Theses and Dissertations from UMD
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Item THE EVOLUTIONARY TRAJECTORY OF METARHIZIUM ROBERTSII ENDOPHYTIC CAPABILITY AND ENTOMOPATHOGENICITY(2024) Sheng, Huiyu; St. Leger, Raymond; Entomology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Metarhizium fungi are a keystone genus of soil-inhabiting ascomycetes providing essential ecosystem services as saprotrophs, plant symbionts and insect pathogens, among other roles. Recent studies have looked at how Metarhizium niches have evolved and shaped genome evolution over large time scales within the Metarhizium genus. This dissertation uses Metarhizium robertsii (M. robertsii) as a model to explore the evolution of its dual roles as an entomopathogen and endophyte by examining phenotypic and genomic differences among eight closely related strains. The study found that early diverged strains, characterized by slow germination on insect cuticles, low virulence, and extensive sporulation, exhibit a biotrophic lifestyle, systemically colonizing living hosts. In contrast, recently diverged strains exhibited rapid germination, high virulence, and reduced sporulation, indicating a shift towards a necrotrophic lifestyle. The study highlighted the influence of host immune responses in shaping M. robertsii-insect interactions, and showed that strong insect virulence correlated with better colonization of plant roots. Comparative genomics revealed that recently diverged strains expanded a small number of gene families related to gene expression as well as carbohydrate-degrading enzymes and proteases enhancing metabolic capabilities, insect virulence, and endophytic potential. Some early diverged strains exhibited high Repeat-Induced Point mutation activity, suggesting cryptic sexual reproduction in their evolutionary past. Overall, M. robertsii strains maintained a conserved genome with similar protein family sizes, with differences in gene expression patterns driving their varied lifestyles. This research provides new insights into M. robertsii’s recent co-evolution with plants and insects, highlighting the importance of understanding the ecological and evolutionary dynamics of these interactions for optimizing its use in sustainable agriculture.Item Roles of Female Sex Hormones in Regulating Neisseria gonorrhoeae Colonization of the Human Cervix(2024) Di Benigno, Sofia; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Neisseria gonorrhoeae (GC) is a human-exclusive pathogen that infects the genital tract. Gonococcal infection may present with or without symptoms and can lead to a variety of serious sequelae if left untreated, especially in female patients. Despite this, there are few models that can effectively mimic GC infection in the female reproductive tract (FRT); of these, even fewer consider the impact of the menstrual cycle, an important feature of the FRT, on GC infection. I used the human cervical tissue explant model previously developed in our lab, which can recapitulate GC infection in vivo. Tissue explants were treated with the sex hormones estradiol and progesterone to mimic various stages of the menstrual cycle and examine its impact on GC infectivity. Estradiol was used to mimic the late proliferative phase, and a combination of estradiol and progesterone was used to mimic the middle of the secretory phase. The effects of hormones on GC infectivity were examined after 72 total hours of hormone treatment and 24 hours of inoculation with GC of strain MS11. My results show that treatment with estradiol and with a combination of estradiol and progesterone both increase the level of GC colonization on the endocervix, but not on the ectocervix, compared to controls that were not treated with hormones. However, the hormone treatment did not affect GC penetration of the cervical epithelium. Both hormone treatments increased the number of GC colonies on the endocervical epithelium, and a combination of estradiol and progesterone produced an additional population of large GC colonies, leading to an increase in the average colony size. These increases in colony number and size were not associated with an increase in the expression of carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), which are the host receptors for GC Opa proteins. In contrast, treatment with estradiol induced a redistribution of CEACAMs from the luminal surface to the inside of epithelial cells. Additionally, estradiol altered the morphology of endocervical epithelial cells from columnar to cuboidal, but the integrity of cell-cell junctions was unchanged. The increase in colonization under high estradiol conditions was correlated with a decrease in levels of certain pro-inflammatory cytokines and chemokines, but this decrease was not sufficient to fully explain the increase in colonization. Next, I investigated the impact of cervical mucus on GC infectivity and interactions, as gel-forming mucin MUC5B but not MUC5AC