2006 Research Fair Archive - Biology Abstracts
Adherence of Trypsonized Enterococcus faecalis
Enterococcus faecalis is a common nosocomial pathogen whose virulence is tied to its ability to adhere to human cells, especially in the urinary tract and lining of the heart. This study examined adherence by known clinical strains to a human cell line, HTB-5. The human cells were grown in DMEM with 10% FBS in an incubator at 37°C and 10% carbon dioxide until confluent. The cells were then plated in chamber slides overnight. The known clinical strains of bacteria were then added to the cell cultures and adherence was tested using a known ¬in vitro method. Adherences of trypsonized and non-trypsonized bacterial cells were compared for any changes in adherent properties. Results from the application of this developed assay will be presented.
Identifying Halophage Hosts from Isolates
Halophage are salt tolerant viruses that infect bacteria in hypersaline waters. They have not been extensively studied. With the discovery of halopahge in Great Salt Lake, questions were raised about what halophiles they attack. In the course of this project, we took different isolates of halophiles and plated them in the presence of halophage pools, isolated by cesium chloride centrifugation. Results will be presented from these assays. Our data will help identify which halophage attacks which halophile.
Thioredoxin Reductase in Artemia franciscana and Its Role
The interaction between selenium and the life in the Great Salt Lake is of great current interest due to the potential ecological impacts of increased selenium levels in the Great Salt Lake. Selenium has long been associated with cytotoxicity in organisms exposed to high selenite concentrations. The multifaceted thioredoxin reductase enzyme family has been implicated in the detoxification of selenium in a wide variety of organisms. While there is some data on the Selenium levels in the lake water, sediments, birds, and brine shrimp (Artemia), little is known about the effect of selenium on the brine shrimp themselves. Our goal is to identify and characterize the Artemia thioredoxin reductase gene and study its regulation in the attempt to provide insights into the reaction of brine shrimp to selenium in their environment. To identify TrxR in Artemia, we have employed Reverse Transcriptase PCR with degenerate primers targeted to conserved. PCR results have demonstrated a potential TrxR fragment and currently the sequence of this band is being investigated via bacterial cloning.
Photoprotection in Halophilic Archaea: Correlation of Carotenoids
Halophilic Archaea are much more resistant to ultraviolet (UV) light damage than Escherichia coli and other species of Bacteria. These extreme halophilic organisms use light-driven ion pumps to maintain an acceptable internal environment despite living in water that has salt concentrations up to 30%. This need for light requires that the organisms experience more UV exposure than most other known microorganisms. For this reason, they have developed survival mechanisms, including efficient DNA repair processes. We propose that halophilic Archaea also employ photoprotective mechanisms such as low adenine-thymine ratios to avoid thymine dimers and pigmentation to protect from UV damage. Methods of analysis included thymine dimer detection as well as isolation and identification of carotenoid compounds using HPLC.
Microbial Diversity of Great Salt Lake
Great Salt Lake (GSL) is a dichotomous ecosystem with a causeway splitting the lake into a hypersaline (30%) North Arm and a less saline (15%) South Arm. This project is a survey of the community of microorganisms dwelling in the North Arm of GSL, using microbial and molecular techniques. To date, no study like this has ever been attempted on this important extreme ecosystem. Microorganisms (Archaea) were isolated and characterized through cultivation, microscopy, biochemistry and 16S rDNA gene sequencing. Through electron microscopy, a number of halo-phage species were discovered, and these were fractionated with a centrifugation gradient. Results from phage characterization will also be discussed. Our studies show that GSL is much more diverse than previously thought. This research will result in a deeper understanding of the ecology of a unique ecosystem in North America.
Searching for an Alternative Energy Source
As fossil fuels are becoming depleted, alternative energy sources must be sought. Past research has shown that green algae naturally produce limited amounts of hydrogen as a byproduct of photosynthesis. However, green algae species inhabiting hypersaline, anerobic environments, are suspected of producing greater amounts of hydrogen. Through DNA analysis, we have been searching for the hydrogenase gene in four species of green algae living in the Great Salt Lake (GSL). PCR primers were designed based on the hydrogenase gene sequence discovered by Dr. Posewitz at the National Renewable Energy Lab. Initial DNA extractions and PCR experiments of GSL samples have not shown a hydrogenase gene thus far. Therefore, our current research has focused on testing the primers against controlled algae samples that are known to possess the gene. If the primers work, we will continue searching for the hydrogenase gene in the GSL samples. If not, primers may need resequencing.
Thymine Dimer Formation in Halophilic DNA
Ultraviolet damage in DNA is responsible for thymine dimer formation within the DNA. Repair mechanisms in halophilic DNA is unique in that, unlike prokaryotes and eukaryotes, they do not show a nucleotide excision repair (NER) system. Rather, it is postulated that halophiles use carotenoids to aid in the photoprotection of cellular DNA. Using halophile DNA, we first subjected it to UV radiation to trigger thymine dimer formation, then extracted and isolated the DNA. Finally, color development was accomplished and the DNA damage was compared to unprotected control DNA. The damage in halophilic DNA was found to be significantly less than the unprotected control, indicating that carotenoids likely aid the halophilic DNA in photoprotection of the cell.
