Abstract
Bacteriophages represent an absolute majority of all organisms in the biosphere and offer a special perspective on the diversity, origins, and evolution of viruses. Over 500 Mycobacteriophages have been isolated and sequenced thus far. These phages can be grouped into clusters, where genomes within a cluster have recognizable sequence similarity that spans over more than 50% of their genome lengths. They are replete with novel genes, which do not have homologs in any database. To predict the function of these genes, it is useful to determine if the genes are required for growth of the phage. Bacteriophage Recombineering of Electroporated DNA (BRED) was developed in the Hatfull laboratory to construct targeted mutations in mycobacteriophages and determine the essentiality of genes for lytic phage growth. However, this method is expensive and time-consuming; therefore, we will develop a high throughput method of determining gene essentiality using ethyl methanesulfonate (EMS) to mutagenize mycobacteriophages. The survivors of this treatment will be pooled, DNA will be extracted, and deep sequencing of phages will be conducted. By analyzing the sequencing results we will be able to determine the number of nonsense and missense mutations, and conclude which genes are essential (survivors not found) and nonessential (survivors sequenced) during lytic growth. Mycobacteriophage Giles was exposed to different amounts of EMS ranging from 5–40 μl; any amount of EMS over 8 μl caused complete phage death and 7 μl of EMS yielded a two-log decrease in phage growth. It was determined that multiple rounds of 7 μl EMS mutagenesis would be required to cause a large amount of mutations. After each round of mutagenesis DNA will be extracted and analyzed to determine gene essentiality. Determination of the set of essential genes in defined infection and growth conditions will provide important insights into the biology of the phage life cycle.