increases with estradiol at the proliferation phase. Under both hormone treatment conditions, GC were able to establish close interaction with the luminal surface of the endocervical epithelial cells, displacing membrane-spanning mucin MUC1 in the membrane. Furthermore, GC were able to diffuse through an artificial mucin hydrogel and diffused more efficiently through a MUC5AC-dominant than a MUC5B-dominant hydrogel. Gel-forming mucins collected from cervical tissue explants enhanced GC aggregation in vitro, even at very low concentrations. However, mucins collected from estradiol-treated tissues showed less impact on GC aggregation than those collected from untreated tissues or tissues treated with both estradiol and progesterone. MUC5B and MUC5AC purified from cows and pigs also increase GC aggregation in vitro with GC aggregating more in a MUC5AC- than a MUC5B-dominant mucin mixture. Taken together, my research reveals for the first time that female sex hormones regulate GC colonization at the human cervix by changing the composition of the cervical mucus, providing a mechanism of hormonal regulation underlying the varying susceptibility of female patients to mucosal GC.Item Impact of salinity on morphology, growth, and pigment profiles of Scenedesmus obliquus HTB1 under ambient air and elevated CO2 (10%) conditions(2024) Jiao, Fanglue; Chen, Feng; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Certain microalgal species tolerate high CO2 concentrations and proliferate faster with elevated CO2 than with ambient air. This feature makes them attractive for carbon sequestration, a tool for mitigating climate change due to increasing atmospheric CO2. Scenedesmus species are among these microalgae. Scenedesmus obliquus strain HTB1 is a microalgal strain isolated from the Baltimore Inner Harbor (brackish water) and has shown a faster growth with 10% CO2 compared to air. However, how HTB1 grows under different salinity and if the salt response is affected by elevated CO2 remains elusive. Two experiments were set up to address these questions. The first experiment tested the impact of salinity gradient (0, 17.5, 20, 22.5, 25, 27.5, and 30 ppt) on HTB1 under ambient air. With increasing salinity, HTB1 cells became smaller, and the cultures changed color from green to brown, yellowish brown, and then to pale white. The pigment analysis showed that HTB1 reduced several pigments (i.e. zeaxanthin, lutein, chlorophyll b) in response to salt stress. However, HTB1 produced higher concentrations of canthaxanthin under the salt stress. The growth of HTB1 decreased with increasing salinity and was inhibited when the salinity was greater than 22.5 ppt. In the second experiment, we compared the impact of salinity (0, 10, and 20 ppt) on HTB1 under air and 10% CO2, respectively. HTB1 cultures showed little color change with increasing salinity under 10% CO2. In contrast, the change of culture color from dark green to brown was observed with increasing salinity when HTB1 was grown with air. Interestingly, the growth of HTB1 was less inhibited with salt under 10% CO2 than with air, suggesting that elevated CO2 mitigates the salt stress of HTB1. Lutein and canthaxanthin increased with increasing salinity when HTB1 was grown with 10% CO2. Our results indicate that increased salinity affects the growth of Scenedesmus obliquus HTB1 more with air than with 10% CO2. This study provides insight into the impact of salt stress on algal morphology, growth, and pigment composition.Item THE STANDALONE REGULATOR ROFA OF STREPTOCOCCUS PYOGENES EXHIBITS CHARACTERISTICS OF A PRD-CONTAINING VIRULENCE REGULATOR(2024) Hart, Meaghan Taylor; McIver, Kevin S; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Streptococcus pyogenes (Group A Streptococcus; GAS) is a human pathogen estimated to cause nearly 790 million cases of disease annually at diverse tissue sites. To successfully infect these sites, GAS must detect nutrient availability and adapt accordingly. One mechanism employed to detect and import carbohydrates is the phosphoenolpyruvate transferase system (PTS), which mediates both carbohydrate uptake and metabolic gene regulation. Gene regulation by the PTS can occur through phosphorylation of transcriptional regulators at conserved PTS-regulatory domains (PRDs). GAS has several stand-alone regulators that contain PRDs, with corresponding regulons encoding both metabolic genes and important virulence factors. These regulators form a family called PRD-Containing Virulence Regulators (PCVRs). RofA is a putative member of this family and is known to regulate the expression of genes important for virulence. It was hypothesized that RofA is phosphorylated by the PTS in response to carbohydrate levels to coordinate appropriate virulence gene expression. In this dissertation, the RofA regulon was determined in strain 5448, a representative strain of the globally disseminated M1T1 serotype. The pilus and capsule operons were