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ItemDevelopment of algorithms for metagenomics and applications to the study of evolutionary processes that maintain microbial biodiversity(Georgia Institute of Technology, 2012-12-20) Luo, ChengweiUnderstanding microbial evolution lies at the heart of microbiology and environmental sciences. Numerous studies have been dedicated to elucidating the underlying mechanisms that create microbial genetic diversity and adaptation. However, due to technical limitations such as the high level of uncultured cells in almost every natural habitat, most of current knowledge is primarily based on axenic cultures grown under laboratory conditions, which typically do not simulate well the natural environment. How well the knowledge from isolates translates to in-situ processes and natural microbial communities remains essentially speculative. The recent development of culture-independent genomic techniques (aka metagenomics) provides possibilities to bypass some of these limitations and provide new insights into microbial evolution in-situ. To date, most of metagenomic studies have been focused on a few reduced-diversity model communities, e.g., acid mine drainage. Highly complex communities such as those of soil and sediment habitats remain comparatively less understood. Furthermore, a great power of metagenomics, which has not been fully capitalized yet, is the ability to follow the evolution of natural microbial communities over time and environmental perturbations, i.e., times-series metagenomics. Although the recent developments in DNA sequencing technologies have enabled (inexpensive) time-series studies, the bioinformatics approaches to analyze the resulting data have clearly fallen behind. Taken together, to scale up metagenomics for complex community studies, three major challenges remain: 1) the difficulty to process and analyze massive short read sequencing data, often at the terabyte level; 2) the difficulty to effectively assemble genomes from complex metagenomes; and 3) the lack of methods for tracking genotypes and mutational events such as horizontal gene transfer (HGT) through time. Therefore, developing efficient bioinformatics approaches to address these challenges represents an important and timely issue. This thesis aimed to develop novel bioinformatics pipelines and algorithms for high performance computing, and, subsequently, apply these tools to natural microbial communities to generate quantitative insights into the relative importance of the molecular mechanisms creating or maintaining microbial diversity. The tools are not specific to a particular habitat or group of organisms and thus, can be broadly used to advance our understanding of microbial evolution in different settings. In particular, the comparative whole-genome analysis of 24 Escherichia isolates form various habitats, including human and non-human associated habitats such as freshwater ecosystems and beaches, showed that organisms with more similar ecologies tend to exchange more genes, which has important implications for the prokaryotic species concept. To more directly test these findings from isolates and quantify the patterns of genetic exchange among co-occurring populations, three years of time-series metagenomics data from planktonic samples from Lake Lanier (Atlanta, GA) were analyzed. For this, it was first important to develop bioinformatics algorithms to robustly assemble population genomes from complex community metagenomes, identify the phylogenetic affiliation of assembled genome and contig sequences, and detect horizontal gene transfer among these sequences. Using these novel algorithms, in situ bacterial lineage evolution was quantitatively assessed, especially with respect to whether or not ecologically distinct lineages evolve according to the recently proposed fragmented speciation model (Retchless and Lawrence, Science 2008). Evidence in support of this model was rarely observed. Instead, it appeared that rampant HGT disseminated ecologically important genes within the population, maintaining intra-population diversity. By expanding the previous approaches to include methods to assess differential gene abundance and selection pressure between samples, it was possible to quantify how soil microbial communities respond to a decade of warming by 2 0C, which simulated the predicted effects of climate change. It was found that the heated communities showed significant shifts in composition and predicted metabolism, reflecting the release of additional soil carbon compared to the unheated (control) communities, and these shifts were community-wide as opposed to being attributable to a few taxa. These findings indicated that the microbial communities of temperate grassland soils play important roles in mediating the feedback responses to climate change. Collectively, the findings presented here advance our understanding of the modes and tempo of microbial community adaptation to environmental perturbations and have important implications for better modeling the microbial diversity on the planet. The bioinformatics algorithms and approaches developed as part of this thesis are expected to facilitate future genomic and metagenomic studies across the fields of microbiology, ecology, evolution and engineering.
