Degree Type

Dissertation

Date of Award

2000

Degree Name

Doctor of Philosophy

Department

Veterinary Microbiology and Preventive Medicine

First Advisor

Donald L. Reynolds

Second Advisor

Gregory J. Phillips

Abstract

The widespread emergence of multi-antibiotic resistant bacteria has increased the need for alternatives to conventional antibiotic therapy. Accordingly, a significant amount of effort has been made to investigate the potential use of bacteriophages as prophylactic and therapeutic agents for bacterial infections. In this study, molecular biological techniques were applied to construct a lysogen of lytic bacteriophage lambda in an attempt to combat with multi-antibiotic resistant bacteria by a novel method of lytic phage delivery;To accomplish this goal, two plasmid-based site-specific recombination (SSR) systems for integration and recovery of DNA constructs from Escherichia coli and Salmonella typhimurium chromosomes were developed. The two systems are mediated by SSR machineries of bacteriophages lambda of E. coli and P22 of S. typhimurium. These systems utilize plasmid vectors with conditional replicating origin of replication and provide stable chromosomal integration of genes at specific bacteriophage attachment sites without disruption of any host gene or a need for antibiotic selection. E. coli contains attachment sites for both bacteriophages. When the two systems are applied consecutively, two different genes can be integrated at two specific locations. The integrated plasmids of both systems can also be completely excised and recovered from the host chromosomes to observe any genetic changes, e.g. by DNA sequencing. Both systems are also very applicable in construction of bacterial strains as well as live E. coli and S. typhimurium recombinant vaccines expressing foreign genes of interest;To construct a lysogen of lytic bacteriophage lambda, both SSR systems were applied. A lytic mutant (cI-) of bacteriophage lambda was marked with an antibiotic resistant gene cassette to facilitate a lysogen selection. The P22 SSR system helped integrate functional lambda repressor gene (cI) into a non-pathogenic E. coli strain and the marked lytic lambda phage lysogenized in the presence of the helper plasmid of the lambda SSR system. The lysogen demonstrated its efficacy in decreasing number of lambda sensitive E. coli. This lytic phage lysogen construction strategy can be applied for other bacteriophages. A pool of different lysogens infects a wider range of bacteria and could be utilized as alternatives to the use of antibiotics to control bacterial infections.

DOI

https://doi.org/10.31274/rtd-180813-15276

Publisher

Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/

Copyright Owner

Ratree Platt

Language

en

Proquest ID

AAI9962838

File Format

application/pdf

File Size

113 pages

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