consistently dysregulated across growth in the absence of RofA. This correlated with increased capsule production and decreased adherence to primary keratinocytes. Purified RofA-His was phosphorylated in vitro by the general PTS components EI and HPr, and phosphorylated species of RofA-FLAG were detected in vivo late in stationary phase in a glucose-dependent manner. Together, these findings support the hypothesis that RofA is a PCVR that may couple sugar detection and utilization with GAS virulence gene regulation. Additionally, a bioluminescent construct was generated for allelic exchange into any S. pyogenes strain. Allelic exchange of this construct into WT 5448 yielded strains that were highly bioluminescent, grew to a similar density as WT, and survived as well as WT when challenged with human neutrophils. This tool could be used to study the contribution of specific proteins on in vivo virulence in a non-invasive manner, including RofA and RofA phosphorylation.Item Three Clostridium species with Health Imparting Properties: In vitro Screening for Probiotic Potential(2024) Mochama, Victor Moronge; Obanda, Diana; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This research aimed to unlock the probiotic potential of the genus Clostridium, which is often overshadowed by the predominant focus on pathogenic species. The study specifically targeted three promising Clostridium species: C. disporicum, C. celatum, and C. vincentii, which have shown potential in mitigating diet-induced obesity. Despite the challenges presented by the anaerobic growth requirements of Clostridium bacteria, the study capitalized on their capacity to sporulate. This characteristic provides an avenue to use them as probiotics, with resilient and dormant spores capable of surviving food processing and harsh stomach conditions. The resilience of these spores was examined by exposing them to oxygen, heat, gastrointestinal juices, and bile salts. The spores survived oxygen exposure, exhibited resilience to both bile salts and gastric acids, and demonstrated a survival temperature of 70°C. When exposed to suitable germination conditions in vitro, the spores successfully germinated. The study assessed the colonization potential of the bacteria by evaluating their adhesion ability, and all bacteria were found to have the adhesion ability. Furthermore, a safety assessment was conducted by examining hemolytic activity and antibiotic susceptibility to selected antibiotics. The bacteria were found to be susceptible to the antibiotics and did not exhibit hemolytic activity. Bile salt hydrolase (BSH) activity and antibacterial activities were also assessed, and none of the bacteria exhibited BSH activity or antibacterial activity. Antioxidant tests revealed that C. vincentii had the highest antioxidant properties. Assessment of anti-inflammatory properties showed that C. celatum downregulated the gene expression of cytokine inflammation markers IL-6, IL-1, and iNOS while upregulating TGF-β expression. In summary all 3 bacterial species showed good probiotic potential from the in vitro tests. Particularly the formation of resistant spores that later germinated to vegetative cells that produced molecular patterns with antioxidant and anti-inflammatory properties. This necessitates further studies on their probiotic properties.Item Methane Biogeochemistry and Microbial Communities in Natural and Restored Freshwater Depressional Wetlands(2024) Hamovit, Nora David; Yarwood, Stephanie A; Behavior, Ecology, Evolution and Systematics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Wetlands are the largest natural source of methane (CH4), a potent greenhouse gas. Wetland CH4 emissions are dependent on rates of microbial CH4 production (methanogenesis) and consumption (methanotrophy). These processes vary spatially and temporally with environmental conditions, edaphic characteristics, and microbial community structure, making it difficult to predict wetland CH4 emissions. This high variability can be further pronounced in restored wetlands that have undergone environmental and edaphic disturbances. The following work aims to understand this variability by assessing patterns of methanogenesis and methanotrophy, and their associated microbial communities, across natural and restored freshwater depressional wetlands on the Delmarva Peninsula (USA). Sites addressed in this work were restored from agricultural land between 1986 and 2004 through multiple programs funded by the United States Department of Agriculture (USDA). In the first set of experiments, we identified a high abundance of active acetoclastic methanogens in intact core incubations from a restored wetland suggesting a higher potential for methanogenesis in situ compared to the natural wetland assessed. The co-occurrence of active methanogens and Fe-reducing bacteria in these restored wetland cores contradicted the hypothesis that loss of competition may allow methanogens to