2005 Research Fair Archive - Biology Abstracts
Biochemical Characterization of Novel Isolates from Great Salt Lake
Great Salt Lake (GSL) is a natural hypersaline ecosystem with enormous environmental significance. It is the fourth largest hypersaline lake in the world and the second-most saline. Additionally, the dramatic fluctuation in water levels and salinity creates an ecological backdrop selective for organisms with a high degree of adaptability. The diversity of a hypersaline ecosystem may not be reflected in the macro-fauna, but instead at the microbial level. We have successfully developed biochemical tests that take into account the optimal growth conditions of these halophilic (salt-tolerant) organisms. Specific protocols were adjusted and applied to five GSL isolates, including the following assays: Gram staining, antibiotic resistance, catalase, oxidase, and gelatinase activity, and Tween-80 hydrolysis. Results from these tests will be presented and their significance as an instrument for taxonomic classification will be discussed.
Effects of Glucose Concentration on
E. faecalis is an opportunistic pathogen and a common cause of nosocomial infections. Adherence to epithelial cells is one of the main virulence factors associated with this microbe. The Esp gene encodes for a surface protein that plays a role in the adherence and pathogenicity of the bacterium. Esp expressing strains have been found to be more virulent (Shankar et al. 2004) and persist longer in murine models of urinary tract infection (Shankar et al. 2001). The appearance and strength of Esp gene expression has been linked to glucose concentration in the growth medium (Tendolkar et al. 2004). Glucose is absorbed by the brush border of the intestinal epithelial cells, and must interact with the E. faecalis that has adhered to those same cells. Our study examined the expression of the Esp gene in glucose media. Various strains of E. faecalis were grown in differing concentrations of glucose, and the expression of the Esp gene was determined.
The Pink Waters of Great Salt Lake:
Halophilic Archaea are much more resistant to ultraviolet (UV) light damage than Escherichia coli and other species of Bacteria. 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.
Several strains of Archaea, including Halobacterium NRC-1 and an unidentified isolate from the Cargill salterns near Great Salt Lake, Utah were examined for sensitivity to UV light. UV survival experiments showed that the LD50 (lethal dose for 50% of the cells) for the Cargill isolate is 24 times that of E. coli. Other halophilic Archaea strains give similar results. Data will be presented from mutant strains deficient in pigment syntheses that support the notion of photoprotection by these carotenoid compounds.
Measuring DNA Damage: A Dot Blot Immunoassay for Thymine Dimers
The purpose of our experiment was to quantify the damage that occurs to DNA when exposed to ultraviolet (UV) light. Following varying amounts of UV exposure, salmon sperm DNA (control) was blotted on a nitrocellulose membrane. The membrane was treated with an antibody to thymine dimers, and then subjected to a detection process. The control irradiated DNA revealed that thymine dimers could be detected and that the response was linear. This assay was then applied to an experimental question: Do carotenoid compounds protect DNA from damage (thymine dimers)? A UV-resistant, carotenoid-containing microorganism from Great Salt Lake, “Cargill,” and a UV-sensitve, carotenoid mutant strain of Cargill were subjected to UV exposure and tested for relative thymine dimer levels under the same protocol. These results and a comparison with controls will be discussed.
Adherence Studies of E. faecalis Using an In Vitro System
Enterococcus faecalis is a G+ bacterium that is the second most common cause of hospital-acquired infections. Our research encompasses adherence assays that we have conducted using several E. faecalis isolates obtained from St. Francis Hospital in Hartford, CT. We are testing the adherence of enterococci to human cells, as well as to canine cells, to determine if the observed adherence is human cell specific. We also wished to determine whether assay media serum concentration had an effect on bacterial adherence, and eukaryotic cell retention. We have tested the adherence of E. faecalis to three cell types: HTB-5, HT-1080 and MDCK, using a previously established in vitro method. HTB-5 was derived from a cancerous human bladder, HT-1080 was isolated from a human fibrosarcoma, and MDCK was established from canine kidney. Our results indicate that E. faecalis adheres preferentially to human cells regardless of tissue origin. Also, assay serum concentration did not alter bacterial adherence to the target cells but also did not significantly increase eukaryotic cell retention during the assay.
