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- Creators: Arizona State University
- Creators: Haydel, Shelley
Description
Phytoplankton comprise the base of the marine food web, and, along with heterotrophic protists, they are key players in the biological pump that transports carbon from the surface to the deep ocean. In the world's subtropical oligotrophic gyres, plankton communities exhibit strong seasonality. Winter storms vent deep water into the euphotic zone, triggering a surge in primary productivity in the form of a spring phytoplankton bloom. Although the hydrographic trends of this "boom and bust" cycle have been well studied for decades, community composition and its seasonal and annual variability remains an integral subject of research. It is hypothesized here that proportions of different phytoplankton and protistan taxa vary dramatically between seasons and years, and that picoplankton represent an important component of this community and contributor to carbon in the surface ocean. Monthly samples from the Bermuda Atlantic Time-series Study (BATS) site were analyzed by epifluorescence microscopy, which permits classification by morphology, size, and trophic type. Epifluorescence counts were supplemented with flow cytometric quantification of Synechococcus, Prochlorococcus, and autotrophic pico- and nanoeukaryotes. Results from this study indicate Synechococcus and Prochlorococcus, prymnesiophytes, and hetero- and mixotrophic nano- and dinoflagellates were the major players in the BATS region plankton community. Ciliates, cryptophytes, diatoms, unidentified phototrophs, and other taxa represented rarer groups. Both flow cytometry and epifluorescence microscopy revealed Synechococcus to be most prevalent during the spring bloom. Prymnesiophytes likewise displayed distinct seasonality, with the highest concentrations again being noted during the bloom. Heterotrophic nano- and dinoflagellates, however, were most common in fall and winter. Mixotrophic dinoflagellates, while less abundant than their heterotrophic counterparts, displayed similar seasonality. A key finding of this study was the interannual variability revealed between the two years. While most taxa were more abundant in the first year, prymnesiophytes experienced much greater abundance in the second year bloom. Analyses of integrated carbon revealed further stark contrasts between the two years, both in terms of total carbon and the contributions of different groups. Total integrated carbon varied widely in the first study year but displayed less fluctuation after June 2009, and values were noticeably reduced in the second year.
ContributorsHansen, Amy (Author) / Neuer, Susanne (Thesis advisor) / Krajmalnik-Brown, Rosa (Committee member) / Sommerfeld, Milton (Committee member) / Arizona State University (Publisher)
Created2010
Description
Pseudomonas aeruginosa is an opportunistic bacterial pathogen commonly associated with increased morbidity and mortality in cystic fibrosis (CF) patients. To adapt to the CF lung environment, P. aeruginosa undergoes multiple genetic changes as it moves from an acute to a chronic infection. The resultant phenotypes have been associated with chronic infection and can provide important information to track the patient’s individualized disease progression. This study examines the link between the accumulation of QS genetic mutations and phenotypic expression in P. aeruginosa laboratory strains and clinical isolates. We utilized several plate-based and colorimetric assays to quantify the production of pyocyanin, rhamnolipids, and protease from paired clinical early- and late-stage chronic infection isolates across 16 patients. Exoproduct production of each isolate was compared to the mean production of pooled isolates to classify high producing (QS-sufficient) and low producing (QS-deficient) isolates. We found that over time P. aeruginosa isolates exhibit a reduction in QS-related phenotypes during chronic infections. Future research of the QS regulatory networks will identify whether reversion of genotype will result in corresponding phenotypic changes in QS-deficient chronic infection isolates.
ContributorsKaranjia, Ava Vispi (Author) / Bean, Heather (Thesis director) / Haydel, Shelley (Committee member) / Davis, Trenton (Committee member) / School of Life Sciences (Contributor) / Chemical Engineering Program (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Cystic Fibrosis (CF) is a genetic disorder that disrupts the hydration of mucous of the lungs, which promotes opportunistic bacterial infections that begin in the affected person’s childhood, and persist into adulthood. One of the bacteria that infect the CF lung is Pseudomonas aeruginosa. This gram-negative bacterium is acquired from the environment of the CF lung, changing the expression of phenotypes over the course of the infection. As P. aeruginosa infections become chronic, some phenotype changes are known to be linked with negative patient outcomes. An important exoproduct phenotype is rhamnolipid production, which is a glycolipid that P. aeruginosa produces as a surfactant for surface-mediated travel. Over time, the expression of this phenotype decreases in expression in the CF lung.