be the primary users of acetate. Following assessments across vegetative-hydrologic zones in a series of restored wetlands of varying ages, and their natural counterparts, highlighted vegetation type and extent as a driver of methanogen community abundance, composition, and activity. In turn, restored wetlands showed elevated potentials rates of methanogenesis compared to natural sites. Potential rates of methanotrophy (aerobic and anaerobic), however, were also elevated in restored wetlands, which could constrain CH4 emissions in situ. Variability of environmental conditions (ie. hydrology and vegetation) and edaphic measures (ie. soil organic matter (SOM)) across all sites sampled are reflected in distinct microbial community composition and CH4 biogeochemistry. Clear patterns of SOC accumulation and CH4 biogeochemistry with restoration age were not observed for these wetlands, and variability in environmental conditions and edaphic measures across the sites (restored and natural), emphasize the need for continued monitoring and maintenance of the wetlands. Our results suggest efforts to manage herbaceous vegetation extent and maintain regular seasonal hydrology in future restorations may help prevent high potentials for CH4 production, and thus emissions.Item DATA-DRIVEN ALGORITHMS FOR CHARACTERIZING STRUCTURAL VARIATION IN METAGENOMIC DATA(2024) Muralidharan, Harihara Subrahmaniam; Pop, Mihai; Computer Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Sequence differences between the strains of bacteria comprising host-associated and environmental microbiota may play a role in community assembly and influence the resilience of microbial communities to disturbances. Tools for characterizing strain-level variation within microbial communities, however, are limited in scope, focusing on just single nucleotide poly-morphisms, or relying on reference-based analyses that miss complex structural variants. In this thesis, we describe data-driven methods to characterize variation in metagenomic data. In the first part of the thesis, I present our analysis of the structural variants identified from metagenomic whole genome shotgun sequencing data. I begin by describing the power of assembly graph analysis to detect over 9 million structural variants such as insertion/deletion, repeat copy-number changes, and mobile elements, in nearly 1,000 metagenomes generated as a part of the Human Microbiome Project. Next, I describe Binnacle, which is a structural variant aware binning algorithm. To improve upon the fragmented nature of assemblies, metagenomic binning is performed to cluster contigs that is likely to have originated from the same genome. We show that binning “graph-based” scaffolds, rather than contigs, improves the quality of the bins, and captures a broader set of the genes of the genomes being reconstructed. Finally, we present a case study of the microbial mats from the Yellowstone National Park. The cyanobacterium Synechococcus is abundant in these mats along a stable temperature gradient from ∼ 50oC to ∼ 65oC and plays a key role in fixing carbon and nitrogen. Previous studies have isolated and generated good quality reference sequences of two major Synechococcus spp. that share a very high genomic content; OS-A and OS-B’. Despite the high abundance of the Synechococcus spp., metagenomic assembly of these organisms is challenging due to the large number of rearrangements between them. We explore the genomic diversity of the Synechococcus spp. using a reference genome, reliant assembly and scaffolding. We also highlight that the variants we detect can be used to fingerprint the local biogeography of the hot spring. In the second part of the thesis, I present our analysis of amplicon sequencing data, specifically the 16S rRNA gene sequences. I begin by describing SCRAPT (Sample, Cluster, Recruit, AdaPt and iTerate), which is a fast iterative algorithm for clustering large 16S rRNA gene datasets. We also show that SCRAPT is able to produce operational taxonomic units that are less fragmented than popular tools: UCLUST, CD-HIT and DNACLUST and runs orders of magnitude faster than existing methods. Finally, we study the impact of transitive annotation on taxonomic classifiers. Taxonomic labels are assigned using machine learning algorithms trained to recognize individual taxonomic groups based on training sequences with known taxonomic labels. Ideally, the training data should rely on experimentally verified-formal taxonomic labels however, the labels associated with sequences in biological databases are most commonly the result of computational predictions– “transitive annotation.” We demonstrate that even a few computationally-generated training data points can significantly skew the output of the classifier to the point where entire regions of the taxonomic space can be disturbed. We also discuss key factors that affect the resilience of classifiers to transitively annotated training