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ItemThe effect of host food quality on host and parasite fitness in an invertebrate-parasite system(Georgia Institute of Technology, 2012-12-18) Snell, Sara JeanneParasites achieve their fitness by reducing the fitness of the hosts they infect. The relationship between host and parasite fitness is often mediated by environmental conditions, such as the quality of food consumed by the host. We used the crustacean Daphnia dentifera and its virulent yeast parasite Metschnikowia bicuspidata to examine how the quality of the food consumed by the Daphnia affected the probability of the host being infected by the parasite and the fitness consequences of infection for the host (measured as offspring production and survival) and for the parasite (measured as the production of transmission spores within the host following infection). We fed Daphnia either high quality food (Ankistrodesmus falcatus) or low quality food (Oocystis sp.) before exposure to Metschnikowia spores, and then either high quality or low quality food after parasite exposure (according to a fully factorial design). We found that when hosts were fed high quality food as juveniles and adults, they were able to invest more energy in preventing loss of fitness due to parasites without limiting parasite growth; high quality food therefore benefits both parasite and host. High food quality benefited both host and parasite when hosts are infected. We saw that infected hosts that produced more offspring also contained more parasite spores. One possible explanation for this is the individuals that produced more offspring and spores were larger, allowing them to take in more resources.
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ItemExpression and purification of internalin a ligand for internalization studies on non-professional phagocytic epithelial cells(Georgia Institute of Technology, 2012-12-18) Haddad, NatalieInfection by intracellular pathogens is a growing concern as these microorganisms can cross their host s cellular barrier and use the host s own cellular proteins for replication, making treatment very difficult (Kenneth et al., 2008). A further restriction for treatment is that a proposed drug must be able to cross the same barriers in order to reach these pathogens. Our study investigates the same protein used by a particular intracellular pathogen, Listeria monocytogenes, as a possible internalization method for intracellular delivery of materials or drugs. A pGEX-6p plasmid vector containing a gene for the bacterial surface protein Internalin A (InlA) and a glutathione S-transferase (GST) tag, was restored and transformed into Escherichia coli MAX Efficiency DH5αF`IQ competent cells in order to increase insert stability and DNA quality. The plasmid was then purified and subsequently transformed into One Shot BL21(DE3)pLysS expression competent E. coli cells. Thereafter, the InlA-GST fusion protein was expressed in the BL21 cells using Isopropyl β-D-1-thiogalactopyranoside (IPTG). It has been shown, through selective plating and gel electrophoresis, that the plasmid was successfully transformed and purified. Our next steps will be to purify the InlA-GST fusion protein using glutathione affinity based chromatography, cleave and remove the GST tag from the InlA protein of interest, fluorescently label InlA using Alexa Flour, and bind InlA to microbeads of various sizes in a range of densities. We will then carry out internalization assays of the InlA-coated beads in different epithelial cell lines to demonstrate the versatility of this method as a possible material or drug delivery option.
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ItemAmazon River influence on nitrogen fixation and export production in the western tropical north Atlantic(Georgia Institute of Technology, 2012-12-17) Weber, Sarah CatherineAs part of the multidisciplinary ANACONDAS program, we characterized the distributions of nutrients, particulate organic matter, phytoplankton, and stable carbon and nitrogen isotopes in the Amazon plume region during the spring high-flow period of May-June 2010. We encountered the lowest salinities (16.6 psu) near the southern end of our study region where the plume showed the greatest spatial coherence, and the highest salinities (36.0 psu) to the east of the plume. The major nutrients showed distinct patterns of variation with salinity, with NO₃- largely absent from the surface plume and SiO₂ and PO₄³⁻ showing different degrees of conservative behavior. SiO₂ distributions were more strongly conservative, but with clear negative deviations that reflected biological consumption. In contrast, PO₄³⁻ concentrations showed clear positive deviations as large as 0.7 µM across a broad range of salinities as particle- and organically-bound P was released. These nutrient distributions resulted in strong nitrogen limitation and delivery of substantial amounts of SiO₂ and PO₄³⁻ to offshore waters, creating conditions that favored diazotrophy rather than simple