UC San Diego

Since their discovery over one hundred years ago, bacteriophages (phages) play a central role in the ongoing molecular biology revolution. As such, model phages like Lambda and T4 are perhaps the most genetically well-understood biological entities. Yet, critical aspects regarding the life histories of these phages, nevertheless non-model phages, remain unaddressed. This dissertation examines the life history of T4 and non-model phages. In chapter 2, the steps involved in the morphogenesis of T4-like phages were reevaluated. During the final stages of morphogenesis, T4 packages DNA into an empty head that is subsequently joined with a tail to make a mature virion. Bioinformatic analysis of one gene involved in this head-to-tail joining process, gene 4 (gp4), identified a previously unrecognized nuclease motif. Biochemical analysis confirmed the DNA-cutting capabilities of gp4. The nuclease function of gp4 is shown to be conserved in T4-like phages. In aggregate, the data contained in chapter 2 of this dissertation suggests gp4 plays a role in the termination of genome packaging to enable head-tail joining. Chapter 3 is dedicated to the analysis of non-model phages, focusing on those that contain diversity-generating retroelements (DGRs). DGRs are genetic cassettes that introduce sequence variation into target genes. Few isolated phages possess a DGR, yet metagenomic analyses reveal their presence in uncultured viral communities. In chapter 3, a survey identified 92 DGRs that were only found in phages exhibiting a temperate lifestyle. One novel temperate phage that possesses a DGR cassette targeting a gene of unknown function was isolated from Bacteroides dorei. This phage, here named Hankyphage, exhibits broad host-range and is capable of infecting at least 13 different Bacteroides species. Sequencing reads from whole-community metagenomes and viromes were recruited to the Hankyphage genome, highlighting the global distribution of Hankyphage. The results in chapter 3 suggest that targeted hypervariation by temperate phages, such as Hankyphage, may be a ubiquitous mechanism underlying phage-bacteria interaction in the human microbiome.