Thesis Topic: Colin Rorex
2016-2018

Abstract

Sponge disease has a number of detrimental effects on aquatic ecosystems and industry. The loss of sponge mass will reduce the amount of free nutrients in the water column and has been linked to an increase in toxic phytoplankton blooms. Previous research has found that multiple organisms are required to trigger a disease state in sponges and that a disease outbreak can result in a significant change of the sponge microbiome. This complex mixture of micro-organism pathogenesis and ecology has made understanding sponge disease difficult. A recently described sponge disease outbreak in Lake Baikal Siberia has resulted in a significant reduction of one of the endemic sponge species present in the lake. Historically, this is the first reported instance of sponge disease in this ecosystem, which allows a unique opportunity to examine archival DNA samples to see the sponge microbiome prior to the causative event of the outbreak against current and compare that against data collected during the event to see how the microbiome has changed.

Thesis
Research Article
dataset@SRA

Thesis Topic: Ray Montez.
2014-2016

Abstract

Global climate change and anthropogenic activity have facilitated the movement and invasive potential of nonnative plants in native environments. These invasions can have negative effects on ecosystem diversity and function. The nonnative and invasive plant, Chinese Tallow (Triadica sebifera), has already invaded much of the south eastern US where it is outcompeting native tree species and changing ecosystem diversity in a variety of habitats. Leaf litter from the Chinese tallow has been shown cause changes in dissolved oxygen and pH in the aquatic environment. Turbidity is also affected when Chinese tallow litter is present in water. A series of experiments were performed to determine the causes of these chemical changes in water when Chinese tallow litter is present. I determined that Chinese tallow litter has a different chemical composition from native litter, a different concentration of essential soluble nutrients from native litter, and a faster decomposition rate compared to native plant litter. Sterilization experiments suggest that Chinese tallow litter is promoting microbial activity through the rapid release of elemental nutrients, which subsequently influences a change in dissolved oxygen thorough stimulated microbial respiration. Changes in pH are not fully understood, but sterilization experiments suggest that unknown secondary chemicals, perhaps tannins and phenolic compounds, are the source of declining pH when Chinese tallow litter is present in water. Using high throughput 16s and 18s rRNA mass parallel gene sequencing, followed by Non-metric Multidimensional Scaling ordination scatter plots, I determined that Chinese tallow litter can promote differences in microbial community composition from that of native plant litter. NMDS ordination scatter plots demonstrate that both bacterial and fungal communities were different in Chinese tallow treatments when compared to native plant litter treatments. These results provide strong evidence that Chinese tallow litter can promote changes in the microbial community composition of an aquatic/wetland habitat.

Thesis

Thesis Topic: Diane Lewis.
2013-2015

Abstract

Habitat loss and pathogens, particularly Batrachochytrium dendrobatidis (Bd), have been identified as primary factors contributing to amphibian decline. These factors may cause decline independently or they may interact with one another. For example, habitat features that affect microclimate such as forest canopy density, natural vegetation and water temperature are good predictors of the presence of Bd but recent evidence suggests that habitat loss is negatively associated with occurrence, prevalence and infection intensity of the fungal pathogen. Anurans are known to possess innate defenses against pathogens in the form of antimicrobial secretions produced in glands on skin of the frogs. In addition, recent evidence suggests that the probiotic role of symbiotic bacteria may be important in the defense against pathogens, specifically Bd. In this study we attempt to determine how habitat, urban vs forested, and microbiome interplay in the context of Bd occurrence and pathogen load. We found that urban frogs had much lower incidence of Bd and the frogs had lower Bd load while forested frogs had lower species richness and diversity of symbiotic bacteria. Symbiotic bacterial communities did not differ between Bd+ and Bd- frogs within habitat type suggesting that habitat, not bacterial community, was the main driver of Bd occurrence in our focal anuran populations with low-level Bd infections.

Thesis

Spatial distribution and structural diversity of bacterial communities in oil-contaminated soils of east Texas.

Thesis Topic: Alexander Fedotov.
2004-2006

Abstract

The purpose of the study is to investigate the microbial diversity of oil-polluted soil communities and to determine the influence of changes in environmental conditions. Soil samples were collected from several oil-contaminated sites and non-contaminated areas in East Texas for bacterial diversity analysis, a type of fingerprinting which produces a genetic profile of the total microbial community. Sequence of 16S rDNA gene was chosen as genetic marker for the analysis due to the low level of evolutionary change and high information content of this gene. PCR amplification of the 16S rDNA fragments was performed on the extracted DNA using several universal and eubacterial primers obtained from literature sources. DGGE analysis of PCR products generated community fingerprints that enabled comparison of the microbial and genetic diversity of several oil-contaminated sites.