Brine Shrimp Nutritional Requirements
Artemia are commonly used to feed economically valuable fish due to their small size, easy transferability, and long shelf life. As a result, there is interest in viable, nutrient dense brine shrimp. It has been noted by local researchers that brine shrimp are unable to reach adulthood in media other than Great Salt Lake water. The logical explanation for this is that there is a nutrient in the Great Salt Lake water that Artemia needs to survive and grow into adulthood. This experiment is designed to isolate the food source that brine shrimp require to grow into full adulthood. The first set of experiments test the shrimp under five nutritional conditions: 1. no algae 2. Dianaliella virdis 3. Carteris 4. Pennate diatoms 5. Coccochloris sp. So far, only the cysts in the Coccochloris have hatched. Experiments are ongoing to verify these preliminary data.
Halite Crystal Dissolution:
Halophilic Archaea have been revived from ancient salt deposits and have been shown to survive for millions of years in halite crystals. These salt crystals are formed around fluid inclusions, which may provide refuge to microorganisms as their environment becomes desiccated. To test the dormancy potential of Great Salt Lake (GSL) halophiles, recently formed hopper-shaped halite crystals from the hypersaline North Arm of GSL were examined. Methods of crystal selection, sterilization, and dissolution were established. Eighty-eight percent of the crystals dissolved contained viable halophiles. Each of these cultures contained all cellular morphotypes present in GSL brine. Archaeal strains were isolated from these crystal cultures. These halite crystals have been shown to be an excellent source of novel strains of GSL microorganisms. Isolated species were characterized using 16S rDNA gene sequencing. Implications of this study included the potential use of this method in isolating novel microorganisms from other hypersaline ecosystems.
Avian DNA Phylogenetic Tree
The classification system of bird species is commonly done by morphology traits such as body size, shape, and color. This type of classification is helpful for grouping but carries two major flaws; cryptic species and convergent evolution. DNA from birds can help clarify misleading classification. We used liver tissue samples from various birds extracts, amplified through PCR procedures, and purified the 16S sequence of avian DNA. Following DNA extraction, samples will be analyzed by DNA sequencing and BLAST searching to align the sequences with database date. In this way the species of the bird is determined. The result for each bird is a more accurate avian phylogenetic tree.
Phylogenic Similarities Based on Avian and Reptilian DNA
Previous phylogenic placements of ratites and reptiles have been based on physical attributes of each family. However, comparisons of 12S ribosomal DNA and 16S ribosomal DNA may show a conserved link between ostrich and alligator. Our primary objective is to determine the phylogenic origin of avian lineages and to help explain the many uncertainties of avian evolution.Through muscle tissue obtained by a meat packaging company, mitochondrial DNA was extracted using the Qiagen DNeasy tissue kit. DNA was then amplified using PCR techniques. Results of these amplifications will be presented.
Thioredoxin Reductase in Artemia and Selenium Cytotoxicity
Great Salt Lake is becoming polluted with selenium. To show how the brine shrimp, Artemia, are surviving in the presence of high selenium, gene expression of this organism was investigated. We are looking closely at levels of gene products in pathways involving selenium. RNA was isolated and some made into CDNA to perform PCR. The primers that were used are sequences within the thioredoxin reductase gene, which is known to be involved in selenium resistance. The rest of the RNA will be tested by PCR using primers from flies whose genes upregulate in response to an increase in selenium. In addition, selenium toxicity exponents will be discussed.
The Genetic Structure
Very little is known about the genetic structure of the brine shrimp (Artemia franciscana) population in Great Salt Lake (GSL). Initially we set out to investigate the polymorphism rate in the Artemia population in general. However, the GSL presents a particularly interesting environment since the salinity of the lake varies depending on the season and location. This is especially true when comparing the north and south arms of the lake that are separated by a railroad causeway. Our next set of experiments will be to examine polymorphisms in different regions of the lake that have different salinity levels. We hope that our data will help us understand the biogeography of the Artemia in GSL and whether the environment of GSL has an effects polymorphism levels.
Examining the Role of External Application of Glutamate to C. elegans
Glutamate has long been thought to play a role in the underline biological mechanisms of learning and memory, specifically long-term potentiation. Researchers have turned to a number of model organisms to explain, the underline mechanisms of learning and memory, one is C. elegans. C. elegans have a number of learning and mutants present to help researchers identify. Using the mechanosensory paradigm and the mutants avr-15, nmr-1, glr-1, eat-4, and the wild-type n2, the focus of this research is to influence the rates at which these worms learn.
Halophites in Salt Crystals
Salt Crystals from the North Arm of Great Salt Lake are rich in dormant life. Microorganisms known as halophiles ("salt loving") were isolated from salt crystals by dissolving these crystals in media, then grown on agar plates. DNA was extracted from these isolated colonies. The 16S rDNA gene was amplified by PCR using primers for halophic archaea. PCR products will be sequenced and compared to a database to reveal the species identity of our isolates. If the species is novel the will be named and classified in their taxonomic genera.