. The addition of mycolactone, hypothesized to inhibit microbial quorum sensing, to environmental water samples altered bacterial community composition and reduced Aedes egg laying. In addition to reducing oviposition of Aedes aegypti (L.) in a dose-dependent manner, mycolactone up- and down-regulated expression levels of multiple taxa (N = 13) and functional groups (N = 13), suggesting the toxin plays an important role in interactions between M. ulcerans, other environmental microbes, and mosquito behavior. These studies highlight the essential role mosquito-microbe interactions play in oviposition behavior and life history and expand our knowledge of the microbial underpinnings of mosquito oviposition behavior. Additionally, experiments testing the relationship between the microbiota of aquatic macroinvertebrates and environmental conditions were conducted in Ostana, Italy using high-throughput amplicon sequencing. While both environmental conditions and macroinvertebrate species influenced the internal microbiome, species had a larger effect than environmental conditions, suggesting that physiological conditions in macroinvertebrate guts plays a role in what microbes are able to colonize and survive.
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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: 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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