Title

The Roles of Moron Genes in the Escherichia Coli Enterobacteria Phage Phi-80

Date of Award

2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biological Sciences

First Advisor

Ray Larsen

Second Advisor

Craig Zirbel (Committee Member)

Third Advisor

Vipa Phuntumart (Committee Member)

Fourth Advisor

Scott Rogers (Committee Member)

Fifth Advisor

George Bullerjahn (Committee Member)

Abstract

The TonB system couples cytoplasmic membrane-derived proton motive force energy to drive ferric siderophore transport across the outer membrane of Gram-negative bacteria. While much effort has focused on this process, how energy is harnessed to provide for transport of ligands remains unknown. Several bacterial viruses ("phage") are known to require the TonB system to irreversibly adsorb (i.e., establish infection) in the model organism Escherichia coli. One such phage is φ80, a "cousin" of the model temperate phage λ. Determining how φ80 is using the TonB system for infection should provide novel insights to the mechanisms of TonB-dependent processes. It had long been known that recombination between λ and φ80 results in a λ-like phage for whom TonB is now required; and this recombination involved the λ J gene, which encodes the tail-spike protein required for irreversible adsorption of λ to E. coli. Thus, we suspected that a φ80 homologue of the λ J gene product was responsible for the TonB dependence of φ80. While φ80 has long served as a tool for assaying TonB activity, it has not received the scrutiny afforded λ. Indeed, only a small portion of its genome (not including a J gene homolog) was known. To facilitate the use of φ80 as a molecular tool, we determined and annotated the full genome of the φ80vir strain commonly used for TonB studies. The 46,285-bp φ80vir genome contained 71 predicted open reading frames, in which the structural genes showed strong synteny with other known lambdoid viruses, with an overall degree of mosaicism indicative of multiple genetic exchanges with other viruses. There was also evidence of unique gene acquisition in the form of morons, one of which, termed cor, when cloned and expressed in E. coli, blocked TonB-dependent transport of ferric siderophore and irreversible adsorption of φ80vir to the TonB-dependent outer membrane protein FhuA. These and other findings reported in this dissertation provide a foundation for the use of φ80vir as a tool to dissect the mechanisms of TonB-dependent energy transduction.