Thesis

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Bacterial Diversity in Soil Communities of East Texas Habitats.

Thesis Topic: Jennifer Edwards.
2007-2009

Abstract

Bacteria are the most diverse component of terrestrial microbial communities and thus greatly influence the quality and health of soil ecosystems. Comprehensive data and information on the classification and ecology of soil bacterial communities is currently lacking due to inadequacy of traditional culturing methods. This study employs molecular techniques to genotypically classify the diversity of bacteria indigenous to east Texas forest habitats, providing a necessary addition to an existing ecological classification system for the West Gulf coastal plain Van Kley et al. (2007; Van kley & Turner in press). Direct extraction of total DNA from environmental soil samples and analysis of 16S rDNA allowed for separation of bacteria from other microbial members within samples and provided simultaneous screening of bacterial communities present. The relative abundance of bacterial operational taxonomic units (OTUs) within soil microbial communities from east Texas forests representing the full range of local ecological types was determined.

Thesis

Dynamics of bacterial community analyzed in poultry litter treated with LitterGuard® using DGGE and pyrosequencing.

Thesis Topic: Akash Kaushik.
2009-2011

Abstract

Bacterial communities present in used poultry litter, a combination of sawdust and manure, largely affects the performance of the birds in poultry houses. The effect of Bacillus subtilis spp. on the bacterial communities in the used poultry litter was studied with DGGE (Denaturing Gradient Gel Electrophoresis) and 454 pyrosequencing of 16S rDNA. DNA was extracted from 8 sets of litter samples collected from 4 different broiler houses. The total DNA samples processed with DGGE were 116 from the first two sets and 96 from the last 6 sets. The total DNA samples pyrosequenced were 20 from each 1 st and 8th sets. Python scripts and multivariate analysis were used to analyze DGGE gel images and pyrosequencing data. The analysis of DGGE gel images showed no significant difference in the richness of the bacterial communities among houses for all 8 sets of samples. The average richness of bacterial communities observed was 234 (approx.) in all the samples. The taxonomical assignment of the sequences from pyrosequencing was done using RDP Classifier. A total of 188 and 199 unique taxa were assigned to the 1st and 8th sets of samples, respectively. In the samples from both the sets, relative abundance of Cornybacterium spp., Stapylococcus spp., Lactobacillus spp., Salinicoccus spp. were found to be more than 10%. The results of the experiment showed that there was no significant change in the richness and the composition of bacterial communities in the poultry litter when treated with Bacillus subtilis spp.

Thesis

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Effect of coccidiostat and natustat^tm on intestinal microflora in caecum of broilers challenged with Eimeria analyzed using bacterial 16s rDNA tag-encoded flx amplicon pyrosequencing.

Thesis Topic: Muthu Savaranan.
2008- 2010

Abstract

Chicken coccidia are prolific parasitic organisms of Eimeria sp. that affect the intestine and reduce the feed conversion ratio (FCR). Among Eimeria sp., Eimeria tenella, Eimeria maxima and Eimeria acervulina are the most problematic in the US. The gut flora (useful and harmful bacteria) of chicken plays a main role in FCR of broilers. Any changes in these levels could affect the chicken's health and FCR drastically. In the present study, fecal sample of vaccinated broilers challenged with combination of Eimeria spp. fed with one of the two food additives: Carina Alternata and Carina Poultry was analyzed for relative percent abundance of bacterial population. The total DNA extracted from the samples was amplified for 16S rRNA using PCR and was sequenced using 454 pyrosequencing. Approximately, 3000-6000 sequences were obtained for each sample from pyrosequencing. The sequences were analyzed using RDP classifier and BLAST for the relative percent abundance of intestinal microflora at different taxa level. The results showed drastic increase in the level of pathogenic organisms like: Clostridium spp. and Salmonella spp. under challenged conditions compared to unchallenged. The feed additives and vaccination had negative impact on the abundance of pathogenic organisms. Significant increase in the levels of beneficial microflora like: E. coli , Lactobacillu was observed in feed additives treatment. Vaccination affected the FCR of chickens. A change in the relative percent abundance of beneficial and pathogenic bacterial populations under different treatment conditions was observed. The study has shone light on the biological importance of intestinal microflora in chickens.

Thesis

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Monitoring of Eimeria in broilers using Real Time PCR.

