UC San Diego

An estimated 10^24 infections per second occur between bacteriophages and bacteria. This dissertation investigates these interactions of E.coli and Salmonella and their prophages. Chapter 1 is an introduction to the history of bacteriophages and their influence on molecular biology and studies of DNA replication. Chapter 2 includes an analysis of the binding of SeqA, a negative regulator of replication, throughout the bacterial chromosome and introduces a new mechanism of how SeqA is potentially involved in transcription regulation and regulating prophage life cycles. This mechanism was found by reanalyzing protein binding data from multiple ChIP-Chip studies and analyzing them with new bioinformatics programs BEDOPS and BEDTools and Markov modeling. The ChIP-Chip data is randomized and both random and observed signals filtered into varying signal strengths. The signals are chopped to 1 bp “regions” in order to get a fine-detailed analysis of the signals and comparison of randomized vs. observed. A method for analyzing GATCs based on pairing is developed and GATCs are randomized GATC locations are compared to observed GATCs. Multiple phage are very low in GATC content. Chapter 3 involves Illumina RNA-Seq analysis of Salmonella and its prophages to observe the Salmonella prophage activity. A workflow was developed using Galaxy and DEBrowser to observe differential expression using DESeq2, EdgeR, and Limma. Chapter 4 involves the investigation and development of an assay to observe genome duplications in vivo using the ParB:parS system and see if prophages recombination proteins are involved in this process. A P1 parS was placed in a region in which duplications occur frequently purH in between rrn operons rrnE and rrnB and compared to a region in which duplications do not occur frequently at argD. A second pMT1 parS is placed in a region in which duplications do not occur frequently at putA. Gifsy-1 prophage recombination genes (recE, recT, ninG) were cloned onto pRHA109-KanR plasmid and duplications can be assayed using pFH4034, which encodes for multiple fluorescent ParB fusions. Chapter 5 is the conclusion and is a perspective on the future of phage-related research. Appendix 1 contains programs and additional RNA-Seq analysis for Chapter 2 and Chapter 3.