data and propose best practices to avoid the artifacts described in this thesis.Item HISTATIN 5 MODIFICATIONS IMPACT PROTEOLYTIC STABILITY IN THE PRESENCE OF FUNGAL AND SALIVARY PROTEASES(2024) Makambi, Wright Kingi; Karlsson, Amy J; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Candida albicans, found in the oral cavities of 30-50% of the global population, can lead to oral candidiasis, particularly in immunocompromised individuals like those with HIV or diabetes. The current treatments, small-molecule antifungals, often fall short due to drug resistance and toxicity. To address these challenges, histatin 5 (Hst5), a 24-amino-acid peptide naturally present in human saliva, has been studied as a potential antifungal therapy. Hst5, however, is susceptible to degradation by secreted aspartyl proteases (Saps) produced by C. albicans and salivary enzymes, limiting its potential efficacy as a therapeutic. We have engineered Hst5 variants utilizing rational design in order to understand the interactions with Saps and Saliva. We have also made advancements in developing a novel screening method utilizing the directed evolution technique yeast surface display. Our study employed rational design to modify Hst5, at its lysine residues (K5, K11, K13, and K17), substituting them with leucine or arginine to examine their influence on interactions with Saps (Sap1, Sap2, Sap3, Sap5, Sap6, Sap9, and Sap10). Sap5, Sap6, and Sap10 did not degrade Hst5 at the tested conditions, while Sap1, Sap2, Sap3, and Sap9 did. Some modifications, such as K13L, are particularly susceptible to proteolysis by Sap1, Sap2, Sap3, and Sap9. In contrast, K17L substantially increases the stability and antifungal activity of Hst5 in the presence of Saps. Additionally, although the K11RK17L variant was degraded more than the K17L variant, their antifungal activities were largely similar. The proteolysis products of were also identified by mass spectrometry identifying the [4-24], [1-17], and [14-24] Sap proteolysis products. We also evaluated the proteolytic stability of these variants in saliva. Both K17L and K5R showed improved stability; however, the enhancements were modest, suggesting that further engineering is required to achieve significant improvements. Further experiments evaluated how additional amino acid substitutions at K13 and K17 affect the peptide’s proteolytic stability in the presence of Saps (with and without zinc). Our findings suggest that the positive charge at K13 is important for the proteolytic stability of Hst5, as all other variants tested except K13R reduce overall proteolytic stability. Furthermore, many substitutions at K17, including tryptophan, significantly enhance proteolytic resistance and antifungal activity following incubation with Saps. The K17W variant showed improved stability and antifungal efficacy, maintaining its function even in the presence of zinc and exhibiting stronger antibiofilm activity than the parent Hst5. In addition to the rational design work, we have advanced the development of a directed evolution yeast surface display platform for screening peptides for proteolytic stability. This would allow for the expression of large peptide libraries on the surface of Saccharomyces cerevisiae. Through optimization of expression and display conditions, we determined an induction media at 30°C with a pH of 3.5 and devoid of glucose improved the expression and display of Hst5 peptides on the surface of S. cerevisiae. We also optimized the degradation conditions for Sap2 37°C, a pH not exceeding 7.4, and a Sap2 concentration of 0.78 µg/mL led to the best discrepancy between proteolytically stable variants. Additionally, we found that a 40 amino acid linker between the peptide and the yeast surface provided the best observing proteolytic degradation. Using the optimized system, we showed that yeast surface display can be used to discriminate between peptide variants with different levels of proteolytic stability. This lays the foundation for future work to screen large libraries of peptides for proteolytic stability. From these results, we have gained a deeper understanding of the interactions between Hst5 and Saps, showing that modification at different lysine residues greatly impacts the proteolytic stability of Hst5. Furthermore, we have shown that the yeast surface display platform can be used to screen the proteolytic stability of peptides. Looking forward, this peptide should be engineered for proteolytic stability in saliva. Furthermore, mock screens should be made before screening a library of peptides using the yeast surface display platform.Item MICROBIAL COMMUNITIES IN COASTAL ECOSYSTEMS(2024) Kim, Carol; Malkin, Sairah Y; Marine-Estuarine-Environmental Sciences; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Firstly, I examined microbial community succession along a chronosequence of constructed salt marshes using the Poplar Island restoration project site as a case