Thesis Topic: Marina Maksimova.
2007-2009

Abstract

Avian coccidiosis is a protozoan disease, which is responsible for significant economic losses in poultry production. It is caused by seven species of the genus Eimeria. It is important to identify and determine the quantity of oocysts for successful treatment of the disease. A real-time PCR assay based on fluorogenic probes species-specific to the 18S rRNA gene was developed to quantify parasite load in fecal samples. The TaqMan probes for the analysis of Eimeria were identified utilizing BLAST, the ClustalW program and custom software developed in our laboratory. The number of oocysts in fecal samples was estimated quantitatively based on a standard curve which was built using plasmid DNA. This technique for quantitative detection of Eimeria species can be used in the poultry industry as a tool for monitoring coccidiosis.

Thesis

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Strategy for detection and identification of Eimeria species infecting chickens.

Thesis Topic: Andrew Syvyk.
2005-2007

WILLIAM R. JOHNSON OUTSTANDING THESIS AWARD.

Abstract

Eimeria is an intracellular protozoan parasite and the causative agent of Coccidiosis – one of the most economically important diseases in modern poultry production. There are at least seven known species of Eimeria. The disease is caused by the replication within the digestive tract by one or several of Eimeria species and leads to the destruction of epithelial cells. Birds with damaged intestine exhibit poor performance, loose weight and in severe cases can die. The total annual cost of coccidial infections in the US has been estimated to be around 700 million dollars annually. Treatment measures may differ depending on the Eimeria species, therefore accurate identification of the species infecting the flock is crucial. Presently, the most advanced system for simultaneous detection and identification of the seven Eimeria species is multiplex PCR. However, concurrent amplification of different target fragments with different primers is not free from bias, introduced by fragment size and primer annealing efficiency. We propose a different approach to the multiplex PCR methodology, the basis of which is the ability of denaturing gradient gel electrophoresis (DGGE) to separate DNA fragments amplified with a single pair of primers. Amplicons are separated not according to their size, but according to the nucleotide composition of amplified fragments, thus eliminating the inherent favoritism of multiplex PCR. This alternative DNA-based approach for simultaneous detection and identification of Eimeria species could potentially satisfy all the requirements for the sensitive and reliable test system and may be accepted by the poultry industry.

Thesis

Research Article

Syvyk A., A. Martynova-Van Kley, A. Nalian. 2008. A positive control for detecting heteroduplexes in DGGE for microbial community fingerprinting. The Texas Journal of Science, 60(1):33-44.

Detecting seasonal changes in arbuscular mycorrhizal communities by denaturing gradient gel electrophoresis and pyrosequencing.

Thesis Topic: Sikta Patnaik.
2008-2010

Abstract

Most land plants have root systems that form symbiosis with Arbuscular Mycorrhizal fungi (AMF) belonging to the phylum Glomeromycota. Symbiotic Arbuscular Mycorrhizal fungi have shown to influence both the diversity and productivity of different plant communities. In this study, the communities of AMF colonizing the roots of two plants species Callicarpa americana and Chasmanthium sessiliflorum through different seasons is characterized. The affect of the environmental factors such as rainfall, soil moisture content, soil nutrient availability is also taken into consideration. The small subunit ribosomal RNA gene (SSU rRNA) is subjected to PCR and Denaturing Gradient Gel Electrophoresis (DGGE) followed by multivariate analysis. PCR results showed the difference between the two plants. In summer, Chasmanthium did not show the presence of AMF whereas Callicarpa showed the presence of AMF for all seasons. The rDNA fragments which potentially represent several AMF species per sample were subsequently subjected to DGGE in order to separate fragments of differing nucleotide sequence. After DGGE the difference between both the plants was obtained in the species level and the affect of seasons on the AMF population was determined. Further, the affect of other environmental factors like soil mineral content, soil moisture content was correlated to the AMF population as soil phosphorus and moisture content are the major determinant of the AMF population in the roots of plants.

Thesis

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Profiling of Arbuscular Mycorrhizal communities by high-throughput sequencing.

Thesis Topic: Elvira Kurmaeva.
2007-2009

Abstract

Arbuscular mycorrhizal fungi (AMF) form a symbiosis with plant roots in more than 80% of terrestrial plant species. AMF enhance plant nutrient absorption, increase soil stability and protect plants against soil pathogens. Samples were collected from 25 sites in the Stephen F. Austin Experimental Forest, Angelina National Forest and Sabine National Forest, Texas, USA and were sequenced using the 454 FLX pyrosequencing and 18S fungal primers. Each sample produced from 2000 to 13,000 reads, from which the exact OTUs presented in the sample were determined. The sequences obtained were processed using a python script. Their origin was determined by Basic Local Alignment Search Tool. Two binary matrixes were constructed: an OTU-by-samples matrix and an environmental matrix. Patterns of AMF diversity as expressed by relative percent abundance were compared with patterns of soils, topography, and vegetation across the sites using multivariate methods including cluster analysis, correspondence analysis, detrended correspondence analysis, and regression of ordination scores/principal components on external (soil vegetative, topographic) factors. Fifty three distinct OTUs of AMF were found and relative percent abundance of Glomus species ranged from 0.3 to 6.8%.Describing the diversity of AMF across several east Texas habitat types will help better understand the genetic organization and taxonomy of AMF and may enhance local forest management and agriculture.

