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- Creators: Arizona State University
- Creators: Haydel, Shelley
Description
The purpose behind this research was to identify unknown transport proteins involved in lactate export. Lactate bioproduction is an environmentally beneficial alternative to petroleum-based plastic production as it produces less toxic waste byproduct and can rely on microbial degradation of otherwise wasted biomass. Coupled with appropriate product refinement, industrial microbial producers can be genetically engineered to generate quantities of bioplastic approaching 400 million metric tons each year. However, this process is not entirely suitable for large investment, as the fermentative bottlenecks, including product export and homeostasis control, limit production metrics. Previous studies have based their efforts on enhancing cellular machinery, but there remain uncharacterized membrane proteins involved in product export yet to be determined. It has been seen that deletion of known lactate transporters in Escherichia coli resulted in a decrease in lactate production, unlike the expected inhibition of export. This indicates that there exist membrane proteins with the ability to export lactate which may have another similar substrate it primarily transports.To identify these proteins, I constructed a genomic library of all genes in an engineered lactate producing E. coli strain, with known transporter genes deleted, and systematically screened for potential lactate transporter proteins. Plasmids and their isolated proteins were compared utilizing anaerobic plating to identify genes through sanger sequencing. With this method, I identified two proteins, yiaN and ybhL-ybhM, which did not show any significant improvement in lactate production when tested. Attempts were made to improve library diversity, resulting in isopropyl-β-D-1-thiogalactopyranoside induction as a likely factor for increased expression of potential fermentation-associated proteins. A genomic library from Lactobacillus plantarum was constructed and screened for transport proteins which could improve lactate production. Results showed that isolated plasmids contained no notable inserts, indicating that the initial transformation limited diversity. Lastly, I compared the results from genomic screening with overexpression of target transporter genes by computational substrate similarity search. Induced expression of ttdT, citT and dcuA together significantly increased lactate export and thus production metrics as well as cell growth. These positive results indicate an effective means of determining substrate promiscuity in membrane proteins with similar organic acid transport capacity.
ContributorsLee-Kin, Jared (Author) / Wang, Xuan (Thesis advisor) / Nielsen, David (Committee member) / Varman, Arul (Committee member) / Arizona State University (Publisher)
Created2022
Description
The biological carbon pump in the ocean is initiated by the photosynthetic fixation of atmospheric carbon dioxide into particulate or dissolved organic carbon by phytoplankton. A fraction of this organic matter sinks to depth mainly in the form of microaggregates (5-60 μm) and visible macroaggregates. These aggregates are composed of cells, minerals, and other sources of organic carbon. Exopolymeric substances (EPS) are exudated by heterotrophic bacteria and phytoplankton and may form transparent exopolymeric particles (TEP) that act as a glue-like matrix for marine aggregates. Heterotrophic bacteria have been found to influence the aggregation of phytoplankton and in some cases result in an increase in TEP production, but it is unclear if marine heterotrophic bacteria can produce TEP and how they contribute to aggregation. Pseudoalteromonas carrageenovora, Vibrio thalassae, and Marinobacter adhaerens HP15 are heterotrophic marine bacteria that were found associated with sinking particles in an oligotrophic gyre station in the subtropical North Atlantic. These bacteria were grown in axenic cultures to determine growth, TEP production, and aggregation. They were also inoculated into roller tanks used to simulate open ocean conditions to determine their ability to form macroaggregates. Treatments with added kaolinite clay simulated aeolic dust input from the Sahara. M. adhaerens HP15 had the highest TEP concentration but the lowest cell-normalized TEP production at all growth stages compared to the other bacteria. Additionally, M. adhaerens HP15 also had the lowest microaggregate formation. The cell-normalized TEP production and microaggregate formation was not significantly different between P. carrageenovora and V. thalassae. All bacteria formed visible macroaggregates in roller tanks with clay addition and exhibited high sinking velocities (150-1200 m d-1) that are comparable to those of aggregates formed by large mineral ballasted phytoplankton. Microaggregates in the clay treatments declined during incubation, indicating that they aggregated to form the macroaggregates. The findings from this study show for the first time that heterotrophic bacteria can contribute to aggregation and the export of organic carbon to depth in the ocean.
