Abstract
Sbash is a temperate bacteriophage, which was isolated on the host, Mycobacterium smegmatis mc2 155 from soil collected in South Africa. It is classified as cluster I and sub-cluster I2. Its genome consists of 55,832 base pairs and 89 protein-coding genes of which only 25 genes were assigned a function by bioinformatic analysis. We are using a genetic screen to uncover the functions of phage genes for which function is unknown. To begin to uncover the functions of the protein products of the genes in Sbash’s genome, we cloned each gene into the pExTra plasmid and assayed each phage gene for two phenotypes: cytotoxicity, the ability to interfere with host cell growth, and defense, the ability to protect the host cell from infection by other phages. In total, we successfully cloned approximately half of the genes in Sbash’s genome with sizes ranging from 90 bp to nearly 1500 bp. We identified two Sbash genes that defend host cells from infection by other mycobacteriophages. We identified six genes that reduced the growth of host cells when expressed. Here, we report our progress on this project. We have also analyzed genes in Mycobacteriophage Island3, a cluster I1 phage, for cytotoxicity and defense to complete the screen of this phage started by students in previous research groups.
Included in
Discovering Gene Functions in Mycobacteriophage Sbash Using a Genetic Screen
Sbash is a temperate bacteriophage, which was isolated on the host, Mycobacterium smegmatis mc2 155 from soil collected in South Africa. It is classified as cluster I and sub-cluster I2. Its genome consists of 55,832 base pairs and 89 protein-coding genes of which only 25 genes were assigned a function by bioinformatic analysis. We are using a genetic screen to uncover the functions of phage genes for which function is unknown. To begin to uncover the functions of the protein products of the genes in Sbash’s genome, we cloned each gene into the pExTra plasmid and assayed each phage gene for two phenotypes: cytotoxicity, the ability to interfere with host cell growth, and defense, the ability to protect the host cell from infection by other phages. In total, we successfully cloned approximately half of the genes in Sbash’s genome with sizes ranging from 90 bp to nearly 1500 bp. We identified two Sbash genes that defend host cells from infection by other mycobacteriophages. We identified six genes that reduced the growth of host cells when expressed. Here, we report our progress on this project. We have also analyzed genes in Mycobacteriophage Island3, a cluster I1 phage, for cytotoxicity and defense to complete the screen of this phage started by students in previous research groups.