Investigating Cytotoxicity and Defense functions of bacteriophage Larva genes in host Mycobacterium smegmatis
Poster Number
34
Session Title
Poster Session 2
College
College of Arts and Sciences
Department
Biology
Faculty Mentor
Victoria Frost, Ph.D.
Abstract
It is estimated that approximately 75% of gene functions within any given phage cluster remain unknown. Two phage traits that can be measured phenotypically were investigated: cytotoxicity, which causes host cell lysis, and superimmunity, which results in protection of the host from infection by similar phages. Five genes (35, 42, 46, 49, and 59), from a subcluster K5 bacteriophage named Larva, were amplified by the polymerase chain reaction (PCR) and assembled into a cloning plasmid (pExTra) using isothermal assembly. Initially, chemical transformation of Escherichia coli with the pExTra + gene insert, enabled amplification of the plasmids. Clonal PCR verified the presence of the inserts that were selected for on Kanamycin plates. Host bacteria Mycobacterium smegmatis cells were electroporated with pExTra + insert and the inducer molecule anhydro-tetracycline (aTc) was used to induce expression of the relevant genes. The expression of Larva genes 35, 42, and 59 were non-toxic to the host. However, Larva gene 46 and 49 were toxic, causing the bacteria to lyse. Evidence of this was further revealed in the defense assay where expression of gene 49 caused an absence of bacterial lawn growth. The expression of Larva genes 42 and 59 highlight a defense mechanism associated with these gene products that protects the host from attack by other phage in the same cluster. Further work to elucidate which host-parasite proteins are in contact to cause the phenotypic changes will be investigated using protein-protein interaction (PPIs) assays and hopefully reveal important clues towards understanding phage gene function.
Type of Presentation
Poster presentation
Grant Support?
South Carolina INBRE Program, HHMI and SEA-GENES
Investigating Cytotoxicity and Defense functions of bacteriophage Larva genes in host Mycobacterium smegmatis
It is estimated that approximately 75% of gene functions within any given phage cluster remain unknown. Two phage traits that can be measured phenotypically were investigated: cytotoxicity, which causes host cell lysis, and superimmunity, which results in protection of the host from infection by similar phages. Five genes (35, 42, 46, 49, and 59), from a subcluster K5 bacteriophage named Larva, were amplified by the polymerase chain reaction (PCR) and assembled into a cloning plasmid (pExTra) using isothermal assembly. Initially, chemical transformation of Escherichia coli with the pExTra + gene insert, enabled amplification of the plasmids. Clonal PCR verified the presence of the inserts that were selected for on Kanamycin plates. Host bacteria Mycobacterium smegmatis cells were electroporated with pExTra + insert and the inducer molecule anhydro-tetracycline (aTc) was used to induce expression of the relevant genes. The expression of Larva genes 35, 42, and 59 were non-toxic to the host. However, Larva gene 46 and 49 were toxic, causing the bacteria to lyse. Evidence of this was further revealed in the defense assay where expression of gene 49 caused an absence of bacterial lawn growth. The expression of Larva genes 42 and 59 highlight a defense mechanism associated with these gene products that protects the host from attack by other phage in the same cluster. Further work to elucidate which host-parasite proteins are in contact to cause the phenotypic changes will be investigated using protein-protein interaction (PPIs) assays and hopefully reveal important clues towards understanding phage gene function.