ContributorsLivar, Britni (Author) / Neuer, Susanne (Thesis advisor) / Hartnett, Hilairy (Committee member) / Cadillo-Quiroz, Hinsby (Committee member) / Arizona State University (Publisher)
Created2022
Description
Megapolitan cities have emerged due to unprecedented urban migration. These changes strain urban resources, especially water distribution and treatment systems. The recent rise of Legionella cases linked to water distribution systems highlights this issue.Bacterial growth and biofilm formation are influenced by factors, such as type and concentration of residual disinfectant, pipe material, water temperature. Experiments were conducted in identical model water distribution systems (WDSs) constructed of three different pipe materials: galvanized steel, copper, and cross-linked polyethylene (PEX) operated under a continuous flow rate of 15 L/min. Each model WDS includes 11 steel coupons screwed to the water distribution pipes. City of Tempe (Arizona) municipal water was used in the experimentation, with no nutrients added. Following biofilm growth, coupons were removed and processed by scrubbing biofilm into
phosphate-buffered saline (PBS). Reasoner's 2A (R2A), Trypticase Soy Agar (TSA), Brilliant, and buffered charcoal yeast extract (BCYE) agar media were used to examine biofilm samples for heterotrophic plate counts (HPC), metabolically active bacteria, E coli,
and Legionella. Simultaneously, water samples from the reservoirs of model WDSs were also collected and examined for the same bacteria.Next, an electrochlorination cell maintained free chlorine residuals in unheated PEX and copper model WDSs. These two systems maintained free chlorine residuals for one week and evaluated biofilm and bacterial kinetics. Higher water temperature increased biofilm development. Bacterial counts in biofilms were higher on new (fresh) coupons compared to the old coupons. Heterotrophic and metabolically active bacteria behaved similarly. Only control and heating systems in copper water reservoirs have Legionella spp. Biofilms
formed less on copper systems than steel and PEX systems. Initially, PEX had more HPC than copper. After electrochlorination, HPC concentration in the PEX system rapidly declined to non-detect, whereas in the copper system dropped to 0.54 log CFU/mL.
Thus, higher temperature increases biofilm growth on all pipe materials and reservoirs bacterial concentration. Electrochlorination is a potential biofilm and microbial disinfection method. This thesis topic investigated how these parameters affect the model distribution system bacterial populations and biofilm growth.
ContributorsKolahi Kouchaki, Bita (Author) / Abbaszadegan, Morteza (Thesis advisor) / Alum, Absar (Committee member) / Perreault, Francois (Committee member) / Arizona State University (Publisher)
Created2022
Description
Embryonic and juvenile development consist of a series of complex and rapid changes driven by a suite of crucially timed developmental cues within the cell. The developmental process begins at the moment of zygote activation, “jump-started” by maternal factors such as mRNA and proteins until transcription can be zygotically-driven. Regulation of transcription initiation plays a crucial role in this process, as minute changes in the timing, density, and characteristics of gene expression can have drastic effects on the zygote’s development. Specific promoter elements can be linked to different patterns of transcription, driving both ubiquitous and sharply regulated gene expression, thus forming the basis for the time-sensitive developmental processes. In order to better understand the genes expressed during different stages of development and the impact of promoter elements on transcription patterns and transcript concentrations within the cell, I created a Gene Expression and Promoter Atlas in two species within the cryptic species complex, Daphnia pulex. I surveyed five embryonic and two juvenile developmental stages in both a North American and mitochondrially European Daphnia pulex utilizing developmental landmarks to visually stages embryos. A total of 17,993 genes were identified in the European species and 15,295 were identified in the North American species, with 11,551 orthologs identified between the two. I utilized the transcription start site (TSS) profiling method STRIPE-seq to identify promoter motifs and RNA-seq to survey mRNA concentration at each stage, generating a wealth of genetic data. The methodology for library construction and the dataset generated therein provide an informative basis for further comparative developmental studies and the elucidation of full gene functionality in an emerging model organism.