Thesis

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Phylogenetic analysis of the partial 18S small subunit gene sequence of arbuscular mycorrhizal fungi in east Texas forest.

Thesis Topic: Kevin Stroup.
2004-2006

Abstract

Arbuscular Mycorrhizal Fungi (AMF) of the Phylum Glomeromycota (Helgason et al. 2003) form symbiotic relationships with the roots of an estimated 90% of the 250,000 known terrestrial plant species. AMF are important to plant resistance to drought and pathogenic organisms, and help in obtaining vital minerals. AMF also aid in the prevention of transplantation shock, a source of plant mortality. Diversity and distribution of AMF is not well known due to difficulties in using traditional morphological methods for identification. The roots of two widely occurring native host plants, Chasmanthium sessiliflorium and Callicarpa americana, collected from three different habitats were used as sources for DNA samples. The 520 bp region of the 18S rDNA served as a genetic marker and was amplified by PCR using Glomeromycota specific primers. Denaturing Gradient Gel Electrophoresis profiles of the AMF population colonizing the roots of these species for each habitat provides the phylogenetic relationships among AMF types.

Thesis

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Analysis of arbuscular mycorrhizal communities in east Texas.

Thesis Topic: Darya Fakhretdinova.
2006-2008

Abstract

An ecological classification of natural habitats based on their vegetation, topographic position, soil texture, hydrology, fire frequency, and plant nutrient availability was recently developed as a management tool for east Texas forests. This classification scheme does not take into account such important components of terrestrial ecosystems as microbes, particularly arbuscular mycorrhizal fungi (AMF) and soil bacteria. AMF facilitate plant mineral uptake, enhance pathogen and disease resistance, and influence plant community structure and productivity. Soil bacteria are important decomposers, improve water infiltration and water-holding capacity of the soil, and amend its stability and aeration. In the current work microbial diversity across different natural east Texas habitat types was studied by molecular techniques: PCR amplification of fragments of the small subunit ribosomal RNA (SSU rRNA) gene with subsequent denaturing gradient gel electrophoresis (DGGE) analysis of obtained amplicons. DGGE profiles show the qualitative microbial diversity of each habitat. Analyzing AMF and soil bacterial diversity across different habitat types using DGGE provides a new and additional component for ecological classification of east Texas natural habitat types.

Thesis

Molecular Dynamics Simulations Provide Insight into Stability of Hyperthermophilic Endoglucanases.

Thesis Topic: Logan E. Sheffield
2019-2021

Abstract

Pyrococcus horikoshii is a hyperthermophilic organism that has been shown to produce a highly thermostable β-(1,4)-endoglucanase (EGPh) that belongs to glycosyl hydrolase family 5. This enzyme possesses a disulfide bond that is distinct from its less thermostable family 5 relatives. A prior study has suggested this single disulfide bond plays a significant role in the thermostability of EGPh (Kim & Ishikawa, 2013). In this study, molecular dynamics simulations are used along with various statistical methods to compare the behavior of a mesophilic endoglucanase produced by Xanthomonas campestris, as well as a moderately thermophilic endoglucanase isolated from Acidothermus cellulolyticus to that of the hyperthermophilic EGPh at various temperature levels. Simulations are carried out using crystal structures obtained from the Protein Data Bank (PDB) to investigate the contribution of disulfide bonding, as well as hydrogen bonding and overall protein structure/organization. Results for each simulation are compared to look at differences between the chosen enzymes.

Thesis

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Comparison of the catalytic activities of the wild-type and mutant endogluconases from Pyrococcus horikoshii.

Thesis Topic: Jorge Del Aguila.
2005-2007

Abstract

Hyperthermophilic enzymes offer many advantages in industrial biotechnology and bioenergy. High temperatures result in low viscosity, high bioavailability, high catalytic rates, and low risk of microbial contamination. There are 45 known families of glycosyl hydrolases (GH) which can break cellulose. Many enzymes of the GH5 family are thermophilic and hyperthermophilic. A recently discovered member is β-1,4 endoglucanase (EGPh) isolated from Pyrococcus horikoshii. Since EGPh is an attractive candidate for industrial use we propose to investigate the effect of site-directed mutants of this enzyme by determining their catalytical properties using avicel as a substrate. Specific sites for mutations are suggested by computational analysis.