eutrophication. We found a variety of diazotrophs in our study area, with interesting regional variation in their distributions. Mesohaline waters to the northwest of the plume axis were strongly dominated by Diatom-Diazotroph Associations (DDAs), particularly the Hemiaulus hauckii Richelia intracellularis association. In contrast, Trichodesmium spp. were most abundant to the southeast of the plume. These two diazotrophs appeared to contribute to the nitrogen and carbon budgets of the upper water column in fundamentally different ways, with H. hauckii making a much greater contribution to the particulate nitrogen pool than Trichodesmium spp. (100% versus 50%, respectively), while contributing to a greater reduction in pCO₂ in the upper water column. These contrasts have important implications for the fate of new production, with DDAs supporting higher export flux than Trichodesmium spp. Our findings provide an important constraint on the role of the Amazon plume in creating distinct niches for diazotrophic phytoplankton, and for their role in the nutrient and carbon budgets of the Western Tropical North Atlantic
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ItemMicrobes and monitoring tools for anaerobic chlorinated methane bioremediation(Georgia Institute of Technology, 2012-12) Justicia-Leon, Shandra D.he chlorinated methanes carbon tetrachloride (CT), chloroform (CF), dichloromethane (DCM) and chloromethane (CM) are widespread groundwater pollutants that pose risks to human and ecosystem health. Although some progress has been made in elucidating the microbiology contributing to the aerobic degradation of DCM and CM, these efforts have had little impact on bioremediation practices aimed at restoring anoxic aquifers impacted by chlorinated methanes. Remaining knowledge gaps include the lack of understanding of the microbial mechanisms and pathways contributing to chlorinated methane transformations under anoxic conditions. Thus, the major goals of this research effort were to identify microbes that can contribute to the transformation of chlorinated methanes in the absence of oxygen, and to develop monitoring tools to assess anaerobic chlorinated methane bioremediation at contaminated sites. To accomplish these goals, freshwater and estuarine sediment samples from 45 geographically distinct locations, including 3 sites with reported chlorinated-methane contamination, were collected and screened for CT-, CF-, DCM- and/or CM-degrading activity. DCM degradation was observed in microcosms established with sediment materials from 15 locations, and the sediment-free, DCM-degrading enrichment culture RM was obtained from Rio Mameyes sediment. 16S rRNA-gene based community analysis characterized consortium RM, and identified a Dehalobacter sp. involved in DCM fermentation to non-toxic products. Organism- and process-specific monitoring tools were designed that target the 16S rRNA gene of the DCM-fermenting Dehalobacter sp. and the consortium’s specific 13C-DCM enrichment factor, respectively. Treatability studies using site materials that showed no chlorinated methane degradation activity demonstrated the feasibility of using CF- and DCM-degrading consortia for bioaugmentation applications. Collectively, this study expands our understanding of bacteria contributing to chlorinated methane degradation, provides new tools for monitoring anaerobic DCM degradation, and demonstrates that microbial remedies at chlorinated methane contaminated sites are feasible.
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ItemOptimization of over-expression and purification of human leukotriene C4 synthase mutant R104A for structure-function studies by two-dimensional crystallization and electron crystallography(Georgia Institute of Technology, 2012-11-15) Kim, Laura YaunheeMembrane proteins are involved in a number of disease pathologies and thus comprise a large number of drug targets. Determination of the high-resolution three-dimensional structure is essential for rational drug design, but several hurdles need to be overcome, primarily the over-expression and purification of said membrane proteins. Human leukotriene C4 synthase (hLTC4S), an 18 kDa integral membrane protein localized in the outer nuclear membrane of eosinophils and basophils, catalyzes the conjugation of LTA4 and reduced glutathione to produce LTC4. LTC4 and its metabolites LTD4 and LTE4 are the cysteinyl leukotrienes implicated in bronchoconstriction and inflammation pathways. The focus of my project involves optimizing the over-expression and purification of hLTC4S, which was heterologously expressed in Schizosaccharomyces pombe, purified by immobilized affinity chromatography, and finally "polished" with a buffer exchange step to remove excess co-purified lipids. The optimized protocol yielded ~1 mg of ~90% homogenous, pure protein per liter of cell culture. The finalized purified protein can then be used for further investigation of two-dimensional crystals by electron crystallography with the overall goal of structure determination.