ContributorsWalls, Sarah (Author) / Lynch, Michael (Thesis advisor) / Raborn, R. Taylor (Committee member) / Mangoni, Marco (Committee member) / Harris, Robin (Committee member) / Arizona State University (Publisher)
Created2022
Description
Electroactive bacteria connect biology to electricity, acting as livingelectrochemical catalysts. In nature, these bacteria can respire insoluble compounds like
iron oxides, and in the laboratory, they are able to respire an electrode and produce an
electrical current. This document investigates two of these electroactive bacteria:
Geobacter sulfurreducens and Thermincola ferriacetica. G. sulfurreducens is a Gramnegative iron-reducing soil bacterium, and T. ferriacetica is a thermophilic, Grampositive bacterium that can reduce iron minerals and several other electron acceptors.
Respiring insoluble electron acceptors like metal oxides presents challenges to a
bacterium. The organism must extend its electron transport chain from the inner
membrane outside the cell and across a significant distance to the surface of the electron
acceptor. G. sulfurreducens is one of the most-studied electroactive bacteria, and despite
this there are many gaps in knowledge about its mechanisms for transporting electrons
extracellularly. Research in this area is complicated by the presence of multiple pathways
that may be concurrently expressed. I used cyclic voltammetry to determine which
pathways are present in electroactive biofilms of G. sulfurreducens grown under different
conditions and correlated this information with gene expression data from the same
conditions. This correlation presented several genes that may be components of specific
pathways not just at the inner membrane but along the entire respiratory pathway, and I
propose an updated model of the pathways in this organism. I also characterized the
composition of G. sulfurreducens and found that it has high iron and lipid content
independent of growth condition, and the high iron content is explained by the large
abundance of multiheme cytochrome expression that I observed. I used multiple
microscopy techniques to examine extracellular respiration in G. sulfurreducens, and in
the process discovered a novel organelle: the intracytoplasmic membrane. I show 3D
reconstructions of the organelle in G. sulfurreducens and discuss its implications for the
cell’s metabolism. Finally, I discuss gene expression in T. ferriacetica in RNA samples
collected from an anode-respiring culture and highlight the most abundantly expressed
genes related to anode-respiring metabolism.
ContributorsHowley, Ethan Thomas (Author) / Torres, César I (Thesis advisor) / Krajmalnik-Brown, Rosa (Thesis advisor) / Nannenga, Brent (Committee member) / Arizona State University (Publisher)
Created2022
Description
The microorganisms that colonize the gastrointestinal tract have been recognized over the last several decades to have a significant bearing on the health trajectories of the hosts that harbor them. The collection of these gut microbes display links with acute and chronic disease, garnering substantial interest in leveraging the microbiome for improved health states. How these microbes assemble as a complex community and interact with each other, and the host depends on a multitude of factors. In adulthood, diet is one of the main moderators, having a significant influence on community composition and the functional output captured in the metabolites produced and/or modified by the gut microbiome. Thus, the assembly of microbes in the gut are tightly intertwined with health. In this dissertation, I examine the impact of diet and feeding behaviors on the gut microbiome and what features may be grounding or responsive under such pressures. Specifically, I first explore the avian gut microbiome as a barometer of nutritional and environmental influence on host health. Birds have continually displayed robust physiology under dietary pressures, placing them in an important, though underutilized, position within the translational science framework. Second, I describe the association of food insecurity on gut microbiome and metabolome profiles in a diverse college-based sample. Food insecurity provides its own set of unique pressures, such as unintentional calorie restriction, and inconsistent dietary intake and access to healthy food options. Third, I examine the effect of a one vs. two-consecutive days of intermittent fasting on the gut microbiome, the plasma metabolome, and associated clinical outcomes in overweight and obese adults. Growing in scientific and lay popularity, dietary fasting has been noted to induce changes in the diversity of gut microflora and gut motility, though different fasting lengths have not been assessed in the context of the human microbiome. Overall, this collection of work underscores that the community of microbes in the gut are individualized, resilient, and baseline composition and functioning are germane to how an individual may react to a particular dietary intervention.