The objective of this investigation is to evaluate how environmental changes that are related to the growth environment in the CF lung alters rhamnolipid production. Thirty-five P. aeruginosa isolates from Dartmouth College and Seattle Children’s Hospital were selected to observe the impact of temperature, presence of Staphylococcus aureus metabolites, and oxygen availability on rhamnolipid production. It was found that the rhamnolipid production significantly decreased for 30C versus 37C, but not at 40C. The addition of S. aureus spent media, in any of the tested conditions, did not influence rhamnolipid production. Finally, the change in oxygen concentration from normoxia to hypoxia significantly reduced rhamnolipid production. These results were compared to swarming assay data to understand how changes in rhamnolipid production impact surface-mediated motility.
The objective of this investigation is to evaluate how environmental changes that are related to the growth environment in the CF lung alters rhamnolipid production. Thirty-five P. aeruginosa isolates from Dartmouth College and Seattle Children’s Hospital were selected to observe the impact of temperature, presence of Staphylococcus aureus metabolites, and oxygen availability on rhamnolipid production. It was found that the rhamnolipid production significantly decreased for 30C versus 37C, but not at 40C. The addition of S. aureus spent media, in any of the tested conditions, did not influence rhamnolipid production. Finally, the change in oxygen concentration from normoxia to hypoxia significantly reduced rhamnolipid production. These results were compared to swarming assay data to understand how changes in rhamnolipid production impact surface-mediated motility.
ContributorsKiermayr, Jonathan Patrick (Author) / Bean, Heather (Thesis director) / Misra, Rajeev (Committee member) / Haydel, Shelley (Committee member) / School of International Letters and Cultures (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
The prrAB two-component system has been shown to be essential for viability in Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. To study this system, several prrAB mutants of Mycobacterium smegmatis, a close relative of Mtb, were created for study. These mutants included a deletion mutant complemented with prrA from Mtb controlled by Pmyc1_tetO, a deletion mutant, and a deletion mutant complemented with prrAB from M. smegmatis controlled by the native prrAB promoter sequence (~167 bp upstream sequence of prrAB). In a previous study, the prrAB deletion mutant clumped excessively relative to the wild-type strain when cultured in a nitrogen-limited medium. To address this irregularity, the lipid profiles of these mutants were analyzed through several experimental methods. Untargeted lipidomic profiles were analyzed by Electrospray Ionization Mass Spectrometry (ESI-MS). The ESI-MS data suggested the deletion mutant accumulates triacylglycerol species relative to the wild-type strain. This data was verified by thin-layer chromatography (TLC) and densitometry of the TLC images. The mycolic acid profile of each mutant was also analyzed by TLC but no noteworthy differences were found. High-throughput RNA-Seq analysis revealed several genes involved in lipid biosynthetic pathways upregulated in the prrAB deletion mutant, thus corroborating the ESI-MS and TLC data.