Avian Phylogeny According to 18S & 12S Gene Sequences
For obvious reasons animals have traditionally been classified according to their morphology. However, with advancements in molecular biology we are now able to classify living things according to genetic criteria. In this study template DNA was obtained from four different bird liver samples (Barn Owl, Turquoisene Parrot, Corvus corvax, and Buteo jamaicensis). PCR was then employed to amplify cytochrome oxidase I and 12S ribosomal gene sequences using the respective primers. This information was then used to distinguish each individual bird type. As this information is continually obtained from numerous other species we hope to be able to substantiate and further reform previously organized phylogenies.
The Revival of Halophilic Archaea From
Halophilic Archaea have been revived from ancient salt deposits and shown to survive for millions of years in hopper-shaped halite crystals. These 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 crystals from the hypersaline North Arm GSL were examined. Methods of crystal selection and dissolution were established. The ability of different cell morphotypes present in GSL to survive crystal formation, dormancy, and dissolution was tested. The ability of halophage to survive was determined using high-resolution electron microscopy. Eighty-eight percent of crystals dissolved had viable halophiles. Each crystal had all morphotypes present in GSL brine. No halophage were detected. Implications of this study are discussed, including the application of these methods in determining the presence of halophiles in the ancient salt lake hypothesized to have existed on Mars.
Transduction of Arginine Synthesis in E. coli by Temperate Bacteriophage P1
In transduction a bacteriophage vector transfers genetic material from one bacterial strain to another. Such transfers can produce observable phenotypes, allowing us to quantify the efficacy of the phage in transducing a particular trait. In this experiment bacteriophage P1 lysate prepared from a wild type donor strain of E. coli was introduced to an arginine negative E. coli auxotroph. The auxotrophic acceptor cells were then plated on selective media to assess whether the gene pertinent to arginine synthesis was transduced. The percentage of transductants was then calculated.
Lipid Analysis as a Method of Differentiating Bacteria from Archaea
Prokaryotes have recently been subdivided into two Domains, Bacteria and Archaea, distinguishing them from the third domain, Eukarya. Since both types of prokaryotes are characterized by a single cell existence and a lack of nucleus, unidentified isolates may be difficult to differentiate. In an attempt to establish methodology for distinguishing Bacteria versus Archaea from unknown Great Salt Lake (GSL) isolates, we developed a protocol adapting the Bligh-Dyer lipid analysis procedure. A control Archaea strain, Halobacterium NRC-1, gave a negative result as predicted, while a control Bacteria strain, Salmonella typhi., gave a positive result as predicted. Data from GSL isolates will be presented.
The Effects of Curcumin on C. elegans <i> sel-12 Mutants
Alzheimer’s disease is a progressive neurological disorder which gradually destroys a person’s memory and ability to learn, make judgments, communicate and carry out daily activities. Curcumin is a spice that has been documented to help relieve symptoms of Alzheimer’s with its anti-inflammatory and antioxidant activities. In this study, C. elegans were used as a model organism to study the effects of curcumin on Alzheimer’s. Mutant sel-12 C. elegans served as a model of Alzheimer’s disease in this study, as they have a mutated protein homologue that has been implicated in Alzheimer’s in humans. Since egg laying and thrashing in sel-12 worms is abnormal, those parameters served as a marker for assessing the effects of curcumin on the symptoms of the sel-12 mutation. Preliminary data suggests that curcumin had no effect on the sel-12 mutants.
Antibody Staining-Assay for Detection
Artemia franciscana, the brine shrimp from the Great Salt Lake, is a potentially interesting organism in which to study development and neurobiology. Much of the information that is known about how genes control the development arthropods has come from studies of the fruit fly Drosophila melanogaster. In order to develop Artemia as a model, we are attempting to use Drosophilia antibodies across species to stain Artemia to identify developmentally important genes. A standard protocol is not available for antibody staining of developing Artemia and a large amount of our time has been devoted to working out an effective protocol. Our results will be discussed.