Date of Award

5-2012

Degree Type

Thesis

Degree Name

Master of Science

Major

Microbiology

Major Professor

Gary S. Sayler

Committee Members

Timothy E. Sparer, Alison Buchan

Abstract

Bacteriophage transduction events play a contributive role in bacterial gene diffusion in ecosystems comprised of natural microbial populations. The rate at which these phage-mediated genetic transfers occur in wastewater ecosystems is not well understood. This work describes the effectiveness of a genetically engineered biotinylated bacteriophage T4 system as a tool for examining transduction event rates and compositions in environments that pose increased risk of antibiotic resistance proliferation.

Antibiotic use has steadily increased over the past century, giving rise to antibiotic resistance in microorganisms. Agricultural, medical, and industrial use of antibiotics produces waste teeming with residual antibiotic compounds that are collected to form novel microbial ecosystems. These wastewater microbial communities include, among others, bacteria and their viral parasites, bacteriophage. Bacteriophage can transfer genetic material between bacterial hosts through a phenomenon known as transduction, thus creating circumstances that can contribute to the spread of antibiotic resistance genes. Wastewater ecosystems that contain vestigial antibiotic compounds may produce selective breeding grounds for the development of antibiotic resistant microorganisms. This study aimed to elucidate the role of bacteriophage in the spread of antibiotic resistant genes under these environmental conditions using a novel biotin-based tagging and recovery system. This system analyzed the unbiased tagging of bacteriophage particles, infection of host bacteria, and the recovery of biotinylated phage progeny as means to investigate phage-mediated transduction rates.

A recombinant bacteriophage T4 with biotin carboxylase carrier protein (BCCP) fused to the small outer capsid (SOC) acted as the tagged bacteriophage in this work. Numerous techniques for phage propagation and purification were optimized for this application, indicating important information about the feasibility of using this engineered bacteriophage in a natural ecosystem. The genetic instability and susceptibility to selective pressures of the biotinylated bacteriophage, even under optimally controlled conditions, resulted in the deactivation of the phage’s biotinylation capabilities. The results of this examination indicate the impracticality of using this genetic-based biotinylation approach as a method for measuring transduction events rates in natural ecosystems.

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