ContributorsOlson, Alexandra Nadine (Author) / Haydel, Shelley (Thesis director) / Bean, Heather (Committee member) / Maarsingh, Jason (Committee member) / School of Social Transformation (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
The spread of antibiotic resistant bacteria is currently a pressing global health concern, especially considering the prevalence of multi-drug resistance. Efflux pumps, bacterial machinery involved in various active transport functions, are capable of removing a broad range of antibiotics from the periplasmic space and the outer leaflet of the inner membrane, frequently conferring multi-drug resistance. Many aspects of efflux machinery’s structure, functions, and inter-protein interactions are still not fully understood; further characterization of these components of efflux will provide a strong foundation for combating this resistance mechanism. In this project, I further characterize the channel protein TolC as a part of the AcrAB-TolC efflux pump complex in Escherichia coli by first determining the specificity of compensatory mutations in TolC against defective AcrA and AcrB, and then identifying TolC residues that might influence TolC aperture dynamics or stability when altered. Specificity of compensatory mutations was determined using an array of TolC mutants, previously generated from defective AcrA or AcrB, against a different mutant AcrB protein; these new mutant combinations were then analyzed by real-time efflux and antibiotic susceptibility assays. A vancomycin susceptible TolC mutant—a phenotype that has been associated with constitutively open TolC channels—was then used to generate vancomycin-resistant revertants which were evaluated with DNA sequencing, protein quantification by Western blots, and real-time efflux assays to identify residues important for TolC aperture dynamics and protein stability and complex activity. Mutations identified in revertant strains corresponded to residues located in the lower half of the periplasmic domain of TolC; generally, these revertants had poorer efflux than wild-type TolC in the mutant AcrB background, and all revertants had poorer efflux activity than the parental mutant strain.
ContributorsMcFeely, Megan Elizabeth (Author) / Misra, Rajeev (Thesis director) / Haydel, Shelley (Committee member) / Stout, Valerie (Committee member) / School of Mathematical and Statistical Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
Globally, about two-thirds of the population is latently infected with herpes simplex virus type 1 (HSV-1). HSV-1 is a large double stranded DNA virus with a genome size of ~150kbp. Small defective genomes, which minimally contain an HSV-1 origin of replication and packaging signal, arise naturally via recombination during viral DNA replication. These small defective genomes can be mimicked by constructing a bacterial plasmid containing the HSV-1 origin of replication and packaging signal, transfecting these recombinant plasmids into mammalian cells, and infecting with a replicating helper virus. The absence of most viral genes in the amplicon vector allows large pieces of foreign DNA (up to 150kbp) to be incorporated. The HSV-1 amplicon is replicated and packaged by the helper virus to form HSV-1 particles containing the amplicon DNA. We constructed a novel HSV-1 amplicon vector system containing lambda phage-derived attR sites to facilitate insertion of transgenes by Invitrogen Gateway recombination. To demonstrate that the amplicon vectors work as expected, we packaged the vector constructs expressing Emerald GFP using the replication-competent helper viruses OK-14 or HSV-mScartlet-I-UL25 in Vero cells and demonstrate that the vector stock can subsequently transduce and express Emerald GFP. In further work, we will insert transgenes into the amplicon vector using Invitrogen Gateway recombination to study their functionality.
ContributorsVelarde, Kimberly (Author) / Hogue, Ian B (Thesis advisor) / Manfredsson, Fredric (Committee member) / Sandoval, Ivette (Committee member) / Varsani, Arvind (Committee member) / Arizona State University (Publisher)
Created2021
Description
The oceanic biological carbon pump is a key component of the global carbon cycle in which dissolved carbon dioxide is taken up by phytoplankton during photosynthesis, a fraction of which then sinks to depth and contributes to oceanic carbon storage. The small-celled phytoplankton (<5 µm) that dominate the phytoplankton community in oligotrophic oceans have traditionally been viewed as contributing little to export production due to their small size. However, recent studies have shown that the picocyanobacterium Synechococcus produces transparent exopolymer particles (TEP), the sticky matrix of marine aggregates, and forms abundant microaggregates (5-60 µm), which is enhanced under nutrient limited growth conditions. Whether other small phytoplankton species exude TEP and form microaggregates, and if these are enhanced under growth-limiting conditions remains to be investigated. This study aims to analyze how nutrient limitation affects TEP production and microaggregate formation of species that are found to be associated with sinking particles in the Sargasso Sea. The pico-cyanobacterium Prochlorococcus marinus (0.8 µm), the nano-diatom Minutocellus polymorphus (2 µm), and the pico-prasinophyte Ostreococcus lucimarinus (0.6 µm) were grown in axenic batch culture experiments under nutrient replete and limited conditions. It was hypothesized that phytoplankton subject to nutrient limitation will aggregate more than those under replete conditions due to an increased exudation of TEP and that Minutocellus would produce the most TEP and microaggregates while Prochlorococcus would produce the least TEP and microaggregates of the three phytoplankton groups. As hypothesized, nutrient limitation increased TEP concentration in all three species, however they were only significant in nitrogen-limited treatments of Prochlorococcus as well as nitrogen- and phosphorus-limited treatments of Minutocellus. Formation of microaggregates was significantly enhanced in Minutocellus and Ostreococcus cultures in distinct microaggregate size ranges. Minutocellus produced the most TEP per cell and aggregated at higher volume concentrations compared to Prochlorococcus and Ostreococcus. Surprisingly, Ostreococcus produced more TEP than Prochlorococcus and Minutocellus per unit cell volume. These findings show for the first time how nutrient limited conditions enhance TEP production and microaggregation of Prochlorococcus, Minutocellus, and Ostreococcus, providing a mechanism for their incorporation into larger, sinking particles and contribution to export production in oligotrophic oceans.