Thesis

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Molecular modeling of thermostable endoglucanases.

Thesis Topic: Jasmine Wong.
2007-2009

Abstract

Hyperthermostable endoglucanase from Pyrococcus horikoshii (EGPh) is expected to be a useful enzyme for cellulitic hydrolysis. The aim of this project is to identify specific residues that contribute to thermostability, pH tolerance, catalytic activity, and substrate specificity of EGPh by using Molecular Dynamics Simulations. A homology model of EGPh was constructed using the crystal structure of the endoglucanase from Acidothermus cellulolyticus (EGAc) since the primary structures of EGPh and EGAc share 45% sequence identity. The findings from these simulations will be used to suggest the specific residues for site-directed mutagenesis.

Thesis

Identification and phylogenetic analysis of southeastern USA earthworm species using 16S rDNA and COI sequences.

Thesis Topic: Yulia Leontieva.
2005-2007

Abstract

Earthworms are very important soil inhabitants which have beneficial influence on soil structure, composition, and properties. Diplocardia, considered to be the most species rich native North American earthworm genus (at least 44 described), belongs to family Megascolecidae the taxonomy of which is recognized as the most controversial in comparison to other Oligochaeta families. With so many Diplocardia, species classification for many of them remains unclear. Identification of earthworms is complicated and requires a number of skills. Currently, Diplocardia species, as well as all Megascolecidae, are identified by dissection and observing internal anatomical features under the microscope. Identification is further problematic because for some species the existing classification is vague. Moreover, some species have just a small number of distinctive features which could be highly variable. DNA-based identification of taxa has great potential and it is a rapidly evolving tool to define the position of different taxa. In this research 20 earthworm species from the southeastern USA, most belonging to the genus Diplocardia were collected and identified by traditional morphological methods. 16S rDNA and COI sequences were determined for each and 125 sequences were submitted to the NCBI database. Comparison of the morphological identification to a molecular phylogenetic approach using DNA sequence data of the 16S rDNA and cytochrome c oxidase subunit I (COI) genes is in progress. These molecular markers have been investigating for their ability to delimit Diplocardia species

Thesis

Thesis Topic: O.A. Martyniuk
2004-2006

Abstract

The systematics of the vascular plant family Amaranthaceae Juss. (Townsend et al. 1993) are still poorly understood. Relationships among subtaxa within the family and its position relative to other families within the order Caryophyllales is still a matter of debate. Both traditional morphological and molecular approaches have been unable to resolve these issues. In order to address taxonomic and phylogenetic questions, pollen wall structure may be used as an additional source of evidence. Such palynomorphological work has been done for the Amaranthaceae but has identified another problem: Many different pollen types were observed for this family while for most families only one type of pollen structure exists. Attempts to classify pollen wall structure based on simple characters which could be easily identified under the microscope and use these for phylogeny and taxonomy within the Amaranthaceae did not appreciably resolve taxonomic problems in this group of plants. In order to find taxonomically relevant features of pollen wall structure we decided to perform phylogenetic analysis on pollen from Amaranthaceae species from within the same geographic area, attempt to identify evolutionarily derived characters (synapomorphies) for various groups of these species, and then develop a phylogenetic system based on pollen types for the Amaranthaceae. Corroboration of this system with molecular phylogenetic data derived from known sequences of two genes--maturase K(matK) and a partial sequence of ribulose-1,5-bisphosphate carboxylase (RBCL) which were previously used as a genetic markers in number of phylogenetic studies of vascular plants (Kadereit et al.2003, Müller & Borsch 2005) would provide us with an indication of the strength of our pollen type system.

Human Intestinal Microbiota in Response to Antibiotics, Nicotine and Caffeine.

Undergraduate Research Topic: Maximiliano Rodriguez.
2017-2019

Abstract

Intestinal microbiota play a number of critical roles in the health of the organism. They assist in nutrient metabolism and play a key role in the host’s immune response. Current research is revealing that there may be a significant impact from intestinal bacteria on the gut-brain axis and that they play a downstream role in several neurological disorders. Here we present data from a 26-day single-person pilot study where we looked at the effects from the use of tobacco products on the microbial diversity during an antibiotic treatment and a 2-week recovery period following the antibiotic course. During the course of the project detailed dietary and behavioral data were collected and later used to construct an environmental matrix for multivariate analysis. The goal of this project was to see how behavioral factors influence both the antibiotic driving decline of gut microbiota as well as the recovery period following an antibiotic course.

Research Article