ContributorsMohr, Alex (Author) / Sweazea, Karen L. (Thesis advisor) / Johnston, Carol S. (Committee member) / Sears, Dorothy D. (Committee member) / Whisner, Corrie M. (Committee member) / Krajmalnik-Brown, Rosa (Committee member) / Arizona State University (Publisher)
Created2022
Description
Mycobacterial infections, as represented by leprosy and tuberculosis, have persisted as human pathogens for millennia. Their environmental counterparts, nontuberculous mycobacteria (NTM), are commodious infectious agents endowed with extensive innate and acquired antimicrobial resistance. The current drug development process selects for antibiotics with high specificity for definitive targets within bacterial metabolic and replication pathways. Because these compounds demonstrate limited efficacy against mycobacteria, novel antimycobacterial agents with unconventional mechanisms of action were identified. Two highly resistant NTMs, Mycobacterium abscessus (Mabs) a rapid-growing respiratory, skin, and soft tissue pathogen, and Mycobacterium ulcerans (MU), the causative agent of Buruli ulcer, were selected as targets. Compounds that indicated antimicrobial activity against other highly resistant pathogens were selected for initial screening. Antimicrobial peptides (AMPs) have demonstrated activity against a variety of bacterial pathogens, including mycobacterial species. Designed antimicrobial peptides (dAMPs), rationally-designed and synthetic contingents, combine iterative features of natural AMPs to achieve superior antimicrobial activity in resistant pathogens. Initial screening identified two dAMPs, RP554 and RP557, with bactericidal activity against Mabs. Clay-associated ions have previously demonstrated bactericidal activity against MU. Synthetic and customizable aluminosilicates have also demonstrated adsorption of bacterial cells and toxins. On this basis, two aluminosilicate materials, geopolymers (GP) and ion-exchange nanozeolites (IE-nZeos), were screened for antimicrobial activity against MU and its fast-growing relative, Mycobacterium marinum (Mmar). GPs demonstrated adsorption of MU cells and mycolactone, a secreted, lipophilic toxin, whereas Cu-nZeos and Ag-nZeos demonstrated antibacterial activity against MU and Mmar. Cumulatively, these results indicate that an integrative drug selection process may yield a new generation of antimycobacterial agents.
ContributorsDermody, Roslyn June (Author) / Haydel, Shelley E (Thesis advisor) / Bean, Heather (Committee member) / Nickerson, Cheryl (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2022
Description
For untargeted volatile metabolomics analyses, comprehensive two-dimensional gas chromatography (GC×GC) is a powerful tool for separating complex mixtures and can provide highly specific information about the chemical composition of a variety of samples. With respect to human disease, the application of GC×GC in untargeted metabolomics is contributing to the development of diagnostics for a range of diseases, most notably bacterial infections. Pseudomonas aeruginosa, in particular, is an important human pathogen, and for individuals with cystic fibrosis (CF), chronic P. aeruginosa lung infections significantly increase morbidity and mortality. Developing non-invasive tools that detect these infections earlier is critical for improving patient outcomes, and untargeted profiling of P. aeruginosa volatile metabolites could be leveraged to meet this challenge. The work presented in this dissertation serves as a case study of the application of GC×GC in this area.Using headspace solid-phase microextraction and time-of-flight mass spectrometry coupled with GC×GC (HS-SPME GC×GC-TOFMS), the volatile metabolomes of P. aeruginosa isolates from early and late chronic CF lung infections were characterized. Through this study, the size of the P. aeruginosa pan-volatilome was increased by almost 40%, and differences in the relative abundances of the volatile metabolites between early- and late-infection isolates were identified. These differences were also strongly associated with isolate phenotype. Subsequent analyses sought to connect these metabolome-phenome trends to the genome by profiling the volatile metabolomes of P. aeruginosa strains harboring mutations in genes that are important for regulating chronic infection phenotypes. Subsets of volatile metabolites that accurately distinguish between wild-type and mutant strains were identified. Together, these results highlight the utility of GC×GC in the search for prognostic volatile biomarkers for P. aeruginosa CF lung infections.