ContributorsShurtleff, Catrina (Author) / Neuer, Susanne (Thesis advisor) / Lomas, Michael W. (Committee member) / Garcia-Pichel, Ferran (Committee member) / Arizona State University (Publisher)
Created2022
Description
Under current climate conditions northern peatlands mostly act as C sinks; however, changes in climate and environmental conditions, can change the soil carbon decomposition cascade, thus altering the sink status. Here I studied one of the most abundant northern peatland types, poor fen, situated along a climate gradient from tundra (Daring Lake, Canada) to boreal forest (Lutose, Canada) to temperate broadleaf and mixed forest (Bog Lake, MN and Chicago Bog, NY) biomes to assess patterns of microbial abundance across the climate gradient. Principal component regression analysis of the microbial community and environmental variables determined that mean annual temperature (MAT) (r2=0.85), mean annual precipitation (MAP) (r2=0.88), and soil temperature (r2=0.77), were the top significant drivers of microbial community composition (p < 0.001). Niche breadth analysis revealed the relative abundance of Intrasporangiaceae, Methanobacteriaceae and Candidatus Methanoflorentaceae fam. nov. to increase when MAT and MAP decrease. The same analysis showed Spirochaetaceae, Methanosaetaceae and Methanoregulaceae to increase in relative abundance when MAP, soil temperature and MAT increased, respectively. These findings indicated that climate variables were the strongest predictors of microbial community composition and that certain taxa, especially methanogenic families demonstrate distinct patterns across the climate gradient. To evaluate microbial production of methanogenic substrates, I carried out High Resolution-DNA-Stable Isotope Probing (HR-DNA-SIP) to evaluate the active portion of the community’s intermediary ecosystem metabolic processes. HR-DNA-SIP revealed several challenges in efficiency of labelling and statistical identification of responders, however families like Veillonellaceae, Magnetospirillaceae, Acidobacteriaceae 1, were found ubiquitously active in glucose amended incubations. Differences in metabolic byproducts from glucose amendments show distinct patterns in acetate and propionate accumulation across sites. Families like Spirochaetaceae and Sphingomonadaceae were only found to be active in select sites of propionate amended incubations. By-product analysis from propionate incubations indicate that the northernmost sites were acetate-accumulating communities.
These results indicate that microbial communities found in poor fen northern peatlands are strongly influenced by climate variables predicted to change under current climate scenarios. I have identified patterns of relative abundance and activity of select microbial taxa, indicating the potential for climate variables to influence the metabolic pathway in which carbon moves through peatland systems.