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ItemComparative genomics reveal ecophysiological adaptations of organohalide-respiring bacteria(Georgia Institute of Technology, 2012-11-13) Wagner, Darlene DarlingtonOrganohalide-respiring Bacteria (OHRB) play key roles in the reductive dehalogenation of natural organohalides and anthropogenic chlorinated contaminants. Reductive dehalogenases (RDases) catalyze the cleavage of carbon-halogen bonds, enabling respiratory energy conservation and growth. Large numbers of RDase genes, a majority lacking experimental characterization of function, are found on the genomes of OHRB. In silico genomics tools were employed to identify shared sequence features among RDase genes and proteins, predict RDase functionality, and elucidate RDase evolutionary history. These analyses showed that the RDase superfamily could be divided into proteins exported to the membrane and cytoplasmic proteins, indicating that not all RDases function in respiration. Further, Hidden Markov models (HMMs) and multiple sequence alignments (MSAs) based upon biochemically characterized RDases identified previously uncharacterized members of an RDase superfamily, delineated protein domains and amino acid motifs serving to distinguish RDases from unrelated iron-sulfur proteins. Such conserved and discriminatory features among RDases may facilitate monitoring of organohalide-degrading microbial communities or improve accuracy of genome annotation. Phylogenetic analyses of RDase superfamily sequences provided evidence of convergent evolution and horizontal gene transfer (HGT) across distinct OHRB genera. Yet, the low frequency of RDase transfer outside the genus level and the absence of RDase transfer between phyla indicate that RDases evolve primarily by vertical evolution or HGT is restricted among related OHRB strains. Polyphyletic evolutionary lineages within the RDase superfamily comprise distantly-related RDases, some exhibiting activities towards the same substrates, suggesting a longstanding history of OHRB adaptation to natural organohalides. Similar functional and phylogenetic analyses provided evidence that nitrous oxide (N₂O, a potent greenhouse gas) reductase (nosZ) genes from versatile OHRB members of the Anaeromyxobacter and Desulfomonile genera comprised a nosZ sub-family evolutionarily distinct from nosZ found in non-OHRB denitrifiers. Hence, elucidation of RDase and NosZ sequence diversity may enhance the mitigation of anthropogenic organohalides and greenhouse gases (i.e., N₂O), respectively. The tetrachloroethene-respiring bacterium Geobacter lovleyi strain SZ exhibited genomic features distinguishing it from non-organohalide-respiring members of the Geobacter genus, including a conjugative pilus transfer gene cluster, a chromosomal genomic island harboring two RDase genes, and a diminished set of c-type cytochrome genes. The G. lovleyi strain SZ genome also harbored a 77 kbp plasmid carrying 15 out of the 24 genes involved in biosynthesis of corrinoid, likely related to this strains ability to degrade PCE to cis-DCE in the absence of supplied corrinoid (i.e., vitamin B₁₂). Although corrinoids are essential cofactors to RDases, the strictly organohalide-respiring Dehalococcoides mccartyi strains are corrinoid auxotrophs and depend upon uptake of extracellular corrinoids via Archaeal and Bacterial salvage pathways. A key corrinoid salvage gene in D. mccartyi, cbiZ, occurs at duplicated loci adjacent to RDase genes and appears to have been horizontally-acquired from Archaea. These comparative genome analyses highlight RDase dependencies upon corrinoids and also suggest mobile genomic elements (e.g., plasmids) are associated with organohalide respiration and corrinoid acquisition among OHRB. In summary, analyses of OHRB genomes promise to enable more complete modeling of metabolic and evolutionary processes associated with the turnover of organohalides in anoxic environments. These efforts also expand knowledge of biomarkers for monitoring OHRB activity in anoxic environments, and will improve our understanding of the fate of chlorinated contaminants.
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ItemDynamic stability of quadrupedal locomotion: animal model, cortical control and prosthetic gait(Georgia Institute of Technology, 2012-11-13) Farrell, Bradley J.The ability to control balance and stability are essential to prevent falls during locomotion. Maintenance of stable locomotion is challenging especially when complicated by amputation and prosthesis use. Humans employ several motor strategies to maintain stability during walking on complex terrain: decreasing walking speed, adjusting stride length and stance width, lowering the center of mass, and prolonging the double support time. The mechanisms of selecting these motor strategies by the primary motor cortex are unknown and cannot be studied directly in humans. There is also little information about dynamic stability of prosthetic gait with bone-anchored prostheses, which are thought to provide sensory feedback to the amputee through osseoperception. Therefore, the Specific Aims of my research were to (1) evaluate dynamic stability and the activity of the primary motor cortex during walking with different constraints on the base of support and (2) develop an animal model to evaluate mechanics and stability of prosthetic gait with a bone-anchored prosthesis. To address these aims, I developed a feline model that allows for investigating (1) the role of the primary motor cortex in regulation of dynamic stability of intact locomotion, (2) skin and bone integration with a percutaneous porous titanium implant facilitating prosthetic attachment, and (3) dynamic stability of walking on a bone-anchored prosthesis. The results of Specific Aim 1 demonstrated that the area and shape of the base of support influence the margins of dynamic stability during quadrupedal walking. For example, I found that the animal is dynamically unstable in the sagittal plane and frontal plane (although to a lesser degree) during a double-support by a forelimb and the contralateral hindlimb. Elevated neuronal activity from the right forelimb representation in the primary motor cortex during these phases suggests that the motor cortex may contribute to selection of paw placement location and thus to regulation of stability. The results of Specific Aim 2 on the development of skin-integrated bone-anchored prostheses demonstrated the following. Skin ingrowth into 3 types of porous titanium pylons (pore sizes 40-100 μm and 100-160 μm and nano-tubular surface treatment) implanted under skin of rats was seen 3 and 6 weeks after implantation, and skin filled at least 30% of available implant space. The duration of implantation, but not implant pore size (in the studied range) or surface treatment statistically influenced skin ingrowth; pore size and time of implantation affected the implant extrusion length (p<0.05). The implant type with the slowest extrusion rate (pore size 40-100 μm) was used in a feline model of prosthetic gait with skin-integrated bone-anchored prosthesis. The developed implantation methods, rehabilitation procedures and feline prostheses allowed 2 animals to utilize skin- and bone-integrated prostheses for dynamically stable locomotion. Prosthetic gait analysis demonstrated that the animals loaded the prosthetic limb, but increased reliance on intact limbs for weight support and propulsion. The obtained results and developed animal model improve the understanding of locomotor stability control and integration of skin with percutaneous implants.