study. By comparing 16S rRNA gene amplicon sequences across 6 constructed low marshes spanning a chronosequence of 1-16 years at Poplar Island (Chesapeake Bay) and a nearby natural reference marsh, I found strong evidence that the development of soil microbial communities is on a trajectory towards natural marsh conditions following marsh restoration with successional rates within timescales expected for soil development. Results from this study showed the value of microbial communities to serve as effective bioindicators for monitoring the recovery of microbially mediated biogeochemical processes in restored or newly constructed salt marshes, as well as potentially for assessing the marsh inundation period and by extension marsh health and resiliency. Secondly, I conducted a manipulation experiment to explore microbial communities associated with cable bacteria using RNA stable isotope probing (RNA-SIP). I traced the uptake of isotopically labeled bicarbonate and acetate in sediments with baseline and with stimulated cable bacteria activity, to test the hypothesis that cable bacteria activity can stimulate chemoautotrophic bacteria in anaerobic sediments. I used 16S rRNA sequencing to identify the active “incorporators” of bicarbonate (as a tracer of chemoautotrophy) and acetate (as a tracer of heterotrophy). I found that estuarine cable bacteria activity stimulated the chemoautotrophic activity of Gammaproteobacteria (Nitrosomonas, Thioalkalispira-Sulfurivermis) and Campylobacterota (Sulfurovum, Sulfurimonas) at anaerobic depths. This result is not explainable with conventional understanding of chemoautotrophic activity. Rather, this study contributes to the emerging concept that cable bacteria activity stimulates metabolic activities at suboxic sediment depths, potentially by serving as an electron sink for other microbes. Furthermore, I found that heterotrophic activity, measured as 13C-acetate assimilation into RNA, was stimulated amongst known chemoautotrophic sulfur oxidizers at depth, highlighting that metabolic flexibility, and specifically mixotrophy, may be widespread in complex natural sediment environments. Lastly, I characterized the composition and metabolic potential of microbial communities in estuarine sediment enriched with cable bacteria. By using metagenomic and 16S rRNA sequencing, I constructed 23 medium- to high-quality metagenome-assembled genomes (MAGs) that span across 9 phyla. I retrieved MAGs exhibiting mixotrophy and a range of capabilities for extracellular electron transport. This study revealed a diverse range of metabolically flexible communities of microbes that contribute to the biogeochemical cycling of carbon, nitrogen, and sulfur.Item EVOLUTION OF THE CRISPR IMMUNE SYSTEM FROM ECOLOGICAL TO MOLECULAR SCALES(2024) Xiao, Wei; Johnson, Philip LF; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Bacteria and archaea inhabit environments that constantly face viral infections and other external genetic threats. They have evolved an arsenal of defense strategies to protect themselves. My research delves into the CRISPR immune system, the only known adaptive immune system of prokaryotes. My work explores three different dimensions of the CRISPR immune system, ranging from ecological to molecular scales.From an evolutionary perspective, CRISPR is widely distributed across the prokaryotic tree, underscoring its immune effectiveness. However, the CRISPR distribution is uneven and some lineages are devoid of CRISPR. Here, I identify two ecological drivers of the CRISPR immune system. By analyzing both 16S rRNA data and metagenomic data, I find the CRISPR system is favored in less abundant prokaryotes in the saltwater environment and higher diverse prokaryote communities in the human oral environment. On the molecular level, the CRISPR system selects and cleaves its “favorite” DNA segments (also known as “spacers”) from invading viral genomes to form immune memories. I explore how the spacer sequence composition affects its acquisition rate by the CRISPR system. I develop a convolutional neural network model to predict the spacer acquisition rate based on the spacer sequence composition in natural microbial communities. The model interpretation reveals that the PAM-proximal end of the spacer is more important in predicting the spacer abundance, which is consistent with previous findings from controlled experimental studies. Combining these scales, CRISPR repeat sequences coevolve with the rest of the genome. Thus, I explore the potential of utilizing CRISPR repeat sequences for taxonomy profiling. I find a strong relationship between unique repeat sequences and taxonomy in both the RefSeq database and a human metagenomic dataset. Then I show high accuracy when utilizing repeat sequences in taxonomy annotation of human metagenomic contigs. This novel method not only aids in annotating CRISPR arrays but also introduces a novel tool for metagenomic sequence annotation.