ContributorsSarno, Analissa Flores (Author) / Cadillo-Quiroz, Hinsby (Thesis advisor) / Garcia-Pichel, Ferran (Committee member) / Krajmalnik-Brown, Rosa (Committee member) / Childers, Daniel (Committee member) / Arizona State University (Publisher)
Created2022
Description
Predatory bacteria are a guild of heterotrophs that feed directly on other living bacteria. They belong to several bacterial lineages that evolved this mode of life independently and occur in many microbiomes and environments. Current knowledge of predatory bacteria is based on culture studies and simple detection in natural systems. The ecological consequences of their activity, unlike those of other populational loss factors like viral infection or grazing by protists, are yet to be assessed. During large-scale cultivation of biological soil crusts intended for arid soil rehabilitation, episodes of catastrophic failure were observed in cyanobacterial growth that could be ascribed to the action of an unknown predatory bacterium using bioassays. This predatory bacterium was also present in natural biocrust communities, where it formed clearings (plaques) up to 9 cm in diameter that were visible to the naked eye. Enrichment cultivation and purification by cell-sorting were used to obtain co-cultures of the predator with its cyanobacterial prey, as well as to identify and characterize it genomically, physiologically and ultrastructurally. A Bacteroidetes bacterium, unrelated to any known isolate at the family level, it is endobiotic, non-motile, obligately predatory, displays a complex life cycle and very unusual ultrastructure. Extracellular propagules are small (0.8-1.0 µm) Gram-negative cocci with internal two-membrane-bound compartmentalization. These gain entry to the prey likely using a suite of hydrolytic enzymes, localizing to the cyanobacterial cytoplasm, where growth begins into non-compartmentalized pseudofilaments that undergo secretion of vesicles and simultaneous multiple division to yield new propagules. I formally describe it as Candidatus Cyanoraptor togatus, hereafter Cyanoraptor. Its prey range is restricted to biocrust-forming, filamentous, non-heterocystous, gliding, bundle-making cyanobacteria. Molecular meta-analyses showed its worldwide distribution in biocrusts. Biogeochemical analyses of Cyanoraptor plaques revealed that it causes a complete loss of primary productivity, and significant decreases in other biocrusts properties such as water-retention and dust-trapping capacity. Extensive field surveys in the US Southwest revealed its ubiquity and its dispersal-limited, aggregated spatial distribution and incidence. Overall, its activity reduces biocrust productivity by 10% at the ecosystem scale. My research points to predatory bacteria as a significant, but overlooked, ecological force in shaping soil microbiomes.
ContributorsBethany Rakes, Julie Ann (Author) / Garcia-Pichel, Ferran (Thesis advisor) / Gile, Gillian (Committee member) / Cao, Huansheng (Committee member) / Jacobs, Bertram (Committee member) / Arizona State University (Publisher)
Created2022
Description
Alkanolamines are useful as building blocks for a variety of applications, ranging from medical applications such as drug and gene delivery. In this work, Escherichia coli was investigated as a viable candidate for the production of 5-amino-1-pentanol (5-AP). Taking advantage of the existing L-lysine degradation pathway, a novel route to 5-AP was constructed by co-expressing the genes cadA (encoding lysine decarboxylase, responsible for the conversion of L-lysine to cadaverine) and patA (encoding putrescine aminotransferase, responsible for the conversion of cadaverine to 5-amino-1-pentanal), followed by the endogenous reduction of 5-amino-pentanal (5-APL) to 5-AP. To avoid the competing conversion of 5-APL to 5-amino-1-pentanoate and avoid accumulation of byproduct 1-Δ-piperideine, further host engineering was performed to delete the gene patD also known as prr (encoding 5-amino-pentanal dehydrogenase). Flask scale fermentation experiments in minimal medium of the newly constructed pathway was conducted where 62.6 mg/L 5-AP was observed to be produced. It was hypothesized that 5-AP production could be boosted by optimizing production medium to M10 media. However, change in the culture medium resulted in the production of just 51 mg/L 5-AP. Shifts observed in HPLC chromatogram peaks made it difficult to conclude exact titers of 5-AP and can be further improved by exploring different analysis methods and optimization of the method currently in place.
ContributorsBrookhouser, Brendan (Author) / Nielsen, David R (Thesis advisor) / Tonkovich, Anna L (Committee member) / Varman, Arul M (Committee member) / Arizona State University (Publisher)
Created2022