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ItemMembrane effects of sex hormones on growth plate chondrocytes(Georgia Institute of Technology, 2012-11-12) ElBaradie, Khairat BahgatUnderstanding and studying the normal bone growth and development is causal. Bone and cartilage tissue provide in addition to their mechanical support, they provide a protection for vital organs such as heart, lung and brain. Longitudinal growth is regulated by the activity of chondrocytes in the epiphyseal growth plates of long bones. Many hormones and growth factors are involved in the regulation of this process. Among these, sex steroids are of crucial importance, especially during puberty. In long bones, endochondral bone formation occurs at the growth plate, a region of developing cartilage located between the epiphysis and the metaphysic. The process of endochondral ossification is regulated in part by sex steroid hormones. Androgens stimulate endochondral bone growth and elongation, while estrogen is known to suppress longitudinal bone growth and accelerate growth plate closure. Studies using rat costochondral growth plate chondrocytes as a model show that the effects of 17β-estradiol (E₂) on apoptosis are found in both male and female cells and the same mechanism is involved. In contrast, E₂ causes rapid activation of PKC in female cells but not in male cells. Dihydroxytestosterone (DHT) also has direct effects on growth plate chondrocytes, increasing matrix synthesis including sulfated glycosaminoglycan production, and enhancing cell maturation by increasing alkaline phosphatase enzymatic activity. Short stature and abnormally slow increase in height is one of the main reasons for referral to endocrinologist. Excessive growth and abnormally tall is also a problem, especially because it increase risk for the trunk abnormalities. Furthermore until now a few growth-promoting therapies are available for clinical use. Therefore future therapies for treating the growth disorders are essential. The overall goal of this project is to investigate the sexual-dimorphic effect of the sex steroid hormone in rat growth plate chondrocytes, the cellular signaling pathways mediating these actions, and their physiological role. The information gleaned from this study will provide new information about the role of sex steroid hormones in chondrogenesis and has implications in the development of new therapies for the treatment of bone fracture healing, and growth plate disorders. The central hypothesis was that sex steroid would play an important and sex-specific role in regulating chondrocytes as a main regulator of longitudinal bone growth.
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ItemUsing blood informative transcripts in geographical genomics: impact of lifestyle on gene expression in Fijians(Georgia Institute of Technology, 2012-11-09) Nath, Artika Praveeta ; Arafat, Dalia ; Gibson, GregIn previous geographical genomics studies of the impact of lifestyle on gene expression inferred from microarray analysis of peripheral blood samples, we described the complex influences of culture, ethnicity, and gender in Morocco, and of pregnancy in Brisbane. Here we describe the use of nanofluidic Fluidigm quantitative RT-PCR arrays targeted at a set of 96 transcripts that are broadly informative of the major axes of immune gene expression, to explore the population structure of transcription in Fiji. As in Morocco, major differences are seen between the peripheral blood transcriptomes of rural villagers and residents of the capital city, Suva.The effect is much greater in Indian villages than in Melanesian high-landers and appears to be similar with respect to the nature of at least two axes of variation. Gender differences are much smaller than ethnicity or lifestyle effects. Body mass index is shown to associate with one of the axes as it does in Atlanta and Brisbane, establishing a link between the epidemiological transition of human metabolic disease, and gene expression profiles.