Finally, the complex data sets acquired from untargeted GC×GC studies pose major challenges in downstream statistical analysis. Missing data, in particular, severely limits even the most robust statistical tools and must be remediated, commonly through imputation. A comparison of imputation strategies showed that algorithmic approaches such as Random Forest have superior performance over simpler methods, and imputing within replicate samples reinforces volatile metabolite reproducibility.
ContributorsDavis, Trenton James (Author) / Bean, Heather D (Thesis advisor) / Haydel, Shelley E (Committee member) / Lake, Douglas F (Committee member) / Runger, George C (Committee member) / Arizona State University (Publisher)
Created2022
Description
Sinking particles are important conduits of organic carbon from the euphotic zone to the deep ocean and microhabitats for diverse microbial communities, but little is known about what determines their origin and community composition. Events in the northwestern Sargasso Sea, such as winter convective mixing, summer stratification, and mesoscale (10–100 km) eddies, characteristic features of this region, affect the vertical and temporal composition and abundance of pelagic and particle-attached microorganisms. To assess the connections of the microbial communities between the euphotic zone and sinking particles, I carried out indicator and differential abundance analyses of prokaryotes and photoautotrophs based on the V4-V5 amplicons of the 16S rDNA from samples collected in the Sargasso Sea during the spring and summer of 2012. I found that gammaproteobacteria such as Pseudoalteromonas sp. and Vibrio sp., common particle-associated bacteria often linked with zooplankton, dominated the sequence libraries of the sinking particles. The analysis also revealed that members of Flavobacteria, particularly the fish pathogen Tenacibaculum sp., as well as Chloropicon sp. and Chloroparvula sp., among the smallest known green algae, were indicators taxa of sinking particles. The cryptophyte Teleaulax and the diatom Chaetoceros were overrepresented in the particle communities during both seasons. Interestingly, I also found that the large centric diatom, Rhizosolenia sp., generally rare in the oligotrophic Sargasso Sea, dominated photoautotrophic communities of sinking particles collected in the center of an anticyclonic eddy with unusual upwelling due to eddy-wind interactions. I hypothesize that the steady contribution by picophytoplankton to particle flux is punctuated by pulses of production and flux of larger-sized phytoplankton in response to episodic eddy upwelling events and can lead to higher export of particulate organic matter during the summer.
ContributorsFontánez Ortiz, Marc Alec (Author) / Neuer, Susanne (Thesis advisor) / Zhu, Qiyun (Committee member) / Trembath-Reichert, Elizabeth (Committee member) / Arizona State University (Publisher)
Created2022
Description
Lophomonas is a genus of flagellated parabasalids that exist as commensal symbionts in the hindguts of a variety of pest cockroaches. The genus contains two species: Lophomonas blattarum and Lophomonas striata. The two species differ by way of bacterial ectosymbionts that attach to the outside of L. striata, giving rise to a striated and spindle-shaped appearance. As the attachment of bacterial symbionts prohibits L. striata from taking up large food particles in the same manner as L. blattarum, it is likely the two species differ in which metabolic genes they possess. Here, a comparison of transcriptomes between the two Lophomonas species show slight differences between the species. Metagenomic analysis of L. striata also presents the possibility of L. striata ectosymbionts as belonging to the genus Parabacteroides.
ContributorsNguyen, Leann (Author) / Gile, Gillian (Thesis advisor) / Dada, Nsa (Committee member) / Wideman, Jeremy (Committee member) / Arizona State University (Publisher)
Created2023