DNA Modification of the Novel Bacteriophage: Hydrafoxglove and the Isolation, DNA Analysis, and Genomic Annotation of the Novel Bacteriophage: Goose

Saturday, February 16, 2013
Auditorium/Exhibit Hall C (Hynes Convention Center)
Molly Jane Robertson , Del Mar College, Corpus Christi, TX
J. Robert Hatherill , Del Mar College, Corpus Christi, TX
Background: Bacteria are thought to be the most abundant life in the biosphere, yet bacteriophages that infect bacteria have a numerical supremacy. The immense quantity of phage has set the stage for developing unique therapeutic methods of treating pathogenic bacterial diseases while the diversity allows for dynamic insights into the lateral gene transfer between bacteria and bacteriophage. The primary objective of this research is to discover, identify, and manipulate the novel architecture of phage genomes and provide to the growing library of knowledge, known as phage genomics. Methods: In the present study, two novel mycobacteriophages, Hydrafoxglove and Goose, were isolated from the environment using Mycobacterium smegmatis, a mutant strain of bacteria with similar cell wall structure to that of Mycobacterium tuberculosis. Plating the infected bacteria on a soft medium and incubating for 24 hours at 37 degrees Celsius, allows for bacterial cell death to be visualized and plaque morphology to be determined. Samples of phage were added to a copper grid, stained with uranyl acetate, and imaged via transmission electron microscopy (TEM). The genomic DNA from Hydrafoxglove and Goose was quantified using nanodrop spectrometry, and analyzed using restriction digests. The Goose DNA was sequenced using the ion-torrent method and evaluated using the bioinformatic programs DNA Master and Phamerator. Results: Plaque morphology suggests that Hydrafoxglove is an active lytic phage, while Goose is a slow growing temperate phage. TEM images of Hydrafoxglove show a distinctively large capsid, 100nm in diameter, which strongly indicates classification as a subcluster C1 Phage. Images of Goose reveal that the subcluster A10 phage has a capsid of 50 nm in diameter and tail 150 nm long. The genomic DNA of Hydrafoxglove, was evaluated several times by restriction digest analysis, and proved to be resistant to many common restriction enzymes known to cut most phage DNA as well as methylated DNA; therefore, we hypothesize the presence of a DNA sulfur modification, known as phosphorothioation along the DNA backbone. Conclusion: Goose was the first mycobacteriophage is South Texas to be sequenced and used for bioinformatics analysis. The genome is approximately 50,645 bp and contains 87 protein coding genes, including one tRNA. Current studies are being conducted in efforts to determine the genetic patterns contained within the Hydrafoxglove genome, but it is clear this highly sophisticated genetic material has been modified on a level never seen before in mycobacteriophage studies. The arms race between bacteria and bacteriophage has been prevalent and critical for life on Earth for millions of years, but analysis of genetic information being exchanged is just beginning to be explored. Since antibiotic resistance has been recognized as a global threat, it is imperative that we investigate bacteriophage as a